JP2009514532A - Methods for adapting mammalian cells - Google Patents

Abstract

  Methods are provided for adapting cells, eg, mammalian cells, to a cell culture process. When adapted cells are genetically modified and used for protein production, these cells can reach higher cell densities and / or achieve a higher overall yield of protein production, etc. Show advantageous features.

Description

This application is co-pending with US Provisional Patent Application No. 60 / 732,818, filed Nov. 2, 2005, sharing at least one common inventor and claiming priority. The contents of which are incorporated herein by reference.

TECHNICAL FIELD The present disclosure relates generally to methods for adapting mammalian cells, eg, non-transfected mammalian cells, with respect to production of a therapeutic protein of interest. Specifically, the present disclosure relates to adapting non-transfected mammalian cells for better performance in a bioreactor.

  Proteins can be produced using well-known recombinant techniques. Usually, transformed cells are cultured in a controlled environment such as a bioreactor. Most large-scale commercial manufacturing strategies utilize suspension cell cultures grown in large stirred tank reactors. However, most cell lines do not readily achieve good performance and / or fail to reach the desired high cell density in high cell density protein production processes (eg, fed-batch processes). . During the production run, many inhibitors are present or accumulate in the cell culture medium. These inhibitors may be by-products arising from metabolic processes, such as lactic acid or ammonia, among others. Typically, cell lines are initially grown and maintained at conditions designed to support the growth and / or survival of these cell lines. However, the conditions used during protein production (eg conditions encountered in production bioreactors) are very different, eg secondary metabolites and / or other components are present as protein production proceeds And / or accumulate, which may have deleterious effects on cell lines. As a non-limiting example, such deleterious effects may include a decrease in live cell density and / or a final titer, and a decrease in the amount and / or quality of the protein produced.

  Numerous experiments are performed, carrying out conventional methods, to determine whether a cell line is adapted for growth and / or production in a protein production process such as a fed-batch process in a bioreactor. . Such experiments often require multiple clonal cell lines and / or transfection pools, which are then typically used in production bioreactors (eg, serum-free suspensions or “known” The cells are adapted for growth in a composition medium "). Once the cell line is adapted, additional rounds of screening and optimization are typically performed in a bench scale bioreactor. An additional experiment was then performed on individual clones that usually showed good expression phenotypes in preliminary studies, and which clones were also in an acceptable range of growth and / or in bioreactors, eg, fed-batch bioreactors. Or determine whether it exhibits survival characteristics. Such an extensive screening process is costly and particularly laborious for personnel.

  Accordingly, what is needed is a method for adapting cells to conditions that produce proteins, including but not limited to bioreactor conditions. Further, what is needed is a feature of one or more cell cultures (e.g., after transfecting non-transfected host cells prior to transformation to conditions where they are transfected to produce protein) (e.g., This method is adapted so that there is no or little adverse effect on titer, survival rate, protein quality, etc.).

  The present disclosure relates to methods for adapting mammalian cells to high density protein production processes such as fed-batch processes. The process of the invention can involve both non-transfected mammalian cells and genetically engineered host cells. In certain embodiments, non-transfected mammalian cells are adapted to conditions consistent with production, such as those used during protein production. For example, non-transfected mammalian cells can be adapted to the production medium used in the bioreactor during the production run.

  A variety of methods can be used to adapt non-transfected mammalian cells to the conditions of production. In certain embodiments, the cells are cultured in production medium and / or adaptation medium. In certain embodiments, the cells are cultured in an adaptive medium and a standard division cycle is repeated. For example, a subpopulation of cells can be passaged once or multiple times, eg, every 3 or 4 days. In certain embodiments, production media and / or adaptation media generally have higher levels of nutrients, vitamins and / or trace elements as compared to standard growth media. In certain embodiments, the cells are then given a recovery period between passages, for example, during one of the cycles, after which the passaged cells are allowed to grow in standard growth media. Return to production medium and / or adaptation medium.

  In certain embodiments, the cells are adapted by passaging the cells in a batch-refeed manner, wherein a subpopulation of cells is split or passaged every 7 or 8 days. In certain embodiments, secondary metabolites accumulate in the cell culture medium when cells are passaged after such longer periods. In certain embodiments, the adapted cells acquire resistance to such secondary metabolites and / or initiate their absorption. In certain embodiments, such secondary metabolites include inhibitors and / or metabolites, such as lactic acid and / or ammonia, that normally accumulate in the bioreactor during the production run. In certain embodiments, the cells are adapted by growing in a medium containing one or more inhibitors, including but not limited to lactic acid, ammonia, alanine, glutamine and / or acetolactate. . In certain embodiments, such inhibitors and / or metabolites are consistent with inhibitors and / or metabolites normally found in a bioreactor during a production run.

  In certain embodiments, non-transfected mammalian cells are cultured in standard growth media supplemented with inhibitors such as alanine, glutamine, acetolactate, ammonia and lactic acid (and optionally every 3-4 days). Can be adapted. In certain embodiments, cells so adapted are passaged every 3 to 4 days. In certain embodiments, the concentration of such inhibitor corresponds to the concentration normally found in the bioreactor during the conditions used for protein production. In certain embodiments, the concentration of some inhibitors is from about 2 to about 10 g / L lactic acid, or from about 0.1 to about 0.5 g / L ammonia, mimicking normal bioreactor conditions. including. In certain embodiments, cells adapted to bioreactor conditions by one or more of the methods of the present invention may exhibit particular characteristics or phenotypes and / or generate such phenotypes. In that case, the cell begins to take up lactate and / or other secondary metabolites, so that lactate and / or other secondary metabolites during one or more time frames during the production run The level of actually decreases. Such phenotypes can be screened for because they exhibit the desired quality for high density fed-batch production bioreactors.

  In certain embodiments, the cells adapted by one or more methods of the invention are host cells that have not been transfected to produce the protein of interest. In certain embodiments, such adapted host cells are then screened for one or more desired characteristics. In certain embodiments, once the subpopulation is screened for one or more desired characteristics, the cells can then be genetically engineered (eg, transfected) to produce a cell line that produces the protein of interest. it can. In certain embodiments, such adapted cell lines are placed in a bioreactor where the cell lines are readily adapted to high cell density protein production processes such as, for example, fed-batch processes. In certain embodiments, as a result of pre-adapting non-transfected host cells, e.g., mammalian host cells, genetically engineered cell lines need not be transferred from standard growth media to production media, and Even migrating is associated with less and / or lesser detrimental effects. In certain embodiments, prior adaptation helps to minimize possible deleterious effects on the cell line and helps to guarantee the performance of the cell line, accelerating the developmental timeline. In certain embodiments, such adapted cell lines can take up secondary metabolites during protein production runs. One skilled in the art of cell culture can readily determine which components constitute a standard growth medium and a standard production medium. In certain embodiments, growth conditions and protein production conditions differ in the composition of the cell culture medium used. Bioreactor conditions can be changed during the growth phase and / or protein production phase, and / or adjuvants may be added to improve productivity and / or maintain cell line viability it can. The supplement can include a feed medium and / or one or more additives. One skilled in the art will be able to select an appropriate media supplement. Additional adjuvants to the media include the desired protein product, cell culture media parameters (eg, ingredients, pH, etc.), methods used throughout the growth and / or production process, and / or various other known to those of skill in the art Will depend on any of the factors.

  In certain embodiments, by a method comprising culturing non-transfected mammalian cells in an adaptive medium and performing one or more repeated splitting cycles (eg, every 3 to 4 days), Adapt the transfected mammalian cells. The iterative splitting cycle involves splitting non-transfected mammalian cells in an adaptive medium, providing a recovery period in which the cells are cultured in standard growth media, screening the cells, and generating bioreactors. Selecting a subpopulation that exhibits a phenotype with improved proliferation and / or viability compared to an unadapted type of cells when cultured under conditions. In certain embodiments, cells are transfected with a gene encoding the protein of interest and cultured in a production bioreactor to express the protein of interest. In certain embodiments, the adaptation medium contains increased levels of nutrients, vitamins and / or trace elements as compared to the standard growth medium of interest. In certain embodiments, the adaptation medium contains an increased amount of an inhibitory metabolite compared to a standard production medium prior to cell culture.

  In certain embodiments, the method comprises culturing non-transfected mammalian cells in an adaptive medium supplemented with carbon by-products of primary metabolism and performing one or more repeated splitting cycles. Adapt the transfected mammalian cells. The iterative splitting cycle involves splitting non-transfected mammalian cells approximately 3 to 4 days in adaptive media, providing a recovery period in which the cells are cultured in standard growth media, and any level of by-products. And a non-transfected mammalian cell that exhibits an improved phenotype when screened in non-transfected mammalian cells and cultured in the adaptive medium. Selecting a population. In certain embodiments, such by-products are one or more of lactic acid, ammonia, alanine, glutamine and / or acetolactate. In certain embodiments, lactic acid 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 about 0.1 to about 0.5 g / L. In certain embodiments, non-transfected mammalian cells are adapted to absorb the byproduct of the primary carbon source.

  In certain embodiments, after the non-transfected mammalian cells are cultured in a preconditioned medium having an accumulation of at least one inhibitory metabolite for a period of about 7 to 8 days, A method comprising: dividing an animal cell; and screening the non-transfected mammalian cell to select a subpopulation that exhibits an improved phenotype in the production bioreactor. Adapt. In certain embodiments, the cells are split after giving them a recovery period by culturing the cells in a standard growth medium for a period of about 3 days to about 4 days. In certain embodiments, the conditioned medium contains an accumulation of inhibitory metabolites from at least one metabolic process of non-transfected mammalian cells. In certain embodiments, such inhibitory metabolites are one or more of ammonia, alanine, glutamine, acetolactate and / or lactic acid. In certain embodiments, such inhibitory metabolites are consistent with inhibitory metabolites found in production bioreactors at the end of normal commercial scale batch-refeed processes.

  In the protein production process, the cells can be cultured using any of a variety of suitable culture procedures and / or media (eg, inoculation media, feed media, etc.). Both serum-containing and serum-free media can be used. For example, in certain embodiments, the cells are grown in a known composition medium. In certain embodiments, the cells are grown in complex media. Moreover, one or more specific culture methods can be used, varied and / or optimized to culture cells suitable for specific cell types and protein products. Such procedures are well known and understood by workers in cell culture technology and ordinary technicians. Other features and advantages of the disclosure will be apparent from the following description of the specific embodiments and from the claims.

Definitions In accordance with long-standing convention, including in the claims, and as used in this application, the terms “a” and “an” mean “one or more”. Although the present invention has been described with a certain degree of particularity, it is apparent that many alternatives, modifications and variations will be apparent to those skilled in the art in light of the present disclosure. Accordingly, all such alternatives, modifications and variations are within the spirit and scope of the present invention and are intended to be encompassed by the following claims.

  The phrase “host cell” refers to a cell that can be genetically engineered and / or a cell that can grow and survive in a cell culture medium. Usually, the cells can express large amounts of the endogenous or heterologous protein of interest and can either retain the protein or secrete it into the cell culture medium. It is.

  Host cells are usually “mammalian cells”, which include baby hamster kidney (BHK) cells, Chinese hamster ovary (CHO) cells, human kidney (293) cells, rhesus monkey fetal normal diploid (FRhL-2). ) Non-limiting examples of vertebrate cells, including cells and mouse myeloma (eg, SP2 / 0 and NS0) cells. Those skilled in the art will recognize other host cells that can be used in accordance with the methods and compositions of the present invention.

  The term “cell culture medium” refers to cells in solution containing nutrients that support cell survival under conditions that allow the cells to grow and / or produce the desired protein. The phrase “inoculation medium” or “inoculum medium” refers to a nutrient-containing solution or substance that initiates cell culture. In certain embodiments, the cell culture is supplemented with “feed medium” one or more times, which is given to the cells after the start of culture. In certain embodiments, the phrase “feed medium” contains nutrients similar to the inoculum medium, but this is the solution or substance that is given to the cells following the start of culture. In certain embodiments, the feed medium contains one or more components that are not present in the inoculation medium. In certain embodiments, the feed medium lacks one or more components present in the inoculation medium. One of ordinary skill in the cell culture art will know which components constitute such inoculum and feed media without undue experimentation. These solutions typically provide essential and non-essential amino acids, vitamins, energy sources, lipids, and / or trace elements that cells need for growth and survival.

  As used herein, the term “cell culture characteristic” 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 the characteristics of one or more cell cultures. In certain embodiments, the improvement in cell culture characteristics includes an increase in the size of the cell culture characteristics. In certain embodiments, the improvement in cell culture characteristics includes a reduction in the size of the cell culture characteristics. As a non-limiting example, cell culture features may be improved growth, increased viability, increased live cell density, increased titer, and / or increased cell specific productivity. it can. Those skilled in the art will appreciate other cell culture characteristics that can be improved using the methods and compositions of the present invention.

  The phrase “growth medium” or “standard growth medium” refers to a medium containing nutrients and adjuvants that allow the cell or cell line to divide and grow. In certain embodiments, the phrase “production medium” refers to an enhanced growth medium that allows high levels of expression of the protein of interest. In certain embodiments, the production medium generally contains higher levels of nutrients, vitamins, trace elements and / or other medium components as compared to a standard growth medium. The phrase “adaptive medium” refers to exposing a cell to protein production conditions present in a bioreactor (eg, a late production bioreactor), preparing the cell for protein production conditions, and / or allowing the cell to grow. Refers to a medium that minimizes the harmful effects that can occur when migrating to protein production conditions. In certain embodiments, the adaptive medium includes metabolites resulting from metabolic processes, including, but not limited to, the presence of one or more secondary metabolites, such as, but not limited to, lactic acid and / or ammonia. In certain embodiments, the adaptive media includes one or more inhibitors, including but not limited to lactic acid, ammonia, alanine, glutamine and / or acetolactate. In certain embodiments, the adaptation medium includes a production medium. In certain embodiments, the adaptive medium mimics one or more characteristics of the production medium. In certain embodiments, adaptation of cells in adaptation medium exhibits a reduced or lesser detrimental effect when such adapted cells are switched from growth medium or adaptation medium to production medium. Cells are obtained. Such an adaptive medium, for example, supplements the production medium with such metabolites and / or inhibitors and / or allows such metabolites and / or inhibitors to accumulate in cell culture over time. Can be matched with the generation.

  As used herein, the term “known medium” refers to a medium whose composition is known and controlled.

  As used herein, the term “complex medium” refers to a medium containing at least one component that is either unknown or uncontrolled, either in essence or amount.

  The phrase “cell line” generally refers to a primary host cell that has been transfected with exogenous DNA, eg, DNA encoding a desired protein of interest. In certain embodiments, cells derived from genetically modified cells from a cell line are placed in cell culture medium and grown to produce the protein product of interest. In some embodiments, exogenous DNA containing a regulatory sequence that encodes a desired protein and / or activates expression of the linking sequence, whether endogenous or heterologous, is converted into primary Transfect host cells. In certain embodiments, the cell line comprises a primary host cell that has been transfected with exogenous DNA and expresses a heterologous protein of interest. In certain embodiments, the cell line comprises a primary host cell that is not transfected with exogenous DNA and expresses the endogenous protein of interest.

  The “growth phase” of a cell culture medium refers to the period of time during which cells divide rapidly and grow exponentially or nearly exponentially. Growth phase conditions can include temperatures of about 35 ° C. to 42 ° C., generally about 37 ° C. The length of the growth phase and the culture conditions during the growth phase can vary, but are generally known to those skilled in the art of cell culture. Usually, cells are grown in a “growth medium” or “standard growth medium” during the growth phase. In certain embodiments, the growth phase cell culture medium is supplemented with a supply medium.

  The “transition phase” occurs during a period in which the cell culture medium is shifted from conditions consistent with the growth phase to conditions consistent with the production phase. During the transition period, factors such as temperature are often changed. In certain embodiments, the transitional cell culture medium is supplemented with a feed medium. The methods of the present invention are useful in minimizing the deleterious effects that can occur when transferring cell culture from growth phase conditions to production phase conditions.

  The “production phase” occurs after both the growth phase and the transition phase. After the exponential growth of cells, protein production is the main objective. Usually, cells are grown in production medium during the production phase. The production medium can be supplemented to initiate production. In certain embodiments, the nascent cell culture medium is supplemented with a feed medium. In certain embodiments, generally, the temperature of the cell culture medium during the production phase is lower than the growth phase. As is known in the art, in many cases such a decrease in temperature facilitates protein production. The generation phase continues until the desired endpoint is reached.

  The phrases “split”, “passage” and “subculture” refer to the process of dividing a cell population into two or more subpopulations. For example, a population of cells growing in cell culture medium can be passaged or subcultured by removing the subpopulation of cells from the cell culture medium and diluting the subpopulation to a lower live cell density. . In certain embodiments, a subpopulation of cells can be diluted to a similar volume using fresh media. In certain embodiments, a subpopulation of cells is diluted with a cell culture medium that is similar or identical to the cell culture medium in which the original cell population was grown. For example, if the original cell population was growing in production medium, the subpopulation can be diluted with the same or similar production medium. In certain embodiments, a subpopulation of cells is diluted with a cell culture medium that is different from the cell culture medium in which the original cell population was grown. For example, if the original cell population was growing in growth media, the subpopulation of cells can be diluted with production media or adaptive media. In certain embodiments, the original cell population has stopped growing (eg, increasing cell number) prior to passage or subculture of the subpopulation. In certain embodiments, the population is passaged more than once during the adaptation process. For example, a population may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times or more during the adaptation process Can be passaged. Standard practice is to use standard growth media to maintain or subdivide cell culture media every 3 to 4 days. In certain embodiments, cells are adapted to protein production conditions by growing a cell population over an extended period of time before being passaged. For example, cells can be allowed to grow for 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days or more prior to passage.

  The phrase “viable cell density” refers to the total number of cells surviving in a particular volume of cell culture medium. The phrase “cell viability” refers to the number of viable cells relative to the total number of both dead and viable cells, expressed as a percentage.

  “Live cell density”, “IVCD”: As used herein, the term “live cell density” or “IVCD” refers to the average density of live cells during the course of culture. This is multiplied by the length of time. If the amount of protein produced is proportional to the number of living cells present while the culture proceeds, the density of living cells is useful for estimating the amount of protein produced while the culture proceeds. It becomes a proper tool.

  As used herein, the term “antibody” refers to at least one, typically two VH domains or portions thereof and / or at least one, typically two VL domains or portions thereof. Contains protein containing. In a particular embodiment, the antibody is a tetramer consisting of two immunoglobulin heavy chains and two immunoglobulin light chains, where the immunoglobulin heavy and light chains are interlinked, for example, by disulfide bonds. Connected. The antibody or portion thereof may be obtained from any source including, but not limited to, rodents, primates (eg, human and non-human primates), camelids, As described in more detail herein, it can be produced recombinantly, eg, as a chimeric or humanized antibody, and / or can be made in vitro.

  Examples of binding fragments encompassed within the term “antigen-binding fragment” of an antibody include, but are not limited to, (i) a Fab fragment, ie, a VL domain, a VH domain, a CL domain, and a CH1 domain. (Ii) F (ab ′) 2 fragment, ie, a divalent fragment comprising two Fab fragments joined by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of a VH domain and a CH1 domain (Iv) an Fv fragment consisting of the single arm VL and VH domains of the antibody; (v) a dAb fragment consisting of the VH domain; (vi) a camel or camelized heavy chain variable domain (VHH); (Vii) a single chain Fv (scFv); (viii) a bispecific antibody; and (ix) fused to the Fc region, One or more fragments of disease immunoglobulin molecule. In addition, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but can be joined together by synthetic linkers using recombinant methods. With this synthetic linker, these domains can be produced as a single protein chain, in which the VL region and the VH region are paired to form a monovalent molecule (this is a single chain) Fv (scFv), for example, Bird et al. (1988) Science 242: 423-26; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-83. See). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment” of an antibody. These fragments can be obtained using conventional techniques known to those skilled in the art, and the function of these fragments is assessed as in the case of untreated antibodies.

  An “antigen-binding fragment” can optionally further comprise a moiety that enhances one or more of, for example, stability, effector cell function, or complement binding. For example, the antigen-binding fragment can further comprise a pegylated moiety, albumin, or a heavy and / or light chain constant region.

  Except for “bispecific” or “bifunctional” antibodies, antibodies are understood to have the same binding site for each. A “bispecific antibody” or “bifunctional antibody” is an artificial hybrid antibody having 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 linking of Fab 'fragments. See, for example, Songsivirai and Lachmann, Clin. Exp. Immunol. 79: 315-321 (1990); Kostelny et al., J. Biol. Immunol. 148, 1547-1553 (1992).

  The phrase “screen” or “screening” refers to a method of selecting a subpopulation of cells having a particular phenotype that exhibits one or more advantageous characteristics. In certain embodiments, proteins include, but are not limited to, improved proliferation, increased viability, increased live cell density, increased titer and / or increased cell specific productivity. Cell lines are screened for one or more cell culture characteristics that are advantageous in the production process. In certain embodiments, screening in cell lines is advantageous for one or more cell culture characteristics, such as strong growth and / or viability, that are advantageous in a fed-batch bioreactor process. is there.

  The phrase “bioreactor” refers to a container that can contain cell culture media and whose internal conditions, such as pH and temperature, can be controlled during the culture period. One skilled in the art will know useful and / or appropriate bioreactor conditions that can be controlled and methods for controlling such bioreactor conditions. “Production bioreactor” refers to a bioreactor utilized during the process of producing a protein. For example, a production bioreactor can include large commercial scale vessels that can produce large amounts of protein, but production bioreactors are not limited to such large commercial scale vessels. In certain embodiments, the volume of the bioreactor is at least 1 liter, 10, 100, 250, 500, 1,000, 2,500, 5,000, 8,000, 10,000, or 12,000 liters. The capacity can be any of the above or between them. Moreover, bioreactors that can be used include, but are not limited to, stirred tank bioreactors, fluidized bed reactors, hollow fiber bioreactors or roller bottles.

  The phrases “secondary byproduct” and “secondary metabolite” refer to small molecules that normally occur as a result of the metabolic activity of a cell. As understood by those skilled in the art, such secondary by-products are often detrimental to cell growth and / or viability. Therefore, it is desirable to minimize or eliminate them from cell culture. Non-limiting examples of secondary by-products include lactic acid and ammonia ions. In certain embodiments, cells are adapted to protein production conditions by growing the cells in the presence of such secondary by-products. In certain embodiments, cells adapted to protein production conditions exhibit the ability to absorb such secondary by-products so that the level of secondary by-products decreases with time. Those skilled in the art of cell culture will be aware of other metabolic byproducts that can be used to adapt cells according to the present invention.

  “Feed-batch culture method” refers to a cell culture method in which cells are first inoculated into a bioreactor having an inoculation medium. The cell culture medium is then supplemented with a feed medium containing nutrients and / or other supplements at one or more times throughout the production run.

  “Batch culture method” refers to a cell culture method in which cells are inoculated into a bioreactor having all of the nutrients and adjuvants required for the entire production run. No nutrients, medium, etc. are added to the cell culture medium after the start of cell culture.

  The “perfusion culture method” refers to a cell culture method different from the batch culture method and the fed-batch culture method, and in this case, the culture is terminated or necessarily terminated prior to the isolation and / or purification of the expressed target protein. Rather, new nutrients and other ingredients are added to the medium periodically or continuously, during which time the expressed protein is collected periodically or continuously. The composition of the added nutrients can be varied during cell culture progression, depending on the needs of the cells, the requirements for optimal protein production and / or a variety of other factors known to those skilled in the art.

  The phrase “batch-refeed” refers to the manner in which the bioreactor operates or the method of passaging cells. In certain embodiments, the batch-refeed process includes passage of cells every 7 to 8 days, whereas in other modes in a bioreactor, cells are not passaged or less frequently. Passed on. At smaller scales utilizing standard splitting methods, cells are generally passaged earlier, eg every 3 to 4 days, compared to batch-refeeds. In certain embodiments, using batch-refeed methods, cell cultures can be used to, but are not limited to, improved growth rate, increased viability, increased live cell density, increased titer and / or The cell culture is adapted so that the characteristics of the cell culture can be achieved, including the specific enhancement of cell productivity. In certain embodiments, the batch-refeed process utilizes a more enriched culture and / or medium containing secondary metabolites and / or other inhibitors to adapt the cells to protein production conditions. . The phrase “secondary metabolite” refers to a byproduct of the metabolic process of cellular function.

  The phrase “expression” refers to transcription and translation occurring in the host cell. The level of expression is generally related to the amount of protein produced by the host cell.

  The phrase “protein” or “protein product” refers to one or more chains of amino acids. As used herein, the term “protein” is synonymous with “polypeptide” and is linked via sequential peptide bonds, as commonly understood in the art. Refers to at least one chain of amino acids. In certain embodiments, a “protein of interest” is a protein encoded by an exogenous nucleic acid molecule transformed into a host cell. In certain embodiments, a “protein of interest” is encoded by exogenous DNA that has been transfected into a host cell, and the nucleic acid sequence of the exogenous DNA determines the sequence of the amino acids. 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, a host cell is transfected with an exogenous nucleic acid molecule that can, for example, contain one or more regulatory sequences and / or encode a protein that enhances expression of the protein of interest. Thereby altering the expression of such endogenous protein of interest. Using the methods and compositions of the present invention, a variety of useful in, but not limited to, pharmaceutical properties, diagnostic properties, agricultural properties and / or commercial, experimental and / or other applications Any protein of interest can be produced, including proteins having any of the other properties. In addition, the protein of interest can be a protein therapeutic. That is, a protein therapeutic is a protein that has a biological effect on the area of the body where it acts or the area of the body where it acts indirectly via an intermediate. Examples of protein therapeutics are discussed in more detail below. In certain embodiments, proteins produced using the methods and / or compositions of the invention can be processed and / or modified. For example, a protein to be produced according to the present invention can be glycosylated.

  As used herein, the term “titer” refers to the total amount of recombinantly expressed protein produced by cell culture in a given volume of media. A titer is usually expressed in units of milligrams or micrograms of protein per milliliter of medium.

  One skilled in the art can use the methods disclosed herein to cultivate many of the well-known mammalian cells routinely used and cultured in the art, ie, as described herein. It will be appreciated that the disclosed method is not limited to use only with respect to the present disclosure.

  For example, mammalian cells, including Chinese hamster ovary cells, such as non-transfected mammalian cells, can be adapted to an environment consistent with the conditions of the production bioreactor during subsequent production of the protein of interest. It has been discovered that cell proliferation can be improved. Such methods also include non-transfected and transfected mammalian cells (eg, mammalian cells transfected with cDNA, or genomes that cause expression of the desired recombinant protein, such as from expression vectors). It can be applied to both of the construction). Using the present invention, for example, pharmaceutically or commercially appropriate enzymes, receptors, antibodies (eg, monoclonal and / or polyclonal antibodies), Fc fusion proteins, cytokines, hormones, regulators, growth factors, coagulation Cells can be adapted for the advantageous production of any therapeutic protein, such as factors, antigen binding substances, and the like. One skilled in the art will recognize other proteins that can be produced in accordance with the present invention and will be able to produce such proteins using the methods disclosed herein.

  The methods of the present invention for adapting non-transfected mammalian cells have the potential to identify cell lines that provide superior productivity and / or exhibit excellent protein production characteristics. Furthermore, the methods of the invention can help to identify suitable candidates for cell lines more quickly and with less effort compared to standard cell line development procedures. For example, a standard process for identifying cell line candidates usually requires numerous experiments at different scales and additional tests on strength.

  Host cells are considered non-transfected before transformation. Traditionally, when creating a new cell line for development, non-transfected host cells were first transfected or transformed with exogenous DNA in order to express the protein of interest. When using such previous methods, in general, prior to transfection, the host cells were not tested or altered. After engineering host cells to produce protein products, experiments and research on cell line development began.

  In certain embodiments, an improvement in the process of batch, fed-batch and / or perfusion bioreactors by adapting non-transfected mammalian cells to protein production conditions according to one or more methods of the present invention. Performance can be obtained. In certain embodiments, such development allows such adapted cells to maintain an improved phenotype, such as more powerful proliferation, increased viability, live cell density, etc. Increase in titer, increase in titer, increased specific productivity of cells and / or higher cell density. Utilizing certain embodiments of the inventive methods described herein, adapting non-transfected mammalian cells to batch, fed-batch and / or perfusion protein production processes for superior performance Can be given.

  For example, any of various commercially available media such as Minimal Essential Medium (MEM, manufactured by Sigma), Ham's F10 (manufactured by Sigma) or Dulbecco's modified Eagle's medium (DMEM, manufactured by Sigma) should be used as the basic medium. Can do. Such basal media can then be supplemented with amino acids, vitamins, inorganic salts, trace elements and / or other components to produce growth and / or production media. In certain embodiments, the cells can be adapted by culturing transfected or non-transfected mammalian cells in a production medium that is similar or identical to the production medium. In certain embodiments, conditions that are similar to or identical to conditions normally encountered under production conditions in a bioreactor operating in a batch, fed-batch and / or perfusion mode (eg, normally found in a bioreactor) The cells are adapted by growing the cells in a medium that matches the medium. In certain embodiments, the cell to be adapted is not transfected. In certain embodiments, the cell to be adapted has been transfected with an exogenous nucleic acid molecule, eg, a nucleic acid molecule that expresses the protein of interest. In certain embodiments, the characteristics of one or more cell cultures during the protein production phase are improved by adapting the cells to protein production conditions prior to transfection with the exogenous nucleic acid molecule. In certain embodiments, such improved cell culture features include, but are not limited to, improved growth, improved overall cell viability, increased live cell density, increased titer and / or Or the specific productivity improvement of a cell is mention | raise | lifted. Typically, the production medium is fortified over the growth medium, for example supplemented with higher levels of nutrients, vitamins, trace elements and / or other medium components compared to the growth medium.

  It is normal practice in the art that cells do not encounter the production medium until the cell line is grown until the cells are continuously cultured in growth medium and a fed-batch production assay is initiated. is there. In certain embodiments, prior to transfection, non-transfected mammalian cells are adapted in a protein production medium and / or under protein production conditions, rather than in growth medium and / or growth conditions, to place the cells in one environment. Minimize adverse effects that may occur when moving from one environment to another, eg, from growth conditions to post-transfection protein production conditions.

  In certain embodiments, the methods of the invention are useful for creating host cell lines adapted to protein production conditions. Such an adapted host cell system can be transfected with any of a variety of proteins of interest. In certain embodiments, such adapted transfected cell lines are placed directly within protein production conditions. In certain embodiments, such adapted transfected cell lines are grown to the desired cell density and / or desired cell number during the growth phase, after which the cell line is transferred to the protein production phase. Let According to the present invention, such adapted transfected cell lines show less and less harmful effects during the transition phase compared to non-adapted transfected cell lines.

  In certain embodiments, an adapted host cell is transfected with an exogenous nucleic acid molecule to produce the protein of interest. In certain embodiments, the nucleic acid molecule introduced into the cell encodes a protein desired to be expressed according to the present invention. In certain embodiments, the nucleic acid molecule encodes a gene product that contains regulatory sequences or that induces or enhances the cell to express the desired protein. As a non-limiting example, such a gene product can be a transcription factor that increases the expression of the protein of interest.

  In certain embodiments, nucleic acids that direct protein expression are stably introduced into host cells. In certain embodiments, nucleic acids that direct protein expression are transiently introduced into the host cell. One skilled in the art will be able to choose whether the nucleic acid should be introduced into the cell in a stable or transient state based on experimental, commercial or other needs.

  The gene encoding the protein of interest can be linked to one or more regulatory regulatory elements of the gene, if desired. In some embodiments, the regulatory elements of the gene direct the structural expression of the protein. In some embodiments, genetic regulatory elements that provide for inducible expression of the gene encoding the protein of interest can be used. Inducible gene regulatory elements (eg, inducible promoters) can be used to regulate protein production in cells. Non-limiting examples of inducible gene regulatory elements that are likely to be useful when used in eukaryotic cells include hormone regulatory regions (eg, Mader, S. and White, JH, Proc Natl.Acad.Sci.USA 90: 5603-5607, 1993), synthetic ligand control regions (see, eg, Spencer, DM, et al., Science 262: 1019-1024, 1993), and ionizing radiation control. Regions (see, eg, Manome, Y. et al., Biochemistry 32: 10607-10613, 1993; Datta, R. et al., Proc. Natl. Acad. Sci. USA 89: 10149-10153, 1992). Additional, cell specific or other control systems known in the art can also be used in accordance with 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 invention. Proteins can be expressed from genes that are endogenous to the host cell or from heterologous genes that have been introduced into the host cell. The protein may be naturally occurring or alternatively may have an artificially manipulated or selected sequence. The protein to be produced can be assembled from individual naturally occurring protein fragments. In addition or alternatively, the engineered protein can include one or more non-naturally occurring fragments.

  Any host cell that is sensitive to cell culture and protein expression can be utilized in accordance with the present invention. In certain embodiments, the host cell is a mammalian cell such as, for example, a Chinese hamster ovary (CHO) cell. Other non-limiting examples of mammalian cells that can be used according to the present invention include the BALB / c mouse myeloma cell line (NSO / I, ECACC number: 85110503); human retinoblastoma (PER.C6) (CruCell, Leiden, The Netherlands)), monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651), human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture) Graham et al., J. Gen Virol., 36:59 (1977)); Baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells +/- DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad). Sci.USA, 77: 4 216 (1980)); mouse Sertoli cells (TM4, Mother, Biol. Reprod., 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL). -1 587); human cervical cancer 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, HB 8065); mouse breast cancer (MMT 060562, ATCC CCL51); TRI cells (Mother et al., Anals NY Acad. Sci., 383: 44-68 (1982)). MRC 5 cells; FS4 cells; and the human hepatocellular carcinoma line (Hep G2).

  In certain embodiments, the cells are adapted by culturing the cells (eg, non-transfected mammalian cells) in a growth medium supplemented with an inhibitor. In certain embodiments, such inhibitors include inhibitors normally found when cells are grown under protein production conditions. Non-limiting examples of such inhibitors include lactic acid, ammonia, alanine, glutamine and / or acetolactate. One skilled in the art will recognize other inhibitors that can be used in accordance with the present invention to adapt cells to protein production conditions.

  In certain embodiments, by culturing cells (e.g., non-transfected mammalian cells) in an adaptive medium that lacks or contains a reduced concentration of one or more components conventionally found in the production medium. Adapt the cells. For example, conventional production media typically contain insulin at a concentration of about 10 mg / L or higher. Thus, in certain embodiments, the cells are adapted by culturing the cells in an adapted medium lacking insulin. In certain embodiments, the cells are adapted by culturing the cells in an adaptive medium containing insulin at a concentration lower than that conventionally found in the production medium. A person skilled in the art knows other components normally present in the production medium and can use the methods of the present invention to adapt the cells to a medium lacking such components or containing a reduced concentration. Will.

  In certain embodiments, the cells are adapted by culturing (eg, continuous culture) non-transfected mammalian cells in a growth medium supplemented with one or more inhibitors. Non-limiting examples of such inhibitors include secondary by-products of primary carbon sources. For example, some secondary by-products include, but are not limited to, lactic acid, alanine, glutamine, acetolactate and / or ammonia. In certain embodiments, the concentration of such secondary by-products present and / or added in the adaptation medium is, for example, from about 2.0 to about 10.0 g / L lactic acid and / or about 0. Mimic concentrations normally encountered in bioreactor conditions, such as from 1 to about 0.5 g / L ammonia. In certain embodiments, conditions wherein the concentration of lactic acid in the adaptive medium is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 g / L or more Under, adapt the cells. In addition or alternatively, in certain embodiments, the concentration of ammonia in the adaptive medium is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. The cells are adapted under conditions that are 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 g / L or higher.

  In certain embodiments, individual non-transfected mammalian cells and / or sub-populations of non-transfected mammalian cells that have been adapted to protein production conditions according to one or more methods of the present invention are such A phenotype consistent with secondary by-product uptake can be shown, so that the level of secondary by-products actually decreases with time. In certain embodiments, such adapted non-transfected cells have the ability to absorb secondary by-products even after transfection of exogenous nucleic acid molecule cells to produce the protein of interest. To do. In certain embodiments, generally, such adapted transfected cells are non-transfected mammalian cells that do not take up by-products in subsequent protein production processes (eg, fed-batch bioreactor processes) and It / or behaves better compared to non-transfected mammalian cells that are not adapted to take up such secondary by-products. For example, such adapted transfected mammalian cells behave better in subsequent batch, fed-batch and / or perfusion protein production processes. In a production reactor (eg, a fed-batch production reactor), such secondary by-products often accumulate to a level that generally inhibits subsequent cell growth and / or viability. In certain embodiments, a subpopulation of non-transfected mammalian cells adapted according to the methods of the invention and grown in supplemented media is an inhibitory and stress-reducing group that is normally encountered in production bioreactors. Can grow well under many conditions.

  As is known in the cell culture art, cell culture is often switched from growth conditions to protein production conditions to lower the temperature of the cell culture. In certain forms, the cells are adapted by culturing the cells at a temperature that promotes the production of the protein product. In certain forms, the cells are adapted by culturing the cells at a temperature of about 31 ° C., but the methods of the invention are not limited to such temperatures. For example, 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 The cells can be adapted by culturing the cells at a temperature of 44, 45 ° C. One skilled in the art will know the appropriate temperature for protein production. Such temperature will depend, at least in part, on the cell line, the protein to be produced, other culture conditions, and / or any other factors deemed important by those skilled in the art.

  In certain embodiments, the cells are adapted by continuously culturing non-transfected mammalian cells in a “batch-refeed” manner for a longer period of time. In certain embodiments, non-transfected cells are adapted by such “batch-refeed” adaptation methods. In certain embodiments, transfected cells are adapted by such “batch-refeed” adaptation methods. Normal cell culture operations can utilize a cell culture management method that consists of dividing, subcultured, or subcultured cell cultures every 3 to 4 days. In certain embodiments of the “batch-refeed” adaptation method, non-transfected mammalian cells are cultured for a longer period, such as 7 or 8 days, prior to subculture. In certain embodiments, the mammalian cells are cultured for 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days or more prior to subculture. Such longer-term cultures bring cells to protein production conditions by increasing cell density and / or accumulating higher levels of secondary metabolites in conditioned media that the cells can adapt. Has a dual effect of adapting.

  In certain embodiments, non-transfected mammalian cells can be continuously cultured under conditions “consistent with production”. In certain embodiments, after cell culture under conditions consistent with production, followed by 3 or 4 days under “recovery” or standard growth conditions, optionally using growth media, Non-transfected cells can be cultured using repeated cycles consisting of passage of a subpopulation of cells. In certain embodiments, the population is passaged multiple times during the adaptation process. For example, a population may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times or more during the adaptation process Can be passaged. In certain embodiments, the cells are adapted to protein production conditions by allowing the cell population to grow for a longer period of time before being passaged. For example, cells can be grown for 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days or more prior to passage. Such passaged subpopulations can be screened for one or more desirable characteristics. In certain embodiments, several cycles of repetitive or continuous adaptation in conditions consistent with production may result in improved growth and / or survival over non-adapted starting populations, for example. There will be a subpopulation of cells that display excellent characteristics, including the beginning. Moreover, in certain embodiments, certain subpopulations begin to absorb by-products, such as lactic acid, so that the level of by-products decreases with time, enhancing the performance of cell lines in production bioreactors. To do. In certain embodiments, one or more screened subpopulations of cells exhibiting one or more desirable characteristics (eg, strong growth) are then transformed to produce the protein of interest. The transfected subpopulation is then placed in protein production conditions (eg, conditions consistent with the conditions to which the cells are adapted).

  In certain embodiments, U.S. Patent Application Nos. 11/213308 and 11/213317, each filed on August 25, 2005, the entire contents of each of which are incorporated herein by reference. And the cells are grown according to any of the cell culture methods described in 11/213633. For example, in certain embodiments, cells can be grown in media where the cumulative amino acid concentration is greater than about 70 mM. In certain embodiments, the cells can be grown in a medium in which the molar ratio of cumulative glutamine to cumulative asparagine is less than about 2. In certain embodiments, the cells can be grown in a medium in which the molar ratio of cumulative glutamine to cumulative total amino acids is less than about 0.2. In certain embodiments, the cells can be grown in a medium in which the molar ratio of accumulated inorganic ions to accumulated total amino acids is between about 0.4 and about 1. In certain embodiments, cells can be grown in a medium in which the sum of cumulative glutamine concentration and cumulative asparagine concentration is between about 16 mM and about 36 mM. In certain embodiments, cells can be grown in media having all two, three, four, or five of the aforementioned media conditions. Use of such a medium allows the production of high levels of protein and reduces the accumulation of certain undesirable factors such as ammonium and / or lactic acid.

  In some embodiments, one or more conditions described in US Provisional Patent Application No. 60/830658, filed July 13, 2006, the entire contents of which are incorporated herein by reference. To grow the cells. For example, in some embodiments, the cells are grown in a medium containing manganese at a concentration between about 10 nM and about 600 nM. In some embodiments, the cells are grown in a medium containing manganese at a concentration between about 20 nM and about 100 nM. In some embodiments, the cells are grown in a medium containing manganese at a concentration of about 40 nM. Glycoproteins with improved glycosylation patterns (eg, a greater number of covalently linked sugar residues in one or more oligosaccharide chains) when such media are used to increase glycoproteins Resulting in the generation of

  The components and / or supplements of the production medium and growth medium can be readily determined by one skilled in the cell culture art. As is known in the art, the components and / or adjuvants can vary depending on the host cell used and the desired protein of interest. Moreover, different bioreactor conditions and cell lines may or may not produce different by-product states and amounts. The present disclosure is further illustrated by the following non-limiting examples. Any modifications that may be required in the course of adapting mammalian cells to cell culture media in producing different proteins are also well covered by the cell culture techniques.

Example 1: Adaptation of untransfected Chinese hamster ovary cells to conditions consistent with production Non-transfected CHOK1 cells were treated with standard 3 days / 4 days in growth medium, the ingredients of which are listed in Table 1 below. Multiple-cycle cultures in either batch-refeed conditions or in a 7-day batch-refeed mode in fortified production medium listed in Table 2 below. The cells were cultured at a working volume of 10 to about 30 ml at 37 ° C. The cell number from the experiment is shown in FIGS. 1a and 1b. Near day 58 of the batch-refeed method of FIG. 1a, cells were passaged for 9 days instead of 7 days, and subsequent passages did not grow well. However, after these 7 days of poor passage, the cells were able to recover.

  Cells were then evaluated in a production assay by fed-batch method. In this assay, the working volume was between 10 and 30 ml. Cells were cultured for 4 days at 37 ° C., then the temperature was shifted to 31 ° C. and kept at this temperature until day 12 when the assay was completed. The cell density and viability were compared and the results are shown in FIG. Cells cultured in the 7-day batch-refeed mode showed higher cell density than cells cultured in the standard 3-day / 4-day batch-refeed mode. Similarly, two different groups of cells whose components were adapted for growth in the production medium listed in Table 3 below achieved a higher cell density than the non-adapted control starting population.

Example 2: Adaptation of transfection product cell line to conditions consistent with production Monoclonal antibody heavy chain and light chain gene expressing cell line components listed in Table 1 in standard growth medium Or using either of the two production media “A” and “B”, and cultured under standard 3/4 day batch feed conditions. The production medium A, whose ingredients are listed in Table 2, is moderately enhanced compared to the growth medium, and the production medium B, whose ingredients are listed in Table 4 below, is markedly different compared to the growth medium. It was strengthened. The effect of methotrexate (MTX) was also tested in production medium “B” and 1.5 μM MTX was added into one culture with production medium “B” and into another culture. Was not added. The cells were cultured at a working volume of 10 to about 30 ml at 37 ° C. The growth rate during adaptation in these media is shown in Figure 2a.

  After continuous culture in these media, the cell lines were evaluated in a production assay by fed-batch method. In this assay, cells are evaluated in production medium B and are shown in FIG. Cells were cultured for 4 days at 37 ° C., then the temperature was shifted to 31 ° C. and kept at this temperature until day 12 when the assay was completed. The results show that cell lines overexpressing the recombinant protein can also adapt to conditions consistent with production and achieve superior growth characteristics in a fed-batch production assay.

Example 3: Adaptation of non-transfected Chinese hamster ovary cells to low insulin conditions Insulin directly affects glucose metabolism by mammalian cells. The rapid consumption of glucose in cell culture is frequently associated with the excretion of lactic acid as a metabolic waste product. Lactic acid can inhibit cell growth and has a negative effect on cell viability. Insulin has also been reported to be a growth factor for mammalian cells.

  Cells from the CHOK1 host cell line were taken from normal growth medium (containing 10 mg / L nucellin) and placed in medium completely lacking insulin. After the initial lag phase when the cells show a decrease in proliferation, the growth rate eventually increases to a rate comparable to that of the CHOK1 control culture in medium containing insulin, which is the non-insulin level. It was suggested that cells cultured in the presence were adapted to these conditions. Cell viability was unaffected and remained mid-90s through adaptation. This adaptation continued for about 100 population doublings. When adapted CHOK1-insulin cells were stored and then thawed, they maintained the ability to grow in media lacking insulin. CHOK1 control cells thawed in medium lacking insulin show a reduced growth rate, which indicates that K1-insulin adaptation is a very cellular change that allows the cells to grow independent of exogenous insulin. Suggested to do. The study will continue to include a combination of a phenotype unaffected by insulin and a phenotype of adaptation by passage for 7 days.

  The production assay by the first fed-batch method was performed in a format in which the pH was not adjusted. Experiments include non-adapted CHOK1 cells in standard production medium (20 mg / mL nucellin) as positive controls and non-adapted CHOK1 cells cultured in medium lacking insulin as negative controls. It is. Adapted CHOK1 cells that were not affected by insulin were cultured in production medium lacking insulin. On days 3 and 7, insulin was included in the feed medium (at a final concentration of 0.006 mg / mL). Examining the data, CHOK1 cells adapted to grow in medium lacking insulin may have very similar characteristics in terms of proliferation, viability and IVCD compared to CHOK1 positive control samples. Has been suggested (see FIGS. 3-5). The negative control CHOK1 specimen showed a decrease in growth rate (see FIG. 3). These results confirm that CHOK1 cells adapted to growth lacking insulin are essentially different from non-adapted CHOK1 cells due to their ability to grow well in medium without insulin. .

  The adapted CHOK1 cells unaffected by insulin were then evaluated under pH-controlled conditions in a second fed-batch experiment. In this experiment, non-adapted CHOK1 cells and adapted CHOK1 cells not affected by insulin were placed in three separate conditions. The first condition contained insulin (50 mg / L and 0.006 mg / mL, respectively) in both the basal medium and the feed and was labeled with the (+ / +) symbol in FIGS. The second condition contains a basal medium lacking insulin with the addition of a feed containing insulin (indicated by the symbol (− / +) in FIGS. 6-9), and the third condition contains insulin. A basal medium lacking and a feed medium lacking insulin were included (indicated by the symbol (− / −) in FIGS. 6-9). When cultured in medium lacking insulin, adapted CHOK1 cells that were not affected by insulin showed better growth, viability and IVCD compared to non-adapted CHOK1 cells (see FIGS. 6-9). ). The CHOK1-insulin adapted cell line produces less lactic acid, and it appears that they are almost completely consumed no matter what lactic acid they produce (see FIG. 9). This disadvantageous by-product is eliminated, resulting in a healthier cell culture, which is considered a very promising phenotype. This prospective phenotype provides better growth conditions and allows the cells to reach a higher density than the relevant controls. When cultured in medium containing insulin, the CHOK1-insulin adapted cell line has a lactic acid production rate similar to that of the control sample, so this phenotype is directly related to the exclusion of insulin from the medium. Note that there are.

  Example 3 demonstrates that adaptation of CHOK1 cells to conditions lacking insulin results in a desirable phenotype when cells are cultured in an industrially suitable manner of production. The improved metabolic phenotype results in increased cell proliferation and viability, which is expected to have a significantly positive impact on productivity with respect to the volume of recombinant CHO cell cultures .

  While specific embodiments of the present disclosure have been described herein, the above description is merely exemplary. Further modifications of the embodiments disclosed herein could be provided by one of ordinary skill in the cell culture arts. All such modifications are considered to fall within the scope of the embodiments, as defined by the appended claims.

FIG. 6 is a diagram showing the density of viable cells in the case of 7-day batch-refeed. It is a figure which shows the density of the living cell at the time of dividing | segmenting normally. It is a figure which shows the density of a living cell. FIG. 5 shows the growth rate of a monoclonal antibody cell line. It is a figure which shows the accumulation density | concentration of a living cell during a fed-batch method. FIG. 6 shows cell densities of cell cultures adapted to lack of insulin and control cell cultures. FIG. 5 shows the viability of cell cultures adapted to lack of insulin and control cell cultures. FIG. 6 shows cumulative IVCD of cell cultures adapted to insulin deficiency and control cell cultures. FIG. 6 shows cell densities of cell cultures adapted to lack of insulin and control cell cultures. FIG. 5 shows the viability of cell cultures adapted to lack of insulin and control cell cultures. FIG. 6 shows cumulative IVCD of cell cultures adapted to insulin deficiency and control cell cultures. FIG. 5 shows specific lactate consumption rates of cell cultures adapted to insulin deficiency and control cell cultures.

Claims (30)

A method for adapting cells to protein production conditions,
Culturing the cells in an adaptive medium;
Screening the cells and selecting a sub-population of cells that exhibit improved cell culture characteristics when grown under protein producing conditions, wherein the characteristics are observed by cell growth in a non-adaptive medium Unlike the corresponding cell culture characteristics, the improved cell culture characteristics include improved proliferation, increased viability, increased live cell density, increased titer, increased cell specific productivity and An adaptation method wherein the method is selected from the group consisting of combinations thereof.   The method according to claim 1, further comprising a cell passage step before being subjected to the screening step.   4. A method according to claim 2 or 3, wherein the passaging step comprises passaging the cell twice or more before subjecting to the screening step.   4. The method of any one of claims 1-3, wherein the passaging step comprises passaging the cells in an adaptive medium after about 3 or 4 days.   4. The method of any one of claims 1-3, wherein the passaging step comprises passaging the cells in an adaptive medium after about 7 or 8 days.   The method according to any one of claims 1 to 5, further comprising a step of culturing the cells in a medium that is not an adaptive medium after the passaging step.   7. The method of claim 6, wherein the medium that is not an adaptive medium is a standard growth medium.   8. A method according to any one of claims 1-7, wherein the cells are not transfected.   The method according to any one of claims 1 to 7, wherein the cell is transfected to express a protein of interest.   The method according to claim 9, wherein the protein of interest is an antibody.   The method according to claim 9, wherein the protein of interest is a proteinaceous therapeutic agent.   12. A method according to any one of claims 1-11, wherein the adaptive medium comprises a production medium.   The production medium comprises a high level of one or more medium components compared to a standard growth medium, wherein the medium components are selected from the group consisting of nutrients, vitamins, trace elements and combinations thereof. Item 13. The method according to Item 12.   14. The method according to any one of claims 1-13, wherein the adaptive medium comprises a secondary metabolite selected from the group consisting of lactic acid, ammonia, and combinations thereof.   15. The method of claim 14, wherein secondary metabolites are added to the adaptation medium when initiating cell culture.   16. A method according to claim 14 or 15, wherein the level of secondary metabolites increases with the progress of cell culture.   17. The method of claim 16, wherein the level of secondary metabolites is increased due to cellular metabolic activity.   18. The method of claim 16 or 17, wherein the level of secondary metabolite is increased through the addition of secondary metabolite to the cell culture.   19. The selected subpopulation of cells takes up a secondary metabolite, so that the level of said metabolite is reduced when adapted cells are grown in production medium. The method according to one item.   20. The method according to any one of claims 1-19, wherein the adaptive medium comprises one or more inhibitors selected from the group consisting of lactic acid, ammonia, alanine, glutamine, acetolactate and combinations thereof.   21. The method of any one of claims 14-20, wherein lactic acid is present at a concentration of about 2 to about 10 g / L.   The method of claim 21, wherein the lactic acid is present at a concentration of about 2 to about 10 g / L at the beginning of the cell culture.   21. The method of any one of claims 14-20, wherein ammonia is present at a concentration of about 0.1 to about 0.5 g / L.   24. The method of claim 23, wherein ammonia is present at a concentration of about 0.1 to about 0.5 g / L at the beginning of cell culture.   25. A method according to any one of claims 1 to 24, wherein the adaptive medium lacks insulin.   26. The method of any one of claims 1 to 25, wherein the adaptive medium comprises insulin at a concentration below about 10 mg / L.   27. The method according to any one of claims 1 to 26, wherein the cell is a mammalian cell.   28. A method according to any one of claims 1 to 27, wherein the protein production conditions comprise conditions used in a bioreactor.   30. The method of claim 28, wherein the bioreactor is a production bioreactor.   30. A method according to any one of claims 1 to 29, wherein the protein production conditions comprise culturing cells in a fed-batch protein production process.

Images