WO2007056062A2 - Procedes pour adapter des cellules de mammifere - Google Patents

Procedes pour adapter des cellules de mammifere Download PDF

Info

Publication number
WO2007056062A2
WO2007056062A2 PCT/US2006/042815 US2006042815W WO2007056062A2 WO 2007056062 A2 WO2007056062 A2 WO 2007056062A2 US 2006042815 W US2006042815 W US 2006042815W WO 2007056062 A2 WO2007056062 A2 WO 2007056062A2
Authority
WO
WIPO (PCT)
Prior art keywords
cells
medium
protein
production
certain embodiments
Prior art date
Application number
PCT/US2006/042815
Other languages
English (en)
Other versions
WO2007056062A3 (fr
Inventor
Gene W. Lee
D. Troy Richards
Timothy S. Charlebois
Mark Melville
Robin A. Heller-Harrison
Martin S. Sinacore
Mark Leonard
Original Assignee
Wyeth
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wyeth filed Critical Wyeth
Priority to EP06827375A priority Critical patent/EP1943333A2/fr
Priority to AU2006312014A priority patent/AU2006312014A1/en
Priority to JP2008539028A priority patent/JP2009514532A/ja
Priority to CA002628340A priority patent/CA2628340A1/fr
Priority to BRPI0618178-3A priority patent/BRPI0618178A2/pt
Publication of WO2007056062A2 publication Critical patent/WO2007056062A2/fr
Publication of WO2007056062A3 publication Critical patent/WO2007056062A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • This disclosure relates generally to methods of adapting mammalian cells, e.g. untransfected mammalian cells, for production of a therapeutic protein of interest. Particularly this disclosure relates to adapting untransfected mammalian cells for superior performance in bioreactors.
  • Proteins can be produced by using well known recombinant techniques.
  • Transformed cells are commonly cultured in a controlled environment, such as a bioreactor.
  • Most large-scale commercial manufacturing strategies employ suspension cell cultures grown in large stirred-tank reactors.
  • Most cell lines do not readily perform well in high cell density protein production processes (e.g., fed-batch processes) and/or cannot reach the desired high cell densities.
  • Many inhibitors are present or accumulate in the cell culture medium during a production run; these inhibitors may be by-products generated from metabolic processes such as lactate or ammonia, among others.
  • the cell lines are typically grown and maintained initially in conditions that are designed to aid in the propagation and/or survival of these cell lines.
  • Conditions used during protein production can be quite different, with e.g., secondary metabolites and/or other components present and/or accumulated as protein production progresses, which can have potential deleterious effects on the cell line.
  • deleterious effects may comprise a decrease in the viable cell density and/or a decrease in the final titer, as well as a decrease in the amount and/or quality of the produced protein.
  • the present disclosure relates to methods for adapting mammalian cells to a high density protein production process such as, for example, a fed-batch process.
  • Inventive processes can involve both untransfected mammalian cells and genetically manipulated host cells.
  • untransfected mammalian cells are adapted to production-matched conditions such as those used during protein production.
  • untransfected mammalian cells may be adapted to a production medium used in a bioreactor during a production run.
  • Different methods may be employed to adapt the untransfected mammalian cells to production conditions.
  • cells are cultured in a production and/or adaptation medium.
  • cells are cultured in an adaptation medium with standard iterative splitting cycles performed. For example, subpopulations of the cells may be passaged one or more times, e.g. every three or four days.
  • a production and/or adaptation medium generally has higher levels of nutrients, vitamins, and/or trace elements compared to a standard growth medium.
  • the cells are then allowed a recovery period between passaging, where the passaged cells are grown in a standard growth medium, e.g. during one of the cycles, before returning the cells to a production and/or adaptation medium.
  • cells are adapted by passaging them in batch-refeed mode, where subpopulations of the cells are split or passaged every seven or eight days. In certain embodiments, passaging the cells after such a longer duration allows an accumulation of secondary metabolites in the cell culture medium. In certain embodiments, adapted cells gain tolerance to and/or begin to take up such secondary metabolites. In certain embodiments, such secondary metabolites comprise inhibitors and/or metabolites that typically accumulate in a bioreactor during a production run,
  • cells are adapted by growth in a medium that includes one or more inhibitors including, but not limited to, lactate, ammonia, alanine, glutamine, and/or acetolactate.
  • inhibitors and/or metabolites are consistent with inhibitors and/or metabolites typically found in a bioreactor during a production run.
  • untransfected mammalian cells may be adapted by being cultured (and optionally split every three or four days) in a standard growth medium which is supplemented with inhibitors such as alanine, glutamine, acetolactate, ammonia, and lactate.
  • cells adapted in such a manner are passaged every three or four days.
  • the concentrations of such inhibitors correspond to concentrations typically found in a bioreactor during conditions used for protein production.
  • concentrations of some inhibitors include about 2 to about 10 g/L of lactate or about 0.1 to about 0.5 g/L of ammonia, mimicking typical bioreactor conditions.
  • cells adapted to bioreactor conditions by one or more methods of the present invention exhibit certain characteristics or phenotypes and/or can develop such phenotypes in which the cells begin to uptake lactate and/or other secondary metabolites, such that the levels of lactate and/or other secondary metabolites actually decrease during one or more time periods during the production run.
  • 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, once a subpopulation is screened for one or more desirable characteristics, after which the cells may be genetically
  • the genetically manipulated cell line does not have to transition from a standard growth medium to a production medium and/or transitions with fewer and/or less severe deleterious effects.
  • prior adaptation minimizes the potential deleterious effects on the cell line, and helps ensure cell line performance and accelerates development timelines.
  • such an adapted cell line may uptake secondary metabolites during the protein production run.
  • a person of ordinary skill in the cell culture art can readily determine what components make-up a standard growth medium and a standard production medium.
  • growth and protein production conditions differ in the composition of the cell culture media used.
  • conditions of a bioreactor may be altered and/or supplements may be added in order to increase the productivity and/or maintain viability of the cell line.
  • Supplements may include a feed medium and/or one or more additives. Those of ordinary skill in the art will be able to select appropriate media supplements.
  • untransfected mammalian cells are adapted by a method comprising culturing untransfected mammalian cells in an adaptation medium, performing one or more iterative splitting cycles (e.g. every 3 or 4 days) comprising
  • an adaptation medium contains increased levels of nutrients, vitamins, and/or trace elements compared to said standard growth medium.
  • an adaptation medium contains an increased amount of inhibitory metabolites as compared to a standard production medium prior to cell culture.
  • untransfected mammalian cells are adapted by a method comprising culturing untransfected mammalian cells in an adaptation medium that has been supplemented with side products of primary carbon metabolism, performing one or more iterative splitting cycles comprising splitting the untransfected mammalian cells about every 3 or 4 days in the adaptation medium, allowing a recovery period where the cells are cultured in a standard growth medium, accumulating levels of the side products, and screening the untransfected mammalian cells and selecting a subpopulation that exhibits an improved phenotype compared to an unadapted version of the untransfected mammalian cells when cultured in said the adaptation medium.
  • such side products are one or more of lactate, ammonia, alanine, glutamine, and/or acetolactate.
  • lactate is initially present at a concentration of about 2 to about 10 g/L.
  • ammonia is initially present in a concentration of about 0.1 to about 0.5 g/L.
  • the side products are one or more of lactate, ammonia, alanine, glutamine, and/or acetolactate.
  • lactate is initially present at a concentration of about 2 to about 10 g/L.
  • ammonia is initially present in a concentration of about 0.1 to about 0.5 g/L.
  • the side products are one or more of lactate, ammonia, alanine, glutamine, and/or acetolactate.
  • lactate is initially present at a concentration of about 2 to about 10 g/L.
  • ammonia is initially present in a concentration of about 0.1 to about 0.5 g/L.
  • the side products are one or more of lactate,
  • 4140522vl untransfected mammalian cells are adapted to take up said side products of primary carbon sources.
  • untransfected mammalian cells are adapted by a method comprising culturing untransfected mammalian cells for a duration of about 7 or 8 days in a previously-conditioned medium having an accumulation of at least one inhibitory metabolite before splitting the untransfected mammalian cells, and screening the untransfected mammalian cells and selecting a subpopulation that exhibits an improved phenotype in a production bioreactor.
  • cells are allowed a recovery period by culturing the cells in a standard growth medium for a duration of about 3 to about 4 days before splitting the cells.
  • a conditioned medium contains an accumulation of the inhibitory metabolites through at least one metabolic process of the untransfected mammalian cells.
  • an inhibitory metabolite is one or more of ammonia, alanine, glutamine, acetolactate, and/or lactate.
  • such inhibitory metabolites are consistent with those found in a production bioreactor at the end of a typical commercial-scale batch re-feed process.
  • any of a variety of suitable culture procedures and/or media may be used to culture the cells in the process of protein production. Both serum-containing and serum-free media may be used.
  • cells are grown in a defined medium.
  • cells are grown in a complex medium.
  • one or more specific culturing methods may be used, altered and/or optimized to culture the cells as appropriate for the specific cell type and protein product. Such procedures are well known and understood by workers and those of ordinary skill within the cell culture art.
  • Other procedures are well known and understood by workers and those of ordinary skill within the cell culture art.
  • Figure 1a shows Seven Day Batch-Refeed Viable Cell Density
  • Figure 1b shows Standard Splitting Viable Cell Density
  • Figure 1c shows Viable Cell Density
  • Figure 2a shows Growth Rate of Monoclonal Antibody Cell Line
  • Figure 2b shows Accumulated Integral Viable Cell Density During Fed-Batch
  • Figure 3 shows Cell Densities of Cell Cultures Adapted to Lack of Insulin
  • Figure 4 shows Viability of Cell Cultures Adapted to Lack of Insulin
  • Figure 5 shows Accumulated IVCD of Cell Cultures Adapted to Lack of Insulin and Control Cell Cultures.
  • Figure 6 shows Cell Densities of Cell Cultures Adapted to Lack of Insulin
  • Figure 7 shows Viability of Cell Cultures Adapted to Lack of Insulin
  • Figure 8 shows Accumulated IVCD of Cell Cultures Adapted to Lack of Insulin and Control Cell Cultures.
  • Figure 9 shows Specific Lactate Consumption Rate of Cell Cultures Adapted to Lack of Insulin and Control Cell Cultures.
  • host cell refers to cells which are capable of being genetically manipulated and/or are capable of growth and survival in a cell culture medium.
  • the cells can express a large quantity of an endogenous or heterologous protein of interest and can either retain the protein or secrete it into the cell culture medium.
  • Host cells are typically "mammalian cells,” which comprise the nonlimiting examples of vertebrate cells, including include baby hamster kidney (BHK), Chinese hamster ovary (CHO), human kidney (293), normal fetal rhesus diploid (FRhL-2), and murine myeloma (e.g., SP2/0 and NSO) cells.
  • BHK baby hamster kidney
  • CHO Chinese hamster ovary
  • human kidney 293
  • normal fetal rhesus diploid fetal rhesus diploid
  • murine myeloma e.g., SP2/0 and NSO
  • cell culture medium refers to cells in a solution containing nutrients to support cell survival under conditions in which the cells can grow and/or produce a desired protein.
  • the phrase "inoculation medium” or “inoculum medium” refers to a solution or substance containing nutrients in which a culture of cells is initiated.
  • a cell culture is supplemented at one or more times with a "feed medium”, with which the cells are fed after initiation of the culture.
  • a "Feed medium” contains similar nutrients as the inoculation medium
  • a feed medium contains one or more components not present in an inoculation medium.
  • a feed medium lacks one or more components present in an inoculation medium.
  • these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and/or trace elements required by a cell for growth and survival.
  • cell culture characteristic refers to an observable and/or measurable characteristic of a cell culture.
  • Methods and compositions of the present invention are advantageously used to improve one or more cell culture characteristics.
  • improvement of a cell culture characteristic comprises increasing the magnitude of a cell culture characteristic.
  • improvement of a cell culture characteristic comprises decreasing the magnitude of a cell culture characteristic.
  • a cell culture characteristic may be improved growth, increased viability, increased integrated viable cell density, increased titer, and/or increased cell specific productivity.
  • One of ordinary skill in the art will be aware of other cell culture characteristics that may be improved using methods and compositions of the present invention.
  • growth medium or “standard growth medium” refers to a medium that contains nutrients and supplements that allows cells or a cell line to divide and grow.
  • production medium refers to an enriched growth medium that permits high levels of a protein of interest to be expressed.
  • a production medium generally, contains higher levels of nutrients, vitamins, trace elements, and/or other medium components when compared
  • an adaptation medium refers to a medium that subjects the cells to protein production conditions that exist in bioreactors (e.g., late- stage production bioreactors), prepares the cells for protein production conditions, and/or minimizes the potentially deleterious effects of transitioning the cells from growth conditions to protein production conditions.
  • an adaptation medium includes the presence of one or more secondary metabolites, e.g. those generated from metabolic processes including but not limited to lactate and/or ammonia.
  • an adaptation medium includes one or more inhibitors including, but not limited to, lactate, ammonia, alanine, glutamine, and/or acetolactate.
  • an adaptation medium comprises a production medium.
  • an adaptation medium mimics one or more characteristics of a production medium.
  • adapting cells in an adaptation medium results in cells that exhibit decreased or less severe deleterious effects when such adapted cells are switched from a growth or adaptation medium to a production medium.
  • Such adaptation media can be production matched, for example, by the supplementation of production media with such metabolites and/or inhibitors and/or by the accumulation of such metabolites and/or inhibitors in an extended duration cell culture.
  • defined medium refers to a medium in which the composition of the medium is both known and controlled.
  • cell line refers to, generally, primary host cells that have been transfected with exogenous DNA, e.g. DNA coding for the desired protein of interest.
  • cells derived from the genetically modified cells form the cell line
  • a cell line comprises primary host cells that have been transfected with exogenous DNA and express an heterologous protein of interest.
  • a cell line comprises primary host cells that have not been transfected with exogenous DNA and express an endogenous protein of interest.
  • Growth phase conditions may include a temperature at about 35°C to 42°C, generally about 37°C.
  • the length of the growth phase and the culture conditions in the growth phase can vary but are generally known to a person of ordinary skill in the cell culture art.
  • cells are grown in a "growth medium” or "standard growth medium”.
  • a cell culture medium in a growth phase is supplemented with a feed medium.
  • the "transition phase” occurs during the period when the cell culture medium is being shifted from conditions consistent with the growth phase to conditions consistent with the production phase. During the transition phase, factors like temperature, among others, are often changed. In certain embodiments, a cell culture medium in a transition phase is supplemented with a feed medium. Methods of the present invention are useful in minimizing the potentially deleterious effect of switching a cell culture from growth phase to production phase conditions.
  • the "production phase” occurs after both the growth phase and the transition phase. The exponential growth of the cells has ended and protein production is the
  • cells are grown in a production medium.
  • a production medium can be supplemented to initiate production.
  • a cell culture medium in a production phase is supplemented with a feed medium.
  • the temperature of the cell culture medium during the production phase is lower, generally, than during the growth phase. As is known in the art, in many instances such a decreased temperature facilitates protein production. The production phase continues until a desired endpoint is achieved.
  • a population of cells growing in a cell culture medium may be passaged or subcultured by removing a subpopulation of cells from the cell culture medium and diluting that subpopulation to a lower viable cell density.
  • a subpopulation of cells may be diluted in a similar volume with fresh medium.
  • the subpopulation of cells is diluted with a cell culture medium that is similar or identical to the cell culture medium in which the original population of cells was growing.
  • the subpopulation may be diluted with the same or similar production medium.
  • the subpopulation of cells is diluted with a cell culture medium that differs from the cell culture medium in which the original population of cells was growing.
  • a subpopulation of cells may be diluted with a production or adaptation medium.
  • the original population of cells has stopped growing (e.g., increasing in cell number) prior to passaging or subculturing a subpopulation.
  • a population is passaged two or more times during the adaptation
  • a population may be passaged, 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.
  • Standard practice maintains passaging or "splitting" a cell culture medium every three or four days using a standard growth medium.
  • cells are adapted to protein production conditions by growing a population of cells for a longer period of time before passaging. For example, cells may be grown 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 days or more before being passaged.
  • viable cell density refers to the total number of cells that are surviving in the cell culture medium in a certain volume.
  • cell viability refers to number of cells that are alive compared to the total number of cells, both dead and alive, expressed as a percentage.
  • Integrated Viable Cell Density The terms “integrated viable cell density” or “IVCD” as used herein refer to the average density of viable cells over the course of the culture multiplied by the amount of time the culture has run. When the amount of protein produced is proportional to the number of viable cells present over the course of the culture, integrated viable cell density is a useful tool for estimating the amount of protein produced over the course of the culture.
  • the term "antibody” includes a protein comprising at least one, and typically two, VH domains or portions thereof, and/or at least one, and typically two, VL domains or portions thereof.
  • the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds.
  • the antibodies, or a portion thereof can be obtained from any origin, including, but not limited to, rodent, primate (e.g., human and non-human primate), camelid, as well as
  • 4140522vl 14 recombinantly produced, e.g., chimeric, humanized, and/or in vitro generated, as described in more detail herein.
  • binding fragments encompassed within the term "antigen-binding fragment" of an antibody include, but are not limited to, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VH domain; (vi) a camelid or camelized heavy chain variable domain (VHH); (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.
  • a Fab fragment a monovalent fragment consisting
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-26; Huston et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:5879-83).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. These fragments may be obtained using conventional techniques known to those skilled in the art, and the fragments are evaluated for function in the same manner as are intact antibodies.
  • the "antigen-binding fragment” can, optionally, further include a moiety that enhances one or more of, e.g., stability, effector cell function or complement fixation.
  • the antigen binding fragment can further include a pegylated moiety, albumin, or a heavy and/or a light chain constant region.
  • bispecific antibodies are understood to have each of its binding sites identical.
  • a “bispecific” 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 1 fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).
  • a cell line is screened for one or more cell culture characteristics that are advantageous in a protein production process including, but not limited to improved growth, increased viability, increased integrated viable cell density, increased titer, and/or increased cell specific productivity.
  • in a cell line for is screened for one or more cell culture characteristics that are advantageous in a fed-batch bioreactor process, e.g., strong growth and/or viability.
  • bioreactor refers to a vessel in which a cell culture medium can be contained and internal conditions of which can be controlled during the culturing period, e.g., pH and temperature.
  • a “production bioreactor” refers to a bioreactor that is utilized during a protein production process.
  • a production bioreactor may comprise a large commercial-scale vessel from which a large amount of protein may be produced, although a production bioreactor is not limited to such large commercial-scale vessels.
  • the volume of a bioreactor is at
  • 4140522V 1 16 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 in between.
  • bioreactors that may be used include, but are not limited to, a stirred tank bioreactor, fluidized bed reactor, hollow fiber bioreactor, or roller bottle.
  • secondary side-products and “secondary metabolites” refer to small molecules typically generated as a result of cellular metabolic activity. As is understood by those of ordinary skill in the art, such secondary side-products are often detrimental to cell growth and/or viability. Thus, their minimization or elimination from a cell culture is desirable.
  • Non-limiting examples of secondary side-products include lactate and ammonium ions.
  • cells are adapted to protein production conditions by growing the cell in the presence of such secondary side- products. In certain embodiments, cells adapted to protein production conditions exhibit the ability to take up such secondary side-products, such that the levels of secondary side-products decrease over time.
  • metabolic side-products that may be used to adapt cells in accordance with the present invention.
  • a “fed batch culture” refers to a method of culturing cells in which cells are first inoculated in a bioreactor with an inoculum medium. The cell culture medium is then supplemented at one or more points throughout the production run with a feed medium containing nutritional components and/or other supplements.
  • a “batch culture” refers to a method of culturing cells in which cells are inoculated in a bioreactor with all the necessary nutrients and supplements for the entirety of the production run. No nutrients, media, etc. are added to the cell culture medium after the cell culture is initiated.
  • a "perfusion culture” refers to a method of culturing cells that is different from a batch or fed-batch culture method, in which the culture is not terminated, or is not necessarily terminated, prior to isolating and/or purifying an 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 harvested.
  • the composition of the added nutrients may be changed during the course of the cell culture, depending on the needs of the cells, the requirements for optimal protein production, and/or any of a variety of other factors known to those of ordinary skill in the art.
  • batch-refeed refers to a mode of operating a bioreactor or a method of passaging cells.
  • a batch-refeed process comprises passaging cells every 7 or 8 days compared to other modes in bioreactors where cells are not passaged or passaged less frequently.
  • cells are generally passaged sooner, e.g. every 3 or 4 days, compared to batch-refeed.
  • batch-refeed methods are used to adapt a cell culture such that it is able to achieve an improved cell culture characteristic including, but not limited to, an improved growth rate, increased viability, increased integrated viable cell density, increased titer, and/or increased cell specific productivity
  • a batch-refeed process utilizes a more enriched culture and/or a medium that contains secondary metabolites and/or other inhibitors in order to adapt cells to protein production conditions.
  • secondary metabolites refers to byproducts of metabolic processes of cell functions.
  • expression refers to the transcription and the translation that occurs within a host cell.
  • the level of expression relates, generally, to the amount of protein being produced by the host cell.
  • protein or “protein product” refers to one or more chains of amino acids.
  • protein is synonymous with “polypeptide” and, as is generally understood in the art, refers to at least one chain of amino acids liked via sequential peptide bonds.
  • a "protein of interest” is a protein encoded by an exogenous nucleic acid molecule that has been transformed into a host cell.
  • the nucleic acid sequence of the exogenous DNA determines the sequence of amino acids.
  • a "protein of interest” is a protein encoded by a nucleic acid molecule that is endogenous to the host cell.
  • expression of such an endogenous protein of interest is altered by transfecting a host cell with an exogenous nucleic acid molecule that may, for example, contain one or more regulatory sequences and/or encode a protein that enhances expression of the protein of interest.
  • Methods and compositions of the present invention may be used to produce any protein of interest, including, but not limited to proteins having pharmaceutical, diagnostic, agricultural, and/or any of a variety of other properties that are useful in commercial, experimental and/or other applications.
  • a protein of interest can be a protein therapeutic.
  • a protein therapeutic is a protein that has a biological effect on a region in the body on which it acts or on a region of the body on which it remotely acts via intermediates. Examples of protein therapeutics are discussed in more detail below.
  • proteins produced using methods and/or compositions of the present invention may be processed and/or modified.
  • a protein to be produced in accordance with the present invention may be glycosylated.
  • the term "titer” as used herein refers to the total amount of recombinantly expressed protein produced by a cell culture in a given amount of medium volume. Titer
  • 4140522vl 19 is typically expressed in units of milligrams or micrograms of protein per milliliter of medium.
  • mammalian cells e.g. untransfected mammalian cells, including, e.g., Chinese hamster ovary cells
  • Such methods are also applicable to both untransfected mammalian cells and to transfected mammalian cells (e.g., mammalian cells transfected with a cDNA or genomic construct causing the expression, as from an expression vector, of a desired recombinant protein).
  • the present invention may be used to adapt cells for the advantageous production of any therapeutic protein, such as, for example, pharmaceutically or commercially relevant enzymes, receptors, antibodies (e.g., monoclonal and/or polyclonal antibodies), Fc fusion proteins, cytokines, hormones, regulatory factors, growth factors, coagulation/clotting factors, antigen binding agents, etc.
  • therapeutic protein such as, for example, pharmaceutically or commercially relevant enzymes, receptors, antibodies (e.g., monoclonal and/or polyclonal antibodies), Fc fusion proteins, cytokines, hormones, regulatory factors, growth factors, coagulation/clotting factors, antigen binding agents, etc.
  • therapeutic protein such as, for example, pharmaceutically or commercially relevant enzymes, receptors, antibodies (e.g., monoclonal and/or polyclonal antibodies), Fc fusion proteins, cytokines, hormones, regulatory factors, growth factors, coagulation/clotting factors, antigen binding agents, etc.
  • Methods of the present invention for adapting untransfected mammalian cells have the potential for identifying cell lines that yield superior productivities and/or exhibit superior protein production characteristics. Furthermore, methods of the present
  • 4140522V 1 20 invention may help in the identification of suitable candidate cell lines more quickly and with less effort as compared to standard cell line development procedures. For example, standard processes for identifying cell line candidates typically require numerous experiments on different scales and additional testing for robustness.
  • Host cells, prior to being transformed are considered untransfected. Traditionally, when creating a new cell line for development, untransfected host cells were initially transfected, or transformed, with exogenous DNA to express of a protein of interest. Using such prior methods, the host cells were not generally experimented with or altered prior to transfection; experimentation and research began on cell line development after the host cells were genetically manipulated to produce a protein product.
  • untransfected mammalian cells can be adapted to protein production conditions with improved performance in batch, fed-batch, and/or perfusion bioreactor processes according to one or more methods of the present invention.
  • such adapted cells are capable of maintaining an improved phenotype, for example exhibiting stronger growth, increased viability, increased integrated viable cell density, increased titer, increased cell specific productivity and/or higher cell densities through such development.
  • Certain embodiments of inventive methods described herein may be employed to adapt untransfected mammalian cells to a batch, fed-batch, and/or perfusion protein production process having a superior performance.
  • any of a variety of commercially available media such as, for example, Minimal Essential Medium (MEM, Sigma), Ham's 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, trace elements,
  • cells may be adapted by culturing transfected or untransfected mammalian cells in a production medium similar or identical to a production medium.
  • cells are adapted by growing the cells under conditions similar or identical to conditions typically encountered under production conditions in a bioreactor (e.g., in a medium consistent with the medium typically found in a bioreactor) operated in batch mode, fed-batch mode, and/or perfusion mode.
  • a bioreactor e.g., in a medium consistent with the medium typically found in a bioreactor operated in batch mode, fed-batch mode, and/or perfusion mode.
  • cells to be adapted are untransfected.
  • cells to be adapted have been transfected with an exogenous nucleic acid molecule, for example a nucleic acid molecule that expresses a protein of interest.
  • one or more cell culture characteristics is improved during a protein production phase by adapting cells to protein production conditions prior to transfection with an exogenous nucleic acid molecule.
  • such an improved cell culture characteristic includes, without limitation, improved growth, improved the overall cell viability, increased integrated viable cell density, increased titer, and/or increased cell specific 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.
  • methods of the present invention are useful for generating a host cells line that is adapted to protein production conditions.
  • Such an adapted host cell line is capable of being transfected with any of a variety of proteins of interest.
  • such an adapted, transfected cell line is placed directly into protein production conditions.
  • such an adapted, transfected cell line is grown to a desired cell density and/or a desired cell number during a growth phase, after which the cell line is transitioned into a protein production phase.
  • such an adapted, transfected cell line exhibits fewer and less severe deleterious effects during the transition phase compared to an non-adapted, transfected cell line.
  • adapted host cells are transfected with an exogenous nucleic acid molecule.
  • a nucleic acid molecule introduced into the cell encodes the protein desired to be expressed according to the present invention.
  • a nucleic acid molecule contains a regulatory sequence or encodes a gene product that induces or enhances the expression of the desired protein by the cell.
  • a gene product may be a transcription factor that increases expression of the protein of interest.
  • a nucleic acid that directs expression of a protein is stably introduced into the host cell. In certain embodiments, a nucleic acid that directs expression of a protein is transiently introduced into the host cell.
  • a nucleic acid that directs expression of a protein is transiently introduced into the host cell.
  • a gene encoding a protein of interest may optionally be linked to one or more regulatory genetic control elements.
  • a genetic control element directs constitutive expression of the protein.
  • a genetic control element that provides inducible expression of a gene encoding the protein of interest can be used.
  • Use of an inducible genetic control element e.g., an inducible promoter
  • potentially useful inducible genetic control elements for use in eukaryotic cells include hormone- regulated elements (see e.g., Mader, S. and White, J. H., Proc. Natl. Acad. Sci.
  • Any protein that is expressible in a host cell may be produced in accordance with methods and compositions of the present invention.
  • the protein may be expressed from a gene that is endogenous to the host cell, or from 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 was engineered or selected by the hand of man.
  • a protein to be produced may be assembled from protein fragments that individually occur in nature. Additionally or alternatively, the engineered protein may include one or more fragments that are not naturally occurring.
  • host cells susceptible to cell culture, and to expression of proteins, may be utilized in accordance with the present invention.
  • host cells are mammalian cells, such as, for example, Chinese hamster ovary (CHO) cells.
  • CHO Chinese hamster ovary
  • non-limiting examples of mammalian cells include BALB/c mouse myeloma line (NSO/I, ECACC No: 85110503); human retinoblasts (PER.C6 (CmCeII, Leiden, The Netherlands)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 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.
  • BALB/c mouse myeloma line NSO/I, ECACC No: 85110503
  • human retinoblasts PER.C6 (CmCeII, Leiden, The Netherlands)
  • monkey kidney CV1 line transformed by SV40 COS-7, ATCC CRL 1651
  • mice Sertoli cells TM4, Mather, 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 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, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.
  • cells are adapted by culturing cells in growth medium supplemented with inhibitors.
  • inhibitors include inhibitors that are typically found when cells are grown under protein production conditions.
  • Non-limiting examples of such inhibitors include lactate, ammonia, alanine, glutamine, and/or acetolactate.
  • cells are adapted by culturing cells in an adaptation medium that lacks or comprises a reduced concentration of one or more components traditionally found in production media.
  • traditional production media typically contain insulin at a concentration of
  • cells are adapted by culturing cells in an adaptation medium that lacks insulin.
  • cells are adapted by culturing cells in an adaptation medium that contains insulin at a concentration lower than an insulin concentration traditionally found in production media.
  • culturing e.g., continuous culturing
  • cells are adapted by culturing (e.g., continuous culturing) of untransfected mammalian cells in growth medium supplemented with one or more inhibitors.
  • Non-limiting examples of such inhibitors include secondary side- products of primary carbon sources.
  • some secondary side-products include, but are not limited to, lactate, alanine, glutamine, acetolactate, and/or ammonia.
  • the concentrations of such secondary side-products present in and/or added to an adaptation medium mimic those typically encountered in bioreactor conditions, such as, for example, about 2.0 to about 10.0 g/L of lactate and/or about 0.1 to about 0.5 g/L of ammonia.
  • cells are adapted under conditions in which the concentration of lactate in the adaptation medium is about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 g/L or higher.
  • cells are adapted under conditions in which the concentration of ammonia 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 higher.
  • individual untransfected mammalian cells and/or subpopulations of untransfected mammalian cells that have been adapted to protein production conditions according to one or more methods of the present invention may exhibit phenotypes consistent with the uptake of such secondary side-products, such
  • such adapted untransfected mammalian cells retain the ability to take up secondary side-products after they are transfected with an exogenous nucleic acid molecule to produce a protein of interest.
  • such adapted, transfected mammalian cells generally perform better in a subsequent protein production process (e.g. a fed-batch bioreactor process) relative to untransfected mammalian cells that do not uptake the side-products and/or that have not been adapted to uptake such secondary side-products.
  • a subsequent protein production process e.g. a fed-batch bioreactor process
  • such adapted, transfected mammalian cells perform better in a subsequent batch, fed-batch and/or perfusion protein production processes.
  • a production reactor e.g., a fed-batch production reactor
  • secondary side-products may often accumulate to levels that are generally inhibitory to further cell growth and/or to viability.
  • subpopulations of untransfected mammalian cells that have been adapted according to methods of the present invention grow in a supplemented medium can grow well under the inhibitory and stressful conditions typically encountered in a production bioreactor.
  • the temperature of a cell culture is decreased the cell culture is switched from growth conditions to protein production conditions.
  • cells are adapted by culturing the cells at a temperature conducive to the production of a protein product.
  • cells are adapted by culturing them at a temperature of about 31 0 C, although methods of the present invention are not limited to such a temperature.
  • cells may be adapted by culturing them 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.
  • temperature(s) suitable for protein production which temperature may depend, at least in part, on the cell line, the protein
  • cells are adapted by continuous culturing of untransfected mammalian cells for a longer duration in a "batch-refeed" mode.
  • untransfected cells are adapted by such "batch-refeed” adaptation methods.
  • transfected cells are adapted by such "batch-refeed” adaptation methods.
  • Typical cell culture operations may employ a cell culture management regimen of splitting, passaging, or sub-culturing a cell culture every three or four days.
  • untransfected mammalian cells are cultured for longer durations, such as seven or eight days, before being sub-cultured.
  • mammalian cells are cultured for 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 days or more before being sub-cultured.
  • Such longer duration cultures have the dual effect of adapting cells for protein production conditions by increasing cell density and/or by accumulating higher levels of secondary metabolites in the conditioned medium to which the cells can adapt.
  • untransfected mammalian cells may be continuously cultured under "production-matched” conditions.
  • untransfected cells may be cultured with iterative cycles of cell culture under production-matched conditions followed by passaging subpopulations of the cells after 3 or 4 days under "recovery" or standard growth conditions with the option of using a growth medium.
  • a population is passaged multiple times during the adaptation process. For example, a population may be passaged, 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.
  • cells are adapted to protein production conditions by growing a population of cells for a longer period of time before passaging. For example, cells may
  • 4140522vl 28 be grown 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 days or more before being passaged. Such passaged subpopulations may be screened for one or more desirable characteristics. In certain embodiments, after several cycles of iterative or continuous adaptation, subpopulations of cells will emerge that exhibit superior characteristics including, for example, improved growth and/or viability relative to the unadapted starting population, in the production-matched conditions. In addition, in certain embodiments, certain subpopulations will begin to take up side products, e.g. lactate, such that the level of that side product decreases over time, enhancing the performance of that cell line in a production bioreactor.
  • side products e.g. lactate
  • one or more screened subpopulations of cells that exhibit one or more desirable characteristics are then transformed such that they produce a protein of interest, after which the transfected subpopulations are placed into protein production conditions (e.g., conditions consistent with those to which the cells have been adapted).
  • cells are grown in accordance with any of the cell culture methods described in United States Patent Application Serial Nos. 11/213,308, 11/213,317 and 11/213,633 each of which was filed August 25, 2005, and each of which is herein incorporated by reference in its entirety.
  • the cells may be grown in a culture medium in which the cumulative amino acid concentration is greater than about 70 mM.
  • the cells may be grown in a culture medium in which the molar cumulative glutamine to cumulative asparagine ratio is less than about 2.
  • the cells may be grown in a culture medium in which the molar cumulative glutamine to cumulative total amino acid ratio is less than about 0.2.
  • the cells may be grown in a culture medium in which the molar cumulative inorganic ion to cumulative total amino acid ratio is between about 0.4 to 1.
  • the cells may be grown in
  • 4140522vl 29 a culture medium in which the combined cumulative glutamine and cumulative asparagine concentration is between about 16 and 36 mM.
  • the cells may be grown in a culture medium that contains two, three, four or all five of the preceding medium conditions. Use of such media allows high levels of protein production and lessens accumulation of certain undesirable factors such as ammonium and/or lactate.
  • the cells are grown under one or more of the conditions described in United States Provisional Patent Application Serial No. 60/830,658, filed July 13, 2006 and incorporated herein by reference in its entirety.
  • cells are grown in a culture medium that contains manganese at a concentration between approximately 10 and 600 nM.
  • cells are grown in a culture medium that contains manganese at a concentration between approximately 20 and 100 nM.
  • cells are grown in a culture medium that contains manganese at a concentration of approximately 40 nM.
  • Use of such media in growing glycoproteins results in production of a glycoprotein with an improved glycosylation pattern (e.g.
  • 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, components and/or supplements may vary depending on 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. The present disclosure is further illustrated by the following, non-limiting examples. Any modifications that might become necessary in the course of the adaptation of
  • 4140522vl 30 mammalian cells to cell culture medium for production of different proteins are well within the art of cell culture.
  • Untransfected CHOK1 cells were cultured either in standard 3 day/4 day batch-refeed conditions in growth medium, components listed in Table 1 below, or cultured in a 7-day batch-refeed mode in enriched production medium, components listed in Table 2 below, for multiple cycles.
  • the cells were cultured at 37 0 C in a working volume from 10 to about 30ml.
  • the cell numbers from the experiment are shown in Figures 1a and 1b.
  • the cells were passaged for 9 days, rather than 7, and the subsequent passaged did not grow well. After the poor 7-day passage, however, the cells were able to recover.
  • Cells were then evaluated in a fed-batch production assay, where the working volume was between 10 and 30ml. The cells were cultured at 37°C for four days and then the temperature was shifted to 31 0 C until day 12, when the assay completed. The cell density and viability were compared and the results are illustrated in Figure 1c. Cells cultured in 7-day batch-refeed mode exhibited higher cell densities than cells cultured in standard 3-day/4 day batch refeed mode. Similarly, two different groups of cells that were adapted for growth in production medium, components listed in Table 3 below, achieved higher cell densities than the unadapted control starting population. TABLE 3
  • a cell line expressing the heavy and light chain genes of a monoclonal antibody was cultured under standard 3 day/4 day batch refeed conditions, using either standard growth medium, components listed in Table 1 , or two versions of production medium, "A" and "B".
  • Production medium A components listed in Table 2
  • production medium B components listed in Table 4 below
  • the effects of methotrexate (MTX) were also tested in production medium "B", 1.5 ⁇ M of
  • the cell lines were evaluated in a fed-batch production assay, in which cells were evaluated in production medium B, illustrated in Figure 2b.
  • the cells were cultured at 37°C for four days and then the temperature was shifted to 31 0 C until day 12, when the assay was complete.
  • the results indicate that cell lines that over express recombinant protein may also be adapted under production-matched conditions to achieve superior growth characteristics in a fed-batch production assay.
  • Insulin directly impacts the metabolism of glucose by mammalian cells.
  • the rapid consumption of glucose in cell culture is frequently coupled with the excretion of lactic acid as a metabolic waste product. Lactic acid can inhibit cell growth and have negative effects on cell viability. Insulin is also reported to be growth factor for mammalian cells.
  • CHOK1 control cells thawed into insulin-free media showed a reduction in growth rate suggesting that the K1 -insulin adaptation indeed altered the cells, allowing them to grow independent of exogenous insulin.
  • Continuing investigations include combining the insulin-free phenotype with the 7-day passage adaptation phenotype.
  • the first fed-batch production assay was set up in a non-pH adjusted format.
  • the experiment included non-adapted CHOK1 cells in standard production media (20 mg/mL nucellin) as a positive control, and non-adapted CHOK1 cells cultured in insulin- free production media as a negative control.
  • the insulin-free adapted CHOK1 cells included non-adapted CHOK1 cells in standard production media (20 mg/mL nucellin) as a positive control, and non-adapted CHOK1 cells cultured in insulin- free production media as a negative control.
  • the insulin-free adapted CHOK1 cells were then evaluated in a second fed batch experiment, under conditions with pH control.
  • the non-adapted CHOK1 cells and insulin-free adapted CHOK1 cells were set up in three separate conditions.
  • the first condition contained insulin in both the base media and the feed (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).
  • the insulin-free adapted CHOK1 cells demonstrate better growth, viability and IVCD when cultured in insulin-free media as compared to the non-adapted CHOK1 cells (see Figures 6-9).
  • the CHOK1- insulin adapted cell lines seem to produce less lactate and almost completely consume whatever lactate they do produce (see Figure 9). This elimination of a detrimental byproduct leads to a healthier cell culture and is seen as a very promising phenotype.
  • This promising phenotype provides better growth conditions and allows the cells to reach higher densities then the associated control. It should be noted that this phenotype is directly related to the elimination of insulin from the media, as the CHOK1-
  • 4140522vl 40 insulin adapted cell lines has similar lactate production rates to the control sample when cultured in media containing insulin.
  • Example 3 demonstrates that adaptation of CHOK1 cells to an insulin-free conditions leads to desirable phenotypes when cells are cultured in industrially relevant production modes.
  • the improved metabolic phenotypes lead to increased cell growth and viability, which are expected to have a significant positive impact on the volumetric productivity of a recombinant CHO cell culture.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne des procédés d'adaptation de cellules, par exemple, de cellules de mammifère, à un processus de culture de cellules. Lorsque les cellules adaptées sont génétiquement modifiées et utilisées pour la production de protéine, elles présentent des caractéristiques bénéfiques, telles que la capacité à atteindre des densités de cellules plus élevées et/ou obtenir un rendement total plus élevé de la protéine produite.
PCT/US2006/042815 2005-11-02 2006-11-02 Procedes pour adapter des cellules de mammifere WO2007056062A2 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP06827375A EP1943333A2 (fr) 2005-11-02 2006-11-02 Procedes pour adapter des cellules de mammifere
AU2006312014A AU2006312014A1 (en) 2005-11-02 2006-11-02 Methods for adapting mammalian cells
JP2008539028A JP2009514532A (ja) 2005-11-02 2006-11-02 哺乳動物細胞を適応させるための方法
CA002628340A CA2628340A1 (fr) 2005-11-02 2006-11-02 Procedes pour adapter des cellules de mammifere
BRPI0618178-3A BRPI0618178A2 (pt) 2005-11-02 2006-11-02 métodos para adaptação de células de mamìferos

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US73281805P 2005-11-02 2005-11-02
US60/732,818 2005-11-02

Publications (2)

Publication Number Publication Date
WO2007056062A2 true WO2007056062A2 (fr) 2007-05-18
WO2007056062A3 WO2007056062A3 (fr) 2007-08-16

Family

ID=37831644

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2006/042815 WO2007056062A2 (fr) 2005-11-02 2006-11-02 Procedes pour adapter des cellules de mammifere

Country Status (7)

Country Link
EP (1) EP1943333A2 (fr)
JP (1) JP2009514532A (fr)
CN (1) CN101300342A (fr)
AU (1) AU2006312014A1 (fr)
BR (1) BRPI0618178A2 (fr)
CA (1) CA2628340A1 (fr)
WO (1) WO2007056062A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100120093A1 (en) * 2008-11-12 2010-05-13 Baxter International Inc. Method of Producing Serum-Free Insulin-Free Factor VII
EP3255141A1 (fr) * 2006-07-13 2017-12-13 Wyeth LLC Production de anticorps avec une gycosylation amelioree

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK3055409T3 (en) * 2013-10-11 2018-07-30 Regeneron Pharma METABOLIC OPTIMIZED CELL CULTURE
US11390663B2 (en) 2013-10-11 2022-07-19 Regeneron Pharmaceuticals, Inc. Metabolically optimized cell culture
JP7317466B2 (ja) * 2017-12-12 2023-07-31 株式会社日立製作所 細胞株及び培養条件のスクリーニング方法、及びその装置

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020182679A1 (en) * 1997-06-20 2002-12-05 Baxter Healthcare Corporation Recombinant cell clones having increased stability and methods of making and using the same

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL87737A (en) * 1987-09-11 1993-08-18 Genentech Inc Method for culturing polypeptide factor dependent vertebrate recombinant cells
JPH03180175A (ja) * 1989-12-07 1991-08-06 Snow Brand Milk Prod Co Ltd 無血清培地
EP2221361A3 (fr) * 1996-08-30 2011-02-09 Life Technologies Corporation Méthode de production d'un polypeptide in vitro dans une cellule de mammifère dans un milieu de culture sans sérum and sans protéines
EP1364002A2 (fr) * 2000-08-23 2003-11-26 Pfizer Products Inc. Procede de preparation de facteur d'inhibition de polynucleaires neutrophiles
US6544072B2 (en) * 2001-06-12 2003-04-08 Berg Technologies Electrical connector with metallized polymeric housing
JPWO2005035740A1 (ja) * 2003-10-09 2006-12-21 協和醗酵工業株式会社 無血清馴化したゲノム改変細胞

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020182679A1 (en) * 1997-06-20 2002-12-05 Baxter Healthcare Corporation Recombinant cell clones having increased stability and methods of making and using the same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SCHUMPP B ET AL: "GROWTH STUDY OF LACTATE AND AMMONIA DOUBLE-RESISTANT CLONES OF HL-60 CELLS" CYTOTECHNOLOGY, vol. 8, no. 1, 1992, pages 39-44, XP009081042 ISSN: 0920-9069 *
SINACORE M S ET AL: "CHO DUKX cell lineages preadapted to growth in serum - free suspension culture enable rapid development of cell culture processes for the manufacture of recombinant proteins" BIOTECHNOLOGY AND BIOENGINEERING, INTERSCIENCE PUBLISHERS, LONDON, GB, 20 November 1996 (1996-11-20), XP002077942 ISSN: 0006-3592 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3255141A1 (fr) * 2006-07-13 2017-12-13 Wyeth LLC Production de anticorps avec une gycosylation amelioree
EP2495307B1 (fr) 2006-07-13 2018-02-21 Wyeth LLC Production du factor IX de coagulation avec une glycosylation ameliorée
EP3255141B1 (fr) 2006-07-13 2021-12-01 Wyeth LLC Production de anticorps avec une gycosylation amelioree
US20100120093A1 (en) * 2008-11-12 2010-05-13 Baxter International Inc. Method of Producing Serum-Free Insulin-Free Factor VII
EP2356247B1 (fr) 2008-11-12 2015-07-15 Baxter International Inc. Procédé de production de facteur vii sans insuline, sans sérum
EP2356247B2 (fr) † 2008-11-12 2019-05-15 Baxalta Incorporated Procédé de production de facteur vii sans insuline, sans sérum

Also Published As

Publication number Publication date
AU2006312014A1 (en) 2007-05-18
BRPI0618178A2 (pt) 2011-08-23
EP1943333A2 (fr) 2008-07-16
JP2009514532A (ja) 2009-04-09
CN101300342A (zh) 2008-11-05
WO2007056062A3 (fr) 2007-08-16
CA2628340A1 (fr) 2007-05-18

Similar Documents

Publication Publication Date Title
CA2627003C (fr) Procedes de production de proteines utilisant des composes anti-senescence
US9388447B2 (en) Method for culturing mammalian cells to improve recombinant protein production
JP6298295B2 (ja) タンパク質発現のための細胞培養培地及びプロセス、ｐａｍインヒビターを含む前記培地及びプロセス
JP2020054388A (ja) 生物薬剤フェドバッチ生産能力及び生産物品質を改善するための灌流シード培養の使用
KR101302904B1 (ko) ｈＣＭＶ 주요 즉각 조기 유전자의 제 1 인트론 및ｍＣＭＶ 프로모터를 포함한 포유동물 발현 벡터
US20070231895A1 (en) Methods for adapting mammalian cells
CN104884467A (zh) 在遗传修饰的哺乳动物细胞中生产治疗性蛋白质
EP3914696A1 (fr) Procédé de culture de semences pour la production d'aav
EP1943333A2 (fr) Procedes pour adapter des cellules de mammifere
JP2022546230A (ja) 細胞培養方法
US9340592B2 (en) CHO/CERT cell lines
CA3175256A1 (fr) Procede de traitement de milieu avant inoculation
JP2023502916A (ja) 細胞の選択方法
Román et al. Enabling HEK293 cells for antibiotic-free media bioprocessing through CRISPR/Cas9 gene editing
JP6697442B2 (ja) 真核細胞の比生産速度を増加させる方法
AU2016268709B2 (en) Cell culturing method using nucleic acid-containing medium
US20170327558A1 (en) Mammalian cell culture processes for protein production
US20050175599A1 (en) Process for producing substance

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200680041039.1

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2006827375

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2006312014

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: MX/a/2008/005686

Country of ref document: MX

WWE Wipo information: entry into national phase

Ref document number: 1759/KOLNP/2008

Country of ref document: IN

ENP Entry into the national phase in:

Ref document number: 2008539028

Country of ref document: JP

Kind code of ref document: A

Ref document number: 2628340

Country of ref document: CA

NENP Non-entry into the national phase in:

Ref country code: DE

ENP Entry into the national phase in:

Ref document number: 2006312014

Country of ref document: AU

Date of ref document: 20061102

Kind code of ref document: A

ENP Entry into the national phase in:

Ref document number: PI0618178

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20080502