BRPI0618178A2 - methods for adapting mammalian cells - Google Patents

Abstract

<B> METHODS FOR ADAPTING MAMMALIAN CELLS <D> Methods of adapting cells, for example, mammalian cells, to a cell culture process are provided. When adapted cells are genetically modified and used for protein production, they exhibit beneficial characteristics such as, for example, being able to reach higher cell densities and / or obtain a higher overall yield of the protein produced.

Description

Patent Descriptive Report for "METHODS FOR MAMMALIAN CELL ADAPTATION".

CROSS REFERENCE WITH RELATED ORDERS

This application is co-pending, shares at least one common inventor, and claims priority for provisional U.S. patent application number 60 / 732,818, filed November 2, 2005, the contents of which are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

This application is generally related to mammalian cell uptake methods, for example, untransfected mammalian cells, for the production of a therapeutic protein of interest. In particular, this application relates to the adaptation of untransfected mammalian cells for superior performance in mammalian cells. bioreactors.

Proteins can be produced using well known recombinant techniques. Transformed cells are commonly cultured in a controlled environment, for example, a bioreactor. Most of the large-scale commercial manufacturing strategies employ suspension cell cultures grown in large stirred tank reactors. Most cell lines, however, do not easily perform well in high cell density protein production processes (e.g., fed batch processes) and / or cannot achieve the desired high cell densities. Many inhibitors are present or accumulate in the cell culture medium during a production cycle; These inhibitors may be byproducts generated by metabolic processes such as lactate or ammonia, among others. Cell lines typically grow and are initially maintained in conditions that are designed to aid in the spread and / or survival of these cell lines. The conditions used during protein production (eg conditions found in production bioreactors), however, may be quite different, with, for example, secondary metabolites and / or other components present and / or accumulated as the production of protein progresses, which may have potential deleterious effects on the cell line. As non-limiting examples, such deleterious effects may comprise a decrease in viable cell density and / or a decrease in final titration, in addition to a decrease in the amount and / or quality of the protein produced.

In the practice of common methods, various experiments are performed to determine if a cell line has adapted to growth and / or production in a protein production process such as a bioreactor fed batch process. This experimentation often requires various clonal cell lines and / or transfection pools, typically followed by adaptation of cells to growth media that will be used in a production bioreactor (for example, a serum-free suspension or a "defined medium"). Following adaptation of the cell line, additional rounds of screening and optimization are typically performed on laboratory scale bioreactors. Non-individual clones that exhibit good expression phenotypes on preliminary screening will then typically be subjected to further experimentation to determine which clones also exhibit acceptable growth and / or viability characteristics in a bioreactor, for example, a batch-fed bioreactor. These extensive tracking processes are expensive and particularly laborious for the trader.

Therefore, methods for adapting cells to protein production conditions that include, without limitation, bioreactor conditions are required. A method of adapting untransfected host cells is also required before they are transformed such that there are no or minimal detrimental effects on one or more cell culture characteristics (eg titration, viability, quality). etc.) after transfection and transition to protein production conditions.

The present application relates to methods for adapting mammalian cells to a high density protein production process such as a fed batch process. The processes of the invention may involve both untransfected mammalian cells and genetically engineered host cells. In some embodiments, untransfected mammalian cells are adapted to the combined production conditions, such as those used during protein production. For example, untransfected mammalian cells may be adapted to a production medium used in a bioreactor during a production cycle.

Different methods may be employed to adapt untransfected mammalian cells to production conditions. In certain embodiments, cells are grown in a medium of production and / or adaptation. In certain embodiments, cells are cultured in a medium adaptation by performing standardized repeat division cycles. For example, cell subpopulations may be passed one or more times, for example, every three to four days. In certain embodiments, a production and / or adaptation medium generally has higher levels of nutrients, vitamins and / or trace elements compared to a standardized growth medium. In certain embodiments, the cells are then allowed to undergo a recovery period between the passages, in which the past cells grow in a standard growth medium, for example, during one of the cycles, before the cells are returned to a production medium. / or adaptation.

In certain embodiments, cells are adapted by their fed-batch mode passage, where the subpopulations of the cells are divided or passed every seven to eight days. In certain ways, the passage of cells after this longer duration allows a buildup of secondary metabolites in the cell culture medium. In certain embodiments, adapted cells gain tolerance and / or begin to collect these secondary metabolites. In certain embodiments, these secondary metabolites comprise inhibitors and / or metabolites that typically accumulate in a bioreactor during a production cycle, such as lactate and / or ammonia. In certain embodiments, cells are adapted for growth in a medium that includes one or more inhibitors that include, without limitation, lactate, ammonia, alanine, glutamine, and / or aceto lactate. In certain embodiments, such inhibitors and / or metabolites are consistent with inhibitors and / or metabolites typically found in an umbiliorator during a production cycle.

In certain embodiments, non-transfected mammalian cells may be adapted by culturing (and optionally dividing every three to four days) in a standard growth medium that is supplemented with inhibitors such as alanine, glutamine, aceto. -lactate, ammonia and lactate. In certain embodiments, cells adapted in this way are passed every three to four days. In certain embodiments, the concentrations of these inhibitors correspond to the concentrations typically found in a bioreactor during the conditions used for protein production. In certain embodiments, concentrations of the same inhibitors include about 2 to about 10 g / l lactate or about 0.1 to about 0.5 g / l ammonia, mimicking typical dobioreactor conditions. In certain embodiments, cells adapted to dobioreactor conditions by one or more methods of the present invention exhibit certain characteristics or phenotypes and / or may develop such phenotypes in which cells begin to pick up lactate and / or other secondary metabolites such that Lactate and / or other secondary metabolites levels actually decrease over one or more periods of time during the production cycle. These phenotypes can be traced, as they exhibit desirable qualities in a high density fed batch production bioreactor.

In certain embodiments, cells adapted by one or more methods of the present invention are host cells that have not been transfected to produce a protein of interest. In certain embodiments, such adapted host cells are then screened for one or more desirable characteristics. In certain embodiments, after a subpopulation is screened for one or more desirable characteristics, cells may be genetically engineered (e.g., transfected) to create a cell line that produces a protein of interest. In certain embodiments, this adapted cell line is placed in a bioreactor where the cell line easily adapts to a high cell density protein production process such as a fed batch process. In certain embodiments, as a result of prior adaptation of untransfected host cells, for example mammalian host cells, the genetically engineered cell line need not transition from a standard growth medium to a production medium and / or transitions. with minor and / or less severe deleterious effects. In certain ways, prior adaptation minimizes the potential deleterious effects on cell alignment, and helps to ensure cell line performance and accelerates developmental chronology. In certain embodiments, such adapted cell line may pick up secondary metabolites during the protein production cycle. Those skilled in the cell culture technique can easily determine which components form a standard growth medium and a standard production medium. In certain embodiments, protein growth and production conditions differ in the composition of the cell culture media used. During the growth and / or protein production stages, the conditions of the umbilioror may be altered and / or supplements may be added to increase productivity and / or maintain the viability of the cell line. Supplements may include a food medium and / or one or more additives. Those skilled in the art will be able to select supplements by appropriate means. Additional supplements for the media will depend on the desired protein product, the parameters (e.g., components, pH etc.) of the cell culture medium, the methods employed throughout the growth and / or production process, and or any of several other factors known to those skilled in the art.

In certain embodiments, untransfected mammalian cells are adapted by a method comprising culturing untransfected mammalian cells in an adaptation medium, performing one or more repetitive cycles of division (e.g., every 3 or 4 days) which comprise the division of untransfected mammalian cells adaptation name, a recovery period in which cells are cultured in a standard growth medium, and the screening of cells and selection of a subpopulation displaying a phenotype. growth and / or viability compared to an unadapted version of cells when cultured under conditions of a production bioreactor. In certain embodiments, cells are transfected with a gene that co-differs a protein of interest, and cultured in a production bioreactor to express the protein of interest. In certain embodiments, a mismatch medium contains increased levels of nutrients, vitamins and / or microelements compared to said standard growth medium. In some embodiments, an adaptation medium contains an increased amount of inhibitor metabolites compared to a standard production medium prior to cell culture.

In certain embodiments, non-transfected mammalian cells are adapted by a method comprising culturing untransfected mammalian cells in an adaptation medium that has been supplemented with by-products of primary carbon metabolism, the performance of one or more repetitive cycles of division comprising dividing untransfected mammalian cells approximately every 3 or 4 days on the adaptation medium, a recovery period in which cells are grown in a standard growth medium, the accumulation of levels of side products, and screening of untransfected mammalian cells and selecting a subpopulation that exhibits an α-primed phenotype compared to an unadapted version of untransfected mammalian cells when cultured in said adaptation medium. In these embodiments, these side products are one or more of Iactate, ammonia, alanine, glutamine and / or acetolactate. In certain embodiments, olactate is initially present at a concentration of about 2 to about 10 g / l. In certain embodiments, ammonia is initially present at a concentration of from about 0.1 to about 0.5 g / l. In certain embodiments, the cells are adapted to collect said side products from primary carbon sources.

In certain embodiments, untransfected mammalian cells are adapted by a method comprising culturing untransfected mammalian cells for a duration of about 7 or 8 days in a pre-conditioned medium that has an accumulation of at least one inhibitory metabolite, prior to division of untransfected mammalian cells, and tracing of untransfected mammalian cells and selection of a subpopulation exhibiting an enhanced phenotype in a production bioreactor. In certain embodiments, the cells undergo a recovery period by culturing the cells in a standard growth medium for a duration of about 3 to about 4 days prior to cell division. In certain embodiments, a conditioned medium contains an accumulation of inhibitory metabolites by at least one metabolic process of untransfected mammalian cells. In certain embodiments, this inhibitor metabolite is one or more of ammonia, alanine, glutamine, acetolactate and / or lactate. In certain embodiments, these inhibitor metabolites are consistent with those found in a production bioreactor at the end of a typical commercial scale feedback batch process.

Any one of a number of suitable procedures and / or culture media (e.g. inoculum media, feed media, etc.) may be used for culturing cells in the protein production process. Both serum-containing media and non-serum-containing media can be used. For example, in certain embodiments, cells grow in a defined medium. In certain embodiments, cells grow in a half complex. In addition, one or more specific culture methods may be used, altered and / or optimized for cell culture, as appropriate for the specific cell type and protein product. These procedures are well known and understood by those skilled in the art and skilled in the cell culture technique. Other features and advantages of this specification will be apparent from the following description of certain embodiments, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1a shows the density of viable seven day fed batch cells.

Figure 1b shows standard dividing viable cell density.

Figure 1c shows the density of viable cells.

Figure 2a shows the growth rate of the monoclonal antibody cell line.

Figure 2b shows accumulated integral density of viable cells during fed batch.

Figure 3 shows cell densities of insulin-free cell cultures and control cell cultures.

Figure 4 shows the viability of cell cultures adapted to the absence of insulin and control cell cultures.

Figure 5 shows accumulated IVCD of insulin-free cell cultures and control cell cultures.

Figure 6 shows cell densities of insulin-free cell cultures and control cell cultures.

Figure 7 shows the viability of cell cultures adapted to the absence of insulin and control cell cultures.

Figure 8 shows accumulated IVCD of insulin-free cell cultures and control cell cultures.

Figure 9 shows Iate specific consumption rate of cell cultures adapted to the absence of Insulin and control cell cultures.

DEFINITIONS

According to a long-established convention, the terms "one" and "one" mean "one or more" when used in this application, including the claims. Although the invention has been described with some degree of particularity, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art in light of this specification. Accordingly, it is intended that all such alternatives, modifications and variations, which fall within the spirit and scope of the invention, be encompassed by the claims.

The term "host cell" refers to cells that are capable of being genetically engineered and / or capable of growing and surviving in a cell culture medium. Typically, cells may express a large amount of an endogenous or heterologous protein of interest, and may either retain the protein or secrete it into the cell culture medium.

Host cells are typically "mammalian cells" comprising non-limiting examples of vertebrate cells, including human hamsterchinese ovary (CHO) hamster (CHO) kidney (293) kidney cells, normal fetal diploids of macaque rhesus (FRhL-2) and murine myeloma (eg, SP2 / 0 and NSO). Those skilled in the art will know other host cells that may be used according to the methods and compositions of this invention. feels invention.

The term "cell culture medium" refers to cells in a nutrient-containing solution to support cell survival under conditions in which cells may grow and / or produce a desired protein. The term "inoculum medium" or "inoculum medium" refers to a nutrient-containing solution or substance in which a cell culture is initiated. In certain embodiments, a cell culture is supplemented one or more times with a "feed medium" with which cells are fed after the start of culture. In certain embodiments, a "feed medium" contains nutrients similar to the inoculation medium, but is a solution or substance with which cells are fed after the start of culture. In certain embodiments, a feed medium contains one or more components that are not present in an inoculation medium. In certain embodiments, a feed medium contains one or more components that are not present in an inoculation medium. In certain embodiments, a feed medium is devoid of one or more components present in an inoculation medium. Those skilled in cell culture will know, without unnecessary experimentation, which components constitute these inoculation and feeding media. Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids and / or trace elements. to a cell for its growth and survival.

The term "cell culture characteristic" as used herein refers to an observable and / or measurable characteristic of a cell culture. The methods and compositions of the present invention are advantageously used to improve one or more cell culture characteristics. In certain embodiments, improving a cell culture trait comprises increasing the magnitude of a cell culture trait. In certain embodiments, the improvement of a characteristic cell culture comprises decreasing the magnitude of a cell culture feature. As non-limiting examples, a feature of cell culture may be increased growth, increased viability, increased integrated viable cell density, increased titration and / or increased specific cell productivity. Those skilled in the art will know other cell culture characteristics that can be improved by using the methods and compositions of the present invention.

The term "growth medium" or "standard growth medium" refers to a medium containing nutrients and supplements that allow cells or a cell line to divide and grow. In certain embodiments, the term "production medium" refers to an enriched growth medium that allows high levels of a protein of interest to be expressed. In certain embodiments, a production medium generally contains higher levels of nutrients, vitamins, microelements, and / or other components of the medium as compared to a standard growth medium. The term "adaptation medium" refers to a medium that submits cells to the conditions of protein production that exist in bioreactors (for example, stadiofinal production bioreactors), which prepares cells for proteinase / or production conditions. which minimizes the potentially deleterious effects of the transition of cells from growing conditions to protein production conditions. In certain embodiments, a means of adaptation includes the presence of one or more secondary metabolites, for example those generated by metabolic processes, including, without limitation, lactate and / or ammonia. In certain embodiments, an adaptation means includes one or more inhibitors which include, without limitation, lactate, ammonia, alanine, glutamine and / or acetolactate. In certain embodiments, a means of adaptation comprises a means of production. In certain embodiments, an adaptation means simulates one or more characteristics of a production medium. In certain embodiments, adaptation of cells to an adaptation medium results in cells that exhibit diminished or less severe deleterious effects when such adapted cells are changed from a growth or adaptation medium to a production medium. This means of production and / or adaptation may be combined with production, for example, by supplementing means of production with these metabolites and / or inhibitors and / or by accumulating these metabolites and / or inhibitors in a cell culture. long lasting.

The term "defined medium" as used herein refers to a means by which the composition of the medium is known and controlled.

The term "complex medium" as used herein refers to a medium containing at least one component whose identity or amount is unknown or uncontrolled.

The term "cell line" generally refers to primary host cells that have been transfected with exogenous DNA, for example, DNA encoding the desired protein of interest. In certain embodiments, cells derived from genetically modified cells form the cell line, and are placed in a cell culture medium to grow and produce the protein product of interest. In some embodiments, primary host cells are transfected with exogenous DNA encoding a desired protein and / or containing control sequences that activate the expression of either endogenous or heterologous linked sequences. In certain embodiments, a cell line comprises primary host cells that have been transfected with exogenous DNA and express a protein of heterologous interest. In certain embodiments, a cell line comprises primary host cells that have not been transfected with exogenous DNA and express an endogenous protein of interest.

The "growth phase" of a cell culture medium refers to the period when cells undergo rapid division and grow exponentially, or almost exponentially. Growing phase conditions may include a temperature of about 35 ° C to 42 ° C, generally around 37 ° C. The duration of the growth phase and growing conditions in the growth phase may vary, but is generally known to those skilled in the cell culture technique. Typically, during growth phase, cells grow in a "growth medium" or a "standard growth medium". In certain embodiments, a cell culture medium in a growth phase is supplemented with a feed medium.

The "transition phase" occurs during the period in which the cell culture medium is being changed from conditions consistent with the growth phase to conditions consistent with the production phase. During the transition phase, factors such as temperature are often quite frequent. changed. In certain embodiments, a cell culture medium in a transition phase is supplemented with a feed medium. The methods of the present invention are useful for minimizing the potentially deleterious effect of changing a cell culture from the conditions of the growth phase to those of the production phase.

The "production phase" occurs after the growth phase and the transition phase. Exponential cell growth is already over, and protein production is the main goal. Typically, during the production phase, cells grow in a production medium. A means of production may be supplemented to start production. In certain embodiments, a cell culture medium in a production phase is supplemented by a common feeding medium. In certain embodiments, the temperature of the cell culture medium during the production phase is generally lower than during the growth phase. As is known in the art, in many cases, this decreased temperature facilitates protein production. The production phase continues until a desired goal is reached.

The phrases "division", "passage" and "subculture" refer to the process of dividing a cell population into two or more subpopulations. For example, a cell population growing in a cell culture medium may be passed or subcultured by removing a subpopulation of cells from the cell culture medium, and diluting those subpopulations to a lower density of viable cells. At certain times, a subpopulation of cells may be diluted to a similar volume with fresh medium. In certain embodiments, the cell subpopulation is diluted with a cell culture medium that is similar or identical to the cell culture medium in which the original cell population was growing. For example, if the original cell population was growing in a production medium, the subpopulation could be diluted with the same or a similar medium. In certain embodiments, cell subpopulation is diluted with a cell culture medium that differs from the cell culture medium in which the original cell population was growing. For example, if the original cell population was growing in a growth medium, a subpopulation of cells could be diluted with a production or adaptation medium. In some embodiments, the original cell population has stopped growing (for example, it has stopped increasing the number of cells) prior to the passage or growth of a subpopulation. In certain modalities, a population spared two or more times during the adaptation process. For example, a population may be passed 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 times or more during the adaptation process. Standardized practice maintains the passage or "division" of a cell culture medium every three to four days using a standard growth medium. In certain embodiments, cells are adapted to the conditions of protein production by growing a cell population for a longer period of time before passage. For example, cells may grow 5, 6, 7, 8, 9, 1G, 11, 12, 13, 14 days or more before they are passed.

The term "viable cell density" refers to the total number of cells that survive in the cell culture medium at a certain volume. The term "cell viability" refers to the number of cells that are alive compared to the total number of both dead and alive cells expressed in percentage.

"Integrated viable cell density", "IVCD": The term "integrated viable cell density" or "IVCD", as used herein, refers to the average viable cell density throughout the culture multiplied by the amount of time in that the culture was performed. When the amount of protein produced is proportional to the number of viable cells present throughout the culture, the integrated viable cell density is a useful tool for estimating the amount of protein produced throughout the culture.

As used herein, the term "antibody" includes a protein that comprises at least one and typically two VH domains or portions thereof, and / or at least one and typically two VL domains or portions thereof. In certain embodiments, the antibody is a tetramer of two immunoglobulin heavy chains and two immunoglobulin light chains, wherein immunoglobulin heavy and light chains are interconnected by, for example, disulfide bonds. Antibodies, or a portion thereof, may be obtained from any source, which include, without limitation, rodents, primates (e.g., human and non-human primate), camelids, as well as recombinantly produced antibodies, for example, antibodies. chimeric, humanized and / or in vitro generated compounds as described in more detail herein.

Examples of binding fragments encompassed by the term "antigen binding fragment" of an antibody include, without limitation, (i) a Fab fragment, a monovalent fragment consisting of the VL1 VH, CL and CH1 domains; (ii) an F (ab ') 2 fragment, a divalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains, (iv) an Fv1 fragment consisting of the VL and VH domains of a single antibody arm, (v) a dAb fragment consisting of a VH domain; vi) a camelid or camelized heavy chain (V-HH) variable domain; (vii) a single chain Fv (scFv); (viii) a bispecific antibody; and (ix) one or more fragments of an immunoglobulin molecule fused to an Fc region. In addition, although the two Fv fragment domains, VL and VH are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that allows them to be made as a single protein chain in which the VL and VH regions separate to form monovalent molecules (known as single-chain Fv (scFv); see, for example, Bird et al (1988) Science 242: 423-26; Huston et al (1988) Proc. NatL Acad. Sci. USA 85: 5,879-83). Such single chain antibodies are also encompassed within the term "antigen binding fragment" of an antibody. Such fragments may be obtained using conventional techniques known to those skilled in the art, and the fragments are evaluated for the same function as that made for intact antibodies.

The "antigen binding fragment" may optionally further include a portion that enhances one or more characteristics, for example, stability, effector cell function or complement fixation. For example, the antigen binding fragment may further include a pegylated portion, albumin or constant region of a heavy and / or light chain.

With the exception of "bispecific" or "bifunctional" antibodies, all antibody binding sites are understood to be identical. A "bispecific" or "bifunctional" antibody is an artificial hybrid antibody that has two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or binding of Fab1 fragments. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79: 315-321 (1990); Kostelny et al., J. Immunol. 148, 1,547-1,553 (1992).

The term "screen" or "screen" refers to a method of deleting a subpopulation of cells with a certain phenotype that exhibits one or more advantageous features. In certain embodiments, a cell line is screened for one or more cell culture characteristics that are advantageous in a protein production process that includes, without limitation, increased growth, increased viability, increased integrated viable cell density, increased titration, and / or increased specific cell productivity. In certain embodiments, a cell line is screened for one or more cell culture characteristics that are advantageous in a fed-bioreactor process, for example, strong growth and / or viability.

The term "bioreactor" refers to a vessel in which a cell culture medium may be contained and whose internal conditions may be controlled during the cultivation period, e.g. pH and temperature. Those skilled in the art will know conditions of the uterine / bioreactor. or appropriate that can be controlled, as well as methods of controlling these bioreactor conditions. A "production bioreactor" refers to a bioreactor that is used during a protein production process. For example, a production bioreactor may comprise a large commercial vessel from which a large amount of protein may be produced, although a protein bioreactor may be produced. production is not limited to these large-scale commercial values. In certain embodiments, the volume of an umbiliorator is at least 1 liter, and may be 10, 100, 250, 500, 1,000,2,500, 5,000, 8,000, 10,000, 12,000 liters or more, or any volume within that range. . In addition, bioreactors which may be used include, without limitation, a stirred tank bioreactor, a fluidized bed reactor, hollow fiber bioreactor or roller bottle.

The terms "secondary side products" and "secondary metabolites" refer to the small molecules typically generated as a result of cellular metabolic activity. As is well known to those skilled in the art, these secondary side products are often detrimental to cell growth and / or viability. Therefore, its minimization or elimination of a cell culture is desirable. Non-limiting examples of by-products include ammonium lactate and ions. In certain embodiments, cells are adapted to the conditions of protein production by cell growth in the presence of these by-products. In certain embodiments, cells adapted to the conditions of protein production exhibit the ability to collect such secondary side products such that the levels of secondary side products decrease over time. Those skilled in the cell culture technique will know other metabolic side products that can be used to adapt cells according to the present invention.

A "fed batch culture" refers to a cell culture method in which cells are initially inoculated into a bioreactor with an inoculum medium. The cell culture medium is then supplemented at one or more points throughout the production cycle with a feed medium containing nutritional components and / or other supplements.

"Batch culture" refers to a method of cell culture in which cells are inoculated into a bioreactor with all the necessary nutrients and supplements for the entire production cycle. No nutrients, media etc. are added. to the cell culture medium after the beginning of the cell culture.

"Perfusion culture" refers to a cell culture method that is different from a batch or fed batch culture method in which the culture is not terminated, or not necessarily determined, prior to isolation and / or purifying the expressed protein of interest, and in which new nutrients and other components are periodically or continuously added to the culture, during which the expressed protein is periodically or continuously collected. The composition of added nutrients may be altered during the evolution of cell culture, depending on the needs of cells, the needs for optimal protein production, and / or any of a number of other factors known to those skilled in them. in technique.

The term "batch-refeed" refers to a mode of operation of a bioreactor or a method of passing cells.

In certain embodiments, a feedback batch process comprises the passage of cells every 7 or 8 days, compared to other bioreactor modes in which cells are not passed or are passed less frequently. On a smaller scale employing standardized division methods, cells are usually passed earlier, for example, every 3 or 4 days, compared to the fed batch. In certain embodiments, batch feeding methods are used to adapt a cell culture to obtain an improvement in cell culture characteristics that includes, without limitation, increased growth rate, increased viability, increased density. viability of integrated viable cells, an increase in titration, and / or an increase in specific cell productivity. In certain embodiments, a feedback batch process utilizes a richer culture medium and / or medium containing secondary metabolites and / or other inhibitors to adapt cells to protein production conditions. The term "secondary metabolites" refers to the byproducts of metabolic processes of cellular functions.

The term "expression" refers to transcription and translation occurring within a host cell. The level of expression is generally related to the amount of protein being produced by the host cell.

The term "protein" or "protein product" refers to one or more amino acid chains. As used herein, the term "protein" is synonymous with "polypeptide" and, as generally understood in the art, refers to at least one amino acid chain linked via sequential peptide bonds. In certain embodiments, a "protein of interest" is a protein encoded by an exogenous nucleic acid molecule that has been transformed into a host cell. In certain embodiments, where the "protein of interest" is encoded by an exogenous DNA with which the host cell has been transformed, the nucleic acid sequence of the exogenous DNA determines the amino acid sequence. In certain embodiments, a "protein of interest" is a protein encoded by a nucleic acid molecule that is endogenous to the host cell. In certain embodiments, the expression of such an endogenous protein of interest is altered by transfecting a host cell with an exogenous nucleic acid molecule which may, for example, contain one or more regulatory sequences and / or encode a protein that enhances expression. of protein interest. The methods and compositions of the present invention may be used to produce any proteins of interest which include, without limitation, proteins having pharmaceutical, diagnostic, agricultural and / or any of several other properties useful in commercial, experimental and / or application. or in other applications. In addition, a protein of interest may be a protein therapeutic substance. Specifically, a protein therapeutic substance is a protein that has a biological effect on a region in the body over which it acted in a region in the body over which it acts remotely through intermediates. Examples of protein therapeutic substances will be discussed in more detail below. In certain embodiments, proteins produced using the methods and / or compositions of the present invention may be processed and / or modified. For example, a protein to be produced according to the present invention may be glycosylated.

The term "titration" as used herein refers to the total amount of recombinantly expressed protein produced by a cell culture in a certain volume volume of medium. Titration is typically expressed in units of milligrams or micrograms of protein per milliliter of medium.

Those skilled in the art will recognize that the methods disclosed herein may be used for culturing many of the known mammalian cells used and routinely cultured in the art, that is, the methods disclosed herein are not limited to use only in the present specification.

DETAILED DESCRIPTION OF CERTAIN MODES OF THE INVENTION

It has been found that mammalian cells, for example, untransfected mammalian cells, including, for example, Chinese hamster ovarian cells, can be adapted to environments consistent with the conditions of a production bioreactor to enhance cell growth during subsequent production. of a protein of interest. All of these methods are also applicable to both untransfected mammalian cells and transfected mammalian cells (e.g., mammalian cells transfected with a cDNA or genomic construct that causes expression, or from an expression vector, of a desired recombinant protein). The present invention may be used to adapt cells to the advantageous production of any therapeutic protein, such as enzymes, receptors, pharmaceutical or commercially relevant antibodies (e.g. monoclonal and / or polyclonal antibodies), fusion proteins. Fc, cytokines, hormones, regulatory factors, growth factors, coagulation factors, antigen-binding agents, etc. Those skilled in the art will know other proteins that may be produced in accordance with the present invention, and will be able to use the methods disclosed herein for the production of such proteins.

The methods of the present invention for adapting untransfected mammalian cells have the potential for identifying cell lines that generate higher yields and / or exhibit superior protein production characteristics. In addition, the methods of the present invention may assist in the identification of suitable stranded cell lines faster and with less effort compared to standard cell line development procedures. For example, standardized cell-line identification processes typically require various experiments on different scales and additional robustness tests.

Host cells, before being transformed, are considered untransfected. Traditionally, when novel cell development is created for development, untransfected host cells were initially transfected, or transformed, with exogenous DNA to express a protein of interest. Using these prior methods, host cells were not generally tried or changed before transfection; Experimentation and research on cell line development began after host cells had been genetically engineered to produce a protein product.

In certain embodiments, non-transfected mammalian cells may be adapted to the conditions of higher performing protein production in batch, feed batch and / or perfusion bioreactor processes according to one or more methods of the present invention. In certain embodiments, such adapted cells are capable of maintaining an improved phenotype, for example, exhibit more vigorous growth, increased viability, increased viable integrated cell density, increased titration, increased specific cell productivity and / or higher cell densities. Certain embodiments of the methods of the invention described herein may be employed to adapt untransfected mammalian cells to a perfusion-producing, batch-fed and / or perfusion protein production process that has superior performance. .

Any of several commercially available media such as Minimum Essential Medium (MEM, Sigma), Ham F10 (Sigma) or Dulbecco's Modified Eagle's Medium (DMEM, Sigma) may be used as the base medium. Such base media may then be supplemented with amino acids, vitamins, inorganic salts, microelements and / or other components for the production of growth and / or production media. In certain embodiments, cells may be adapted by culturing transfected or untransfected mammalian cells in a production medium similar to or identical to a production medium. In certain embodiments, cells are adapted by cell growth under conditions similar to or identical to conditions typically encountered under production conditions in a bioreactor (for example, in a medium consistent with the medium typically found in a bioreactor) operated in batch mode, in batch mode fed and / or in perfusion mode. In certain embodiments, the cells to be adapted are untransfected. In certain embodiments, the cells to be adapted were transfected with an exogenous nucleic acid molecule, for example, a nucleic acid molecule expressing a protein of interest.

In certain embodiments, one or more cell culture characteristics are enhanced during the protein production phase by adapting cells to protein production conditions prior to transfection with an exogenous nucleic acid molecule. In certain embodiments, this enhanced cell culture feature includes, without limitation, increased growth, increased overall cell viability, increased integrated viable cell density, increased titration, and / or increased specific cell productivity. A production medium is typically more enriched than a growth medium and is, for example, supplemented with higher levels of nutrients, vitamins, trace elements and / or other media components compared to a growth medium.

It is typical practice in the art for cells to be continuously grown in growth medium during cell line development and not to contact production medium until the beginning of a fed batch production assay. In certain embodiments, the uptake of untransfected mammalian cells into a protein production medium and / or under protein production conditions prior to transfection, rather than into growth medium and / or under growing conditions. , minimizes the potentially deleterious effects of cell transition from one environment to another, for example from growing conditions to post-transfection protein production conditions.

In certain embodiments, the methods of the present invention are useful for generating a host cell line that is adapted to the conditions of protein production. This adapted host cell line is capable of being transfected with any of several proteins of interest. In certain embodiments, this adapted transfected cell line is placed directly under conditions of protein production. In certain embodiments, this transfected cell-adapted lineage grows to a desired cell density and / or a desired number of cells during a growth phase, when the cell line then transitions to a protein production phase. In accordance with the present invention, such a transfected cell-adapted strain exhibits minor and less severe deleterious effects during the transition phase compared to an un-adapted transfected cell strain.

In certain embodiments, in order to produce a protein of interest, the adapted host cells are transfected with an exogenous nucleic acid molecule. In certain embodiments, a nucleic acid molecule introduced into the cell encodes the protein to be expressed according to the present invention. In certain embodiments, a nucleic acid molecule contains a regulatory sequence or coding for a gene product that induces or enhances the expression of the desired protein by the cell. As a non-limiting example, such a gene product may be a transcription factor that increases expression of the protein of interest.

In certain embodiments, a nucleic acid that directs the expression of a protein is stably introduced into the host cell. In certain embodiments, a nucleic acid that directs the expression of a protein is transiently introduced into the host cell. Those skilled in the art will be able to choose when stable or transiently introducing nucleic acid into the cell based on experimental, commercial or other needs.

A gene encoding a protein of interest may optionally be linked to one or more gene control regulatory elements. In some embodiments, a gene control element directs the constitutive expression of the protein. In some embodiments, a genetic control element may be used which provides inducible expression of a gene encoding the protein of interest. The use of an inducible control element (eg, an inducible promoter) allows the modulation of protein production in the cell. Non-limiting examples of potentially useful inducible genetic control elements for use in eukaryotic cells include hormone-regulated elements (see, for example, Mader, S. and White, JH, Proc. NatL Acad. Sci. USA 90: 5.603-5.607 , 1993), Synthetic Ligand Regulated Elements (see, for example, Spencer, DM et al., Science 262: 1.019-1.024, 1993) and Ionizing Radiation Regulated Elements (see, for example, Manome1Y. Et al., Biochemistry 32 : 10,607-10,613, 1993; Datta, R. et al., Proc. Nati Acad. Sci. USA 89: 10,149-10,153, 1992). Additional regulatory systems with cell specificity or other regulatory systems known in the art may be used according to the methods and compositions described herein.

Any protein that can be expressed in a host cell can be produced according to the methods and compositions of the present invention. The protein may be expressed by a gene that is endogenous to the host cell, or by a heterologous gene that is introduced into the host cell. The protein may be one that occurs in nature, or may alternatively have a sequence that has been designed or selected by the hand of man. A protein to be produced may be assembled from protein fragments that occur individually in nature. Additionally or alternatively, the engineered protein may include one or more non-naturally occurring fragments.

Any host cell susceptible to cell culture and protein expression may be used in accordance with the present invention. In certain embodiments, the host cells are mammalian cells such as Chinese hamster ovary (CHO) cells. Other nonlimiting mammalian cell examples that may be used in accordance with the present invention include the BALB / c mouse myelomade cell line (NSO / I, ECACC Nos. 85110503); human retino blast cells (PER.C6 (CruCell, Leiden1Pasies Netherlands)); SV40-transformed CV1 monkey kidney cell line (COS-7, ATCC CRL1651); human kidney embryological cell line (293 or 293 subcloned cells for growth in suspension culture, Graham et al., J. Gen Virol., 36: 59 (1977)); hamster pup kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells +/- DHFR (CHO, Urlaube Chasin, Proc. Natl. Acad. Sci. U.S.A., 77: 4,216 (1980)); mouse sertolide cells (TM4, Mather, Biol. Reprod., 23: 243-251 (1980)); monkey derim cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB8065); mouse mammary tumor cells (MMT 060562, ATCC C-CL51); TRI cells (Mather et al., Annals, Y. Acad. Sci., 383: 4.468 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma (Hep G2) cell line.

In certain embodiments, cells (e.g., untransfected mammalian cells) are adapted by culturing cells in growth medium supplemented with inhibitors. In certain embodiments, these inhibitors include inhibitors that are typically found when cells grow under protein production conditions. Non-limiting examples of such inhibitors include lactate, ammonia, alanine, glutamine and / or acetolactate. Those skilled in the art will know other inhibitors which may be used in accordance with the present invention to adapt cells to the conditions of protein production.

In certain embodiments, cells (e.g., untransfected mammalian cells) are adapted by culturing cells in an adaptation medium that lacks or has a reduced concentration of one or more components traditionally found in production media. For example, traditional means of production typically contain insulin at a concentration of about 10 mg / l or more. Thus, in certain embodiments, cells are adapted by culturing the cells in an adaptation medium that lacks insulin. In certain embodiments, cells are adapted by culturing cells in an adaptation medium containing insulin at a concentration lower than a concentration of insulin traditionally found in production media. Those skilled in the art will know other components. which are typically present in production media, and will be able to use methods of the present invention to adapt cells to media that do not possess or have a reduced concentration of such components.

In certain embodiments, cells are adapted by culturing (e.g., continuous cultivating) untransfected mammalian cells in growth medium supplemented with one or more inhibitors. Non-limiting examples of such inhibitors include secondary carbon-free side products. For example, some secondary side products include, without limitation, lactate, alanine, glutamine, acetolactate and / or ammonia. In certain embodiments, the concentrations of such side products present and / or added to an adaptation medium are similar to those typically found under bioreactor conditions such as, for example, about 2.0 to about 10.0 g / l. lactate and / or about 0.1 to about 0.5 g / l ammonia. In certain embodiments, cells are adapted under conditions in which the lactate concentration in the adaptation medium is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 g / l or larger. Additionally or alternatively, in certain embodiments, cells are adapted under conditions in which the ammonia concentration in the adaptation medium is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1,3,1.4, 1.5 g / l or greater.

In certain embodiments, individual untransfected mammalian cells and / or sub-populations of untransfected mammalian cells that have been adapted to the conditions of protein production according to one or more methods of the present invention may exhibit phenotypes consistent with their uptake. Secondary side products, such that the levels of secondary side products actually decreased over time. In certain embodiments, such untransfected mammalian adapted cells retain the ability to collect secondary side products after being transfected with an exogenous nucleic acid molecule to produce a protein of interest. In some embodiments, these adapted transfected mammalian cells generally perform better in a subsequent protein production process (e.g., a batch-fed bioreactor process) over untransfected mammalian cells that do not capture side products and / or have not been tailored to capture these secondary side products. For example, such transfected mammalian adapted cells perform better in subsequent batch protein, fed batch and / or perfusion processes. In a production reactor (for example, a batch fed reactor), these by-products can often accumulate to levels that are generally inhibitory for further growth and / or viability of the cells. In certain embodiments, untransfected mammalian cell populations that have been adapted according to the methods of the present invention grow in a supplemented medium and may grow well under the inhibitory and stressful conditions typically found in a production bioreactor.

As is known in the cell culture technique, in many cases the temperature of a cell culture is decreased when the cell culture is changed from growing conditions to protein producing conditions. In certain embodiments, cells are adapted by culturing the cells at a temperature that leads to the production of a protein product. In certain embodiments, cells are adapted to be cultured at a temperature of about 31 ° C, although the methods of the present invention are not limited to that temperature. For example, ascells can be adapted by growing at a temperature of about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37. 38, 39, 40, 41, 42, 43, 44 or 45 ° C. Those skilled in the art will know temperatures suitable for protein production, and these temperatures may depend, at least in part, on the cell line, protein produced, other culture conditions, and / or any other factor considered important by those skilled in the art. in technique.

In certain embodiments, the cells are adapted by continuous cultivation of untransfected mammalian cells for a longer duration in a "fed batch" mode. In certain embodiments, non-transfected cells are adapted by such "batch feed" adaptation methods. In certain embodiments, transfected cells are adapted by such "enhanced batch" adaptation methods. Typical cell culture operations may employ a cell culture management regimen by dividing, passing, or subculturing a cell culture every three to four days. In certain embodiments of the "fed batch" adaptation methods, untransfected mammalian cells are cultured for longer durations, mammalian cells are cultured for 5, 6, 7, 8, 9, 10, 11, 12,13, 14 days or more before being subcultured. These longer-lasting cultures have the dual effect of adapting cells to protein production conditions by increasing cell density and / or by accumulating higher levels of secondary metabolites in the conditioned medium to which cells can grow. be adapt.

In certain embodiments, untransfected mammalian cells can be grown continuously under "combined with production" conditions. In certain embodiments, untransfected cells may be cultured with repetitive cell culture cycles under conditions combined with production, followed by passage of cell subpopulations after 3 or 4 days under "recovery" or standard growth conditions with growth. the option of using a growth medium. In certain styles, a population is passed several times during the process of adaptation. For example, a population may be passed 2, 3, 4, 5, 6, 7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more during the process of adaptation. In certain embodiments, cells are adapted to protein production conditions by growing a cell population for a longer period of time before passage. For example, cells may grow 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more before they are passed. These past subpopulations may be screened for one or more desirable characteristics. In certain embodiments, after several cycles of repetitive or continuous adaptation, cell subpopulations will appear which exhibit superior characteristics including, for example, improved growth and / or viability over the unadapted starting population under the conditions combined with production. In addition, in certain embodiments, certain subpopulations will begin to collect side products such as lactate, such that the level of this side product decreases over time, increasing the performance of that cell line in a production bioreactor. In certain embodiments, one or more screened subpopulations of cells exhibiting one or more desirable characteristics (eg, strong growth) are then transformed into the production of a protein of interest, and then transfected subpopulations are placed under protein-producing conditions (e.g. conditions consistent with those to which cells were adapted).

In certain embodiments, cells grow in accordance with any of the cell culture methods described in US Patent Serial Nos. 11 / 213,308, 11 / 213,317 and 11 / 213,633, each of which was deposited on August 25, 2005, and each of which is incorporated herein by reference in its entirety. For example, in certain embodiments, cells may grow in a culture medium in which the cumulative amino acid concentration is greater than about 70 mM.

In certain embodiments, cells may grow in a culture medium in which the molar ratio of cumulative glutamine concentration to cumulative concentration of asparagine is less than about 2. In certain embodiments, cells may grow in a culture medium. wherein the molar ratio between the cumulative concentration of glutamine and the cumulative concentration of total amino acids is less than about 0.2.

In certain embodiments, cells may grow in a culture medium in which the molar ratio between the cumulative concentration of inorganic ions and the cumulative concentration of total amino acids is between about 0.4 to 1. In certain embodiments, the cells may grow in a medium. culture medium in which the combined concentration of cumulative glutamine and asparagin accumulative is between about 16 and 36 mM. In certain embodiments, cells may grow in a culture medium containing two, three, four, or all five of the above media conditions. The use of these media allows the presence of high levels of protein production and reduces the accumulation of certain undesirable factors such as ammonium and / or lactate.

In some embodiments, cells grow under one or more of the conditions described in U.S. Provisional Patent Application Serial Nos. 60 / 830,658, filed July 13, 2006 and incorporated herein by reference in its entirety. For example, in some embodiments, cells grow in a manganese-containing culture medium at a concentration between approximately 10 and 600 nM. In some embodiments, cells grow in a manganese-containing culture medium at a concentration of approximately 20 to 100 nM. In some embodiments, cells grow in a manganese-containing culture medium at a concentration of approximately 40 nM. The use of these media in glycoprotein growth results in the production of a glycoprotein with an improved glycosylation pattern (eg, a larger number of covalently linked sugar residues in one or more oligosaccharide chains).

The components and / or supplements of the production medium and growth medium can be readily determined by those skilled in the cell culture technique. As is known in the art, components and / or supplements may vary depending upon the host cell used and the desired protein of interest. In addition, the conditions and amount of side products produced may or will vary with different bioreactor conditions and with each different cell line. This application is further illustrated by the non-limiting examples which will be presented below. Any modifications that may be necessary throughout the adaptation of mammalian cells to the cell culture medium for the production of different proteins are included in the cell culture technique.

EXAMPLES

Example 1: Adaptation of untransfected hamsterchinese ovarian cells to conditions combined with production

Untransfected CHOK1 cells were cultured under standard 3-day / 4-day fed-batch conditions with decreasing medium, with components listed in Table 1 below, or cultured in a 7-day-fed batch mode in enriched production medium. components listed in Table 2 below for several cycles. Ascellules were cultured at 37 ° C in a working volume of 10 to about 30 ml. The cell numbers of the experiment are shown in Figures 1a and 1b. Around day 58 of the feedback batch method of Figure 11a, cells were passed for 9 days instead of 7, and the subsequent sequence passage did not grow well. After the bad 7-day spell, however, ascells were able to recover.

TABLE 1

<table> table see original document page 32 </column> </row> <table> <table> table see original document page 33 </column> </row> <table> <table> table see original document page 34 < / column> </row> <table>

Table 2

<table> table see original document page 34 </column> </row> <table> <table> table see original document page 35 </column> </row> <table> <table> table see original document page 36 < / column> </row> <table>

The cells were then evaluated in a fed-fed production run assay, where the workload was between 10 and 30 ml. Cells were cultured at 37 ° C for four days, and then the temperature was changed to 31 ° C until day 12, when the assay was over. Cell density and viability were compared, and the results are illustrated in Figure 1c. Cells grown in a 7-day feedback batch mode showed higher cell densities than cells grown in a standard 3-day / 4-day feedback batch mode. Similarly, two different groups of cells that were adapted for growth in production medium, with the components listed in Table 3 below, obtained higher cell densities than the unadapted starting control population. <table> table see original document page 37 </column> </row> <table> <table> table see original document page 38 </column> </row> <table> <table> table see original document page 39 </column> </row> < table>

Example 2: Adaptation of transfected cell line to conditions combined with production

A cell line expressing the heavy and light chain genes of a monoclonal antibody was cultured under standard 3 day / 4 day fed batch conditions using standard growth medium with the components listed in Table 1, or two versions of medium. production, "A" and "Β". Production medium A, with the components listed in Table 2, was moderately enriched relative to the decreasing medium, and production medium B, with the components listed in Ta-Bela 4 below, was significantly enriched relative to the decreasing medium. The effects of methotrexate (MTX) were also tested as production name "B; 1.5 μΜ MTX was added in one culture with" B "production, not in the other. Cells were grown at 37 ° C. from 10 to about 30 ml Growth rates during adaptation in these media are shown in Figure 2a.

TABLE 4

<table> table see original document page 39 </column> </row> <table> <table> table see original document page 40 </column> </row> <table> <table> table see original document page 41 < / column> </row> <table>

Following continuous culture in these media, cell lines were evaluated in a fed batch production assay, in which cells were evaluated in production medium B, illustrated in Figure 2b. Ascells were cultured at 37 ° C for four days, and then the temperature was changed to 31 ° C until day 129, when the assay was terminated. The results indicate that cell lines that overexpress the recombinant protein can also be adapted under production-matched conditions to obtain superior growth characteristics in a fed batch production assay.

Example 3: Adaptation of untransfected hamsterchinese ovary cells to low insulin conditions

Insulin has a direct impact on the metabolism of glycoseps by mammalian cells. Rapid consumption of glucose in cell culture is often associated with the excretion of lactic acid as dejetometabolic. Lactic acid can inhibit cell growth and has negative effects on cell viability. Insulin is also reported to be a growth factor for mammalian cells.

The cells of the CHOK1 host cell line were taken from normal growth media (containing 10 mg / l nucellin), and placed in completely insulin-free media. After an initial Iog phase in which cells demonstrated decreased growth, the growth rate eventually increased to a rate comparable to that of the control CHOK1 culture in insulin-containing media, suggesting that cells cultured in the absence of insulin adapted to growth. conditions. The viability of the cells was unaffected and remained at 90 ° C throughout the adaptation. This adaptation continued for approximately 100 population doublings. When the adapted CHOK1-insulin cells were stacked and then thawed, they retained their ability to grow on non-insulin media. Defrosted CHOK1 control cells in non-insulin media showed one of the growth rate, suggesting that K1-insulin adaptation actually altered cells, allowing them to grow independently of exogenous insulin. Ongoing investigations include the combination of the non-insulin phenotype. with the 7-day passage adaptation phenotype.

The first batch fed production assay was organized in an unadjusted pH format. The experiment included unadapted CHOK1 cells in standardized production media (20 mg / ml denucelin) as a positive control, and unadapted CHOK1 cells grown in non-insulin producing media as a negative control. Adapted CHOK1 cells without insulin were cultured in insulin-free production media. Insulin was included in the 39 and 7th day feeding media (at a final concentration of 0.006 mg / ml). Examination of the data suggests that adapted CHOK1 cells grow in insulin-free media and have very similar growth, viability, and IVCD characteristics as compared to the CHOK1 positive control sample (see Figures 3-5). The negative control sample of CHOK1 showed a reduction in growth rate (see figure 3). These results confirm that insulin-free growth-adapted CHOK1 cells are inherently different from unadapted CHOK1 cells, considering their ability to grow well in non-insulin-containing media.

The insulin-free CHOK1 cells were then evaluated in a second fed batch experiment under pH controlled conditions. In this experiment, non-adapted CHOK1 cells and non-insulin adapted CHOK1 cells were organized into three separate conditions. The first condition contained insulin in both the middle and the feeding medium (50 mg / l and 0.006 mg / ml, respectively), symbolized in Figures 6-9 as (+ / +). The second condition contained insulin-free base media with an insulin-containing feed (symbolized by (- / +) in Figures 6-9), and the third condition contained insulin-free base media and insulin-free feed media (symbolized by (- / -) in Figures 6-9). Insulin-free CHOK1 cells show better growth, viability, and IVCD when cultured on less insulin, compared with unadapted CHOK1 cells (see Figures 6-9). It appears that CHOK1-insulin adapted cell lines produce less lactate and almost completely consume all lactate they produce (see Figure 9). This elimination of a harmful byproduct produces a healthier cell culture and is seen as a very promising phenotype. This promising phenotype provides better growth conditions and allows cells to reach higher densities than the associated control. It should be noted that this phenotype is directly related to the elimination of insulin from the media, since CHOK1-insulin adapted cell lines have lactate production rates similar to the control sample when grown in media containing insulin.

Example 3 demonstrates that adaptation of CHOK1 cells to an insulin-free condition leads to desirable phenotypes when cells are grown in industrially relevant production modes. Enhanced metabolic phenotypes lead to increased cell growth and viability, which are supposed to have a significant positive impact on the volumetric productivity of a recombinant CHO cell culture.

While certain embodiments of the specification have been described above, the above description is illustrative only. Further modifications of the embodiments herein may be made by those skilled in the art of cell culture, and it is contemplated that all such modifications are within the scope of the embodiments as defined by the appended claims.

Claims (30)

1. A method for adapting cells to protein production conditions which comprises: - culturing cells in an adaptation medium, - screening cells and selecting a subpopulation of cells that exhibits an enhanced culture culture characteristic. cells when subpopulation grows under protein production conditions, the characteristic of which differs from a corresponding cell culture characteristic that would be observed in cells growing in a medium that is not an adaptation medium, where the enhanced cell culture characteristic is selected from the group consisting of: increased growth, increased viability, increased density of integrated viable cells, increased titration, increased specific cell productivity and their combination. The method of claim 1, further comprising the cell passaging step prior to the screening step. The method of claim 2 or 3, wherein the passage step comprises passing the cells two or more times before the stripping step. The method of any one of claims 1-3, wherein the passage step comprises passage of the cells after approximately 3 or 4 days in the adaptation medium. The method of any one of claims 1-3, wherein the passage step comprises passage of the cells after approximately 7 or 8 days in the adaptation medium. The method of any of claims 1-5, further comprising the step of culturing the cells in a medium that is not a means of adaptation after the step of passage. The method of claim 6, wherein the medium which is not a medium of adaptation is a standard growth medium. The method of any one of claims 1-7, wherein cell cells are not transfected. The method of any one of claims 1-7, wherein cell cells have been transfected to express a protein of interest. The method of claim 9, wherein the protein of interest is an antibody. The method of claim 9, wherein the protein of interest is a protein therapeutic substance. The method of any one of claims 1-11, wherein the adaptation method comprises a production medium. The method of claim 12, wherein the production medium comprises an increased level of one or more medium components as compared to a standard growth medium, the medium component selected from the group consisting of: nutrients, vitamins, microelements, and combinations thereof. The method of any one of claims 1-13, wherein the adaptation method comprises a secondary metabolite selected from the group consisting of: lactate, ammonia, and combinations thereof. The method of claim 14, wherein the secondary metabolite is added to the adaptation medium at the beginning of cell culture. The method of claim 14 or 15, wherein the secondary metabolite level is increased as the cell culture progresses. The method of claim 16, wherein the secondary metabolite level increases as a result of the metabolic activity of cells. The method of claim 16 or 17, wherein the secondary metabolite level increases by adding the secondary metabolite to the cell culture. The method of any one of claims 14-18, wherein the selected cell population picks up a secondary metabolite such that the metabolite level decreases when adapted cells grow in a production medium. The method of any one of claims 1-19, wherein the method of adaptation comprises one or more selected group inhibitors consisting of: lactate, ammonia, alanine, glutamine, acetolactate, and echombinations thereof. The method of any of claims 14-20, wherein chelactate is present in a concentration of from about 2 to about 10 g / l. The method of claim 21, wherein the lactate is present at the beginning of cell culture at a concentration of from about 2 to about 10 g / l. The method of any one of claims 14-20, wherein cheamonia is present at a concentration of from about 0.1 to about 0.5 g / l. The method of claim 23, wherein ammonia is present at the beginning of cell culture at a concentration of about 0.1 to about 0.5 g / l. The method of any preceding claim, wherein the adapter means is devoid of insulin. The method of any preceding claim, wherein the adapter means comprises insulin at a concentration of less than about 10 mg / l. The method of any preceding claim, wherein the cells are mammalian. The method of any preceding claim, wherein the protein production conditions comprise the conditions used in a bioreactor. The method of claim 28, wherein the bioreactor is a production bioreactor. The method of any preceding claim, wherein the conditions for protein production comprise culturing the cells in a batch process of protein production.