Serum-free cell culture medium

Abstract

To provide an improved serum-free animal cell culture medium that can be used for production of a protein of interest.SOLUTION: Inclusion of ornithine, either with or without putrescine, in serum-free cell culture medium ("OS" medium) increases cell viability and density, decreases cell doubling time, and allows for high titer protein production by these cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to media for the cultivation of cells and for the production of recombinant proteins. The present invention particularly relates to serum-free media for the cultivation of recombinant CHO cells for the production of protein biotherapeutics. [Background technology]

[0002] Cell culture media containing serum or protein hydrolysate components (i.e., peptone and tryptone) have a long history of use in the production of recombinant proteins from cultured cells. These components contain growth factors and a wide variety of other uncharacterized elements that are beneficial to cell growth and culture. However, they also contain uncharacterized elements that reduce growth or otherwise negatively affect recombinant protein production. These can also be an unwelcome potential source of variability. Despite their drawbacks, the advantages of using serum and hydrolysates outweigh some of the drawbacks, and they have been widely used in many cell culture applications.

[0003] Human biological therapeutics (biopharmaceuticals) are commonly produced in mammalian cell cultures, particularly CHO cell cultures. The presence of uncharacterized or partially characterized components in these cell cultures is highly undesirable for the manufacture of biopharmaceuticals for human use. The use of such uncharacterized or partially characterized components not only introduces production and regulatory conflicts but also increases the likelihood of viral or fungal infection of the production culture.

[0004] Reducing lot-to-lot variability in drug product yield and composition is another important factor when selecting a culture process. Serum, hydrolysates, and other undefined elements introduce variability in the yield, composition, and quality of biopharmaceutical production lots. The quality and purity of medium components can also affect yield, since drug potency often depends in part on maintaining a specific balance of nutrients. If the relative amounts of nutrients vary between medium lots, drug yields may vary, and this variation may be unacceptable or uneconomical.

[0005] The use of serum-containing or hydrolysate-based media introduces downstream processing challenges. The concentration of the desired biopharmaceutical in the culture is generally on the order of grams per liter. The presence of serum and hydrolysates in the medium can add more than 10 g / L of uncharacterized peptides and proteins that must be removed in subsequent processing steps. Serum and hydrolysates can also introduce variability in the amount of metals and other trace elements in the medium. Therefore, excluding serum and hydrolysates from the culture medium eliminates these variations and potential interference with drug substance production and processing.

[0006] Among other benefits, the use of serum-free, hydrolysate-free media includes reduced costs, reduced variability between drug lots, and minimized risk of introducing adventitious agents from undefined and unrefined components. Furthermore, if the media is defined and uniform between batch runs, assay runs to test new culture batches against the current media are similarly minimized. Thus, there is a need in the art for media for culturing mammalian cells that are chemically defined and serum- and hydrolysate-free, or serum-free with low, manageable levels of hydrolysates, yet still allow for the growth and maintenance of healthy, robust cells and the production of high-titer biopharmaceutical drug substances. Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors have surprisingly discovered that including ornithine, either with or without putrescine, in serum-free cell culture medium ("OS" medium) increases cell viability and density, reduces cell doubling time, and enables high-titer protein production by these cells. The present inventors have also discovered that OS medium containing low or trace amounts of protein hydrolysates or chemically defined (i.e., protein hydrolysate-free) OS medium provides particularly restored cell viability and density, cell doubling time, and high-titer protein production. [Means for solving the problem]

[0008] In one aspect, the present invention provides a cell culture medium that is serum-free and includes at least 0.09 mM ± 0.014 mM ornithine. In one embodiment, the ornithine is present in the medium at a concentration ranging from 0.09 ± 0.014 mM to 0.9 ± 0.14 mM, e.g., 0.09 ± 0.014 mM, 0.3 ± 0.05 mM, 0.6 ± 0.09 mM, or 0.9 ± 0.14 mM. In some embodiments, the medium also includes at least 0.20 ± 0.03 mM putrescine. In some embodiments, the additional putrescine is at a concentration ranging from 0.20 ± 0.03 mM to 0.714 ± 0.11 mM, e.g., 0.20 ± 0.03 mM, 0.35 ± 0.06 mM, or 0.714 ± 0.11 mM. In some embodiments, the medium contains ≦7.5 g / L of hydrolysate. In some embodiments, the medium does not contain any hydrolysate.

[0009] In one embodiment, the medium comprises a chemically defined basal medium, e.g., a custom-formulated or commercially available basal medium, hi one embodiment, the complete medium is chemically defined, serum-free, and hydrolysate-free.

[0010] In some embodiments, the medium at its useful concentration (i.e., 1×) comprises at least 40±6 mM or at least 70±10.5 mM of a mixture of amino acids or amino acid salts. In one embodiment, the medium comprises at least 40 mM of the mixture of amino acids. In this or another embodiment, the medium comprises at least 70 mM of the mixture of amino acids. In one embodiment, the mixture of amino acids (obviously excluding glutamine, which may be later added to the medium as a point-of-use addition) comprises alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0011] In some embodiments, the medium comprises one or more fatty acids. In a particular embodiment, the medium comprises a mixture of fatty acids (or fatty acid derivatives) and alpha-tocopherol. The fatty acid or fatty acid derivative is selected from the group consisting of linoleic acid, linolenic acid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and octanoic acid.

[0012] In some embodiments, the medium comprises a mixture of nucleosides, hi one embodiment, the medium comprises adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

[0013] In some embodiments, the medium comprises a mixture of salts. The salts include divalent cations, such as calcium and magnesium. In one embodiment, the medium comprises calcium chloride and magnesium sulfate. Other salts may include salts of phosphate.

[0014] In a specific embodiment, the medium (1) comprises ≦7.5 g / L of hydrolysate, (2) is serum-free, (3) comprises 0.09±0.014 mM, 0.3±0.05 mM, 0.6±0.09 mM, or 0.9±0.14 mM ornithine, (4) optionally further comprises 0.20±0.03 mM, 0.35±0.06, or 0.714±0.11 mM putrescine, (5) comprises at least about 40 mM or at least about 70 mM of alanine, arginine, asparagine, asparagi, or guanylate. (5) a mixture of amino acids including phospholipids, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; (6) a mixture of tocopherols and fatty acids; (7) a mixture of nucleosides including adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine; and (8) salts of calcium, magnesium, and phosphate.

[0015] In another aspect, the present invention provides a method of cultivating cells in a cell culture medium, such as any embodiment of the medium described in the previous aspect. In one embodiment, the method employs growing or maintaining a cell(s) in a medium that (1) contains ≦7.5 g / L of hydrolysate or is hydrolysate-free, (2) is serum-free, (3) contains ornithine at a concentration of at least 0.09 mM±0.014 mM, and (4) optionally contains putrescine, such as at least 0.20±0.03 mM.

[0016] In some embodiments, the cell(s) are mammalian cells, avian cells, insect cells, yeast cells, or bacterial cells. In one embodiment, the cells are mammalian cells useful in the production of recombinant proteins, such as CHO cells or derivatives such as CHO-K1. In some embodiments, the cells express a protein of interest, such as a biotherapeutic protein. The biotherapeutic protein can be an antigen-binding protein that may include an Fc domain. In some embodiments, the protein of interest is a receptor-Fc-fusion protein, such as an ScFv molecule, or a trap molecule. Trap molecules include VEGF trap protein and IL-1 Trap protein. In some embodiments, the protein of interest is an antibody, such as a humanized monoclonal antibody, a bispecific antibody, or an antibody fragment.

[0017] Considering the positive effect on cell proliferation of including ornithine or a combination of ornithine and putrescine in serum-free medium, cells cultured according to this method have an average doubling time of 30 hours or less. In one embodiment, this cell doubling time is 24 hours or less. In one embodiment, when compared to cells grown in a medium containing less than 0.09±0.014 mM ornithine (or less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine), cells grown according to this method have an average doubling time that is at least one-third of the doubling time of a comparator control culture.

[0018] Similarly, the inclusion of ornithine alone or a combination of ornithine and putrescine in a serum-free medium allows cultured cells to reach a higher viable cell count density than without ornithine or the combination of ornithine and putrescine. In one serum-free, hydrolysate-free embodiment of OS medium, the cell culture is capable of reaching a viable cell count density that is at least 15% greater than a similar cell culture in a similar cell culture medium containing less than 0.09±0.014 mM ornithine (or less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine). In another serum-free, hydrolysate-free embodiment of OS medium, the cell culture is capable of reaching a viable cell count density that is at least three times greater than a similar cell culture in a similar cell culture medium containing less than 0.09±0.014 mM ornithine (or less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine).

[0019] In another embodiment, the method includes adding one or more point-of-use additives to the cell culture medium. In some embodiments, the point-of-use additives are any one or more of NaHCO3, glutamine, insulin, glucose, CuSO4, ZnSO4, FeCl3, NiSO4, Na4EDTA, and Na3 citrate. In one embodiment, the method employs adding each of the following point-of-use chemicals to the cell culture medium: NaHCO3, glutamine, insulin, glucose, CuSO4, ZnSO4, FeCl3, NiSO4, Na4EDTA, and Na3 citrate. In some embodiments, these point-of-use additives may be included in the medium at the start.

[0020] In a specific embodiment, this aspect relates to a cell culture medium comprising: (1) any of 0.09±0.014 mM, 0.3±0.05 mM, 0.6±0.09 mM, or 0.9±0.14 mM ornithine; (2) optionally further comprising any of 0.20±0.03 mM, 0.35±0.06, or 0.714±0.11 mM putrescine; (3) at least about 40 mM or at least about 70 mM of a mixture of amino acids comprising alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; (4) a mixture of tocopherols and fatty acids; (5) a mixture of adenomycin, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; (6) a mixture of adenomycin, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; (7) a mixture of adenomycin, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalan A method of cultivating cells in a serum-free medium containing a mixture of nucleosides, including cytidine, guanosine, cytidine, uridine, thymidine, and hypoxanthine, and (9) salts of calcium, magnesium, and phosphate, is provided, wherein cells cultured according to this method have an average doubling time of 24 hours or less or an average doubling time that is at least one-third of the doubling time of a comparator control culture; and the cultured cells are capable of reaching a viable cell count density that is at least 15% greater or at least three-fold greater than a similar cell culture in a similar cell culture medium containing less than 0.09±0.015 mM ornithine (or less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine). In another embodiment, the cell culture is capable of reaching a viable cell count density that is at least three times greater than a similar cell culture in a similar cell culture medium containing less than 0.09±0.014 mM ornithine (or less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine). In one embodiment, the medium contains ≦7.5 g / L of hydrolysate; in another embodiment, the medium is hydrolysate-free.

[0021] In another aspect, the present invention provides a method for producing a protein of interest by using the steps of: (1) introducing a nucleic acid sequence encoding the protein of interest into cells; (2) selecting cells that carry the nucleic acid sequence; (3) culturing the selected cells in an embodiment of the serum-free cell culture medium described in the first aspect or according to any embodiment of the method described in the second aspect; and (4) expressing the protein of interest in the cells, wherein the protein of interest is secreted into the medium. In some embodiments, the cells used in the production of the protein are mammalian cells capable of producing biotherapeutics, such as CHO, 293, and BHK cells, or any derivatives thereof. In one embodiment, the cells are CHO cells, such as CHO-K1 cells.

[0022] In some embodiments, the protein of interest is an antigen-binding protein. In some embodiments, the protein of interest is a protein having an Fc domain. In some cases, such as receptor-Fc-fusion proteins, antibodies, and ScFv proteins, the two proteins of interest may overlap. Thus, in some embodiments, the protein of interest is an antibody, such as a human or humanized antibody, an antibody fragment, such as Fab or F(ab')2, a bispecific antibody, a trap molecule, such as VEGF-Trap or IL-1-Trap, an ScFv molecule, a soluble TCR-Fc fusion protein, or the like.

[0023] In one embodiment, the protein of interest can be produced at an average day 7 titer that is at least 7% greater, at least 14% greater, at least 80% greater, at least 2-fold greater, or at least 3-fold greater than the average day 7 titer produced by similar cells in serum-free cell culture medium ("non-OS" medium) containing less than 0.09±0.014 mM ornithine (or less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine).

[0024] In a specific embodiment, the protein of interest is prepared by (1) introducing into CHO cells a nucleic acid sequence encoding the protein of interest, e.g., an antibody or other antigen-binding protein; (2) selecting cells harboring the nucleic acid sequence; and (3) selecting the cells harboring the nucleic acid sequence by: (a) 0.09±0.014, 0.3±0.05, 0.6±0.09, or 0.9±0.14 mM ornithine; (b) optionally further comprising 0.20±0.03, 0.35±0.06, or 0.714±0.11 mM putrescine; (c) at least 40 mM or at least 70 mM of alanine, arginine, asparagine, aspartic acid, cysteine, glycine, guan ... The protein of interest is produced by culturing the selected cells in a serum-free cell culture medium containing (a) an amino acid mixture containing glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; (b) a tocopherol and fatty acid mixture; (c) a nucleoside mixture containing adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine; and (d) calcium, magnesium, and phosphate salts; and (d) expressing the protein of interest in the CHO cells, where the protein of interest is secreted into the medium. In some embodiments, the serum-free cell culture medium may contain ≦7.5 g / L of hydrolysate; or in other embodiments, it may contain no hydrolysate at all. [The present invention 1001] Cell culture medium that is serum-free and contains ≧0.09 mM±0.014 mM ornithine. [The present invention 1002] 1001. The cell culture medium of the present invention, comprising ≧0.20±0.03 mM putrescine. [The present invention 1003] The cell culture medium of any of the present inventions 1001 and 1002, comprising 0.09±0.014 mM to 0.9±0.14 mM ornithine. [The present invention 1004] 10. The cell culture medium of any one of claims 1001 to 1003, comprising ornithine at 0.09±0.014 mM, 0.3±0.05 mM, 0.6±0.09 mM or 0.9±0.14 mM. [The present invention 1005] The cell culture medium of any one of claims 1001 to 1004, comprising 0.20±0.03 mM to 0.714±0.11 mM putrescine. [The present invention 1006] 10. The cell culture medium of any one of claims 1001 to 1004, comprising putrescine at 0.20±0.03 mM, 0.35±0.06 mM, or 0.714±0.11 mM. [The present invention 1007] 1006. The cell culture medium of any one of claims 1001 to 1006, wherein the cell culture medium is hydrolysate-free. [The present invention 1008] 1006. The cell culture medium of any one of claims 1001 to 1006, wherein the cell culture medium is chemically defined. [The present invention 1009] 1009. The cell culture medium of any of claims 1001 to 1008, comprising ≧40±6 mM of a mixture of amino acids or salts thereof. [The present invention 1010] 1009. The cell culture medium of claim 10, wherein said mixture of amino acids consists of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. [The present invention 1011] 10. The cell culture medium of any of claims 1001 to 1010, comprising one or more fatty acids. [The present invention 1012] 1011. The cell culture medium of the present invention, wherein the one or more fatty acids are selected from the group consisting of linoleic acid, linolenic acid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and octanoic acid. [The present invention 1013] 10. The cell culture medium of any of claims 1001 to 1012, comprising a mixture of nucleosides. [The present invention 1014] 1013. The cell culture medium of claim 10, wherein said mixture of nucleosides comprises one or more of adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine. [The present invention 1015] 10. The cell culture medium of any of claims 1001 to 1014, comprising adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine. [The present invention 1016] 10. The cell culture medium of any of claims 1001 to 1015, comprising one or more divalent cations. [The present invention 1017] 1016. The cell culture medium of claim 10, wherein the divalent cation is magnesium, calcium, or both. [The present invention 1018] Ca 2+ and Mg 2+ 10. The cell culture medium of any of claims 1001 to 1017, comprising: [The present invention 1019] A method for culturing cells, comprising: (a) providing a cell culture medium according to any one of claims 1001 to 1018; and (b) growing or maintaining cells in the cell culture medium to form a cell culture. [The present invention 1020] 1019. The method of claim 1019, wherein said cell is selected from the group consisting of a mammalian cell, an avian cell, an insect cell, a bacterial cell, and a yeast cell. [The present invention 1021] The method of claim 1019 or claim 1020, wherein said cell is a CHO cell. [The present invention 1022] 1022. The method of any of claims 1019 to 1021, wherein said cells express a protein of interest. [The present invention 1023] 1023. The method of claim 1022, wherein said protein of interest is an antigen-binding protein. [The present invention 1024] 1024. The method of claim 1022 or 1023, wherein said protein of interest comprises an Fc domain. [The present invention 1025] 1025. The method of any of claims 1022 to 1024, wherein said protein of interest is a receptor-Fc-fusion protein. [The present invention 1026] 1026. The method of claim 1025, wherein said receptor-Fc-fusion protein is a trap protein. [The present invention 1027] 1027. The method of claim 1026, wherein said trap protein is an IL-1 antagonist or a VEGF antagonist. [The present invention 1028] 1024. The method of claim 1022 or 1023, wherein said protein of interest is an antibody or an antibody fragment. [The present invention 1029] 1028. The method of claim 1028, wherein said antibody or said antibody fragment is a recombinant human antibody or a fragment thereof. [The present invention 1030] 1029. The method of any of claims 1019 to 1029, wherein the cells have a mean doubling time of ≦30 hours. [The present invention 1031] 1031. The method of any of claims 1019 to 1030, wherein the cells have a mean doubling time of ≦24 hours. [The present invention 1032] 1032. Any of the methods of claims 1019 to 1031, wherein the cells have an average doubling time that is at least one-third of the average doubling time of cells grown in a cell culture medium containing <0.3±0.045 mM ornithine and <0.2±0.03 mM putrescine. [The present invention 1033] 10. The method of any of claims 1019 to 1032, wherein the cell culture is capable of reaching a viable cell count density that is at least 15% greater than a similar cell culture in a medium containing <0.09±0.014 mM ornithine and <0.2±0.03 mM putrescine. [The present invention 1034] 10. The method of any of claims 1019 to 1033, wherein said cell culture is capable of reaching a viable cell count density that is at least three times greater than a similar cell culture in a similar cell culture medium comprising <0.09±0.014 mM ornithine and <0.2±0.03 mM putrescine. [This invention 1035] 1035. The method of any of claims 1019 to 1034, comprising adding one or more point-of-use additives to said cell culture medium. [The present invention 1036] 1035. The method of claim 1035, wherein the point-of-use additive comprises one or more of NaHCO3, glutamine, insulin, glucose, CuSO4, ZnSO4, FeCl3, NiSO4, Na4EDTA, and Nacitrate. [This invention 1037] 1035 or 1036, wherein each of NaHCO3, glutamine, insulin, glucose, CuSO4, ZnSO4, FeCl3, NiSO4, Na4EDTA, and Nacitrate is added to said medium as a point-of-use additive. [The present invention 1038] 1. A method for producing a protein, the method comprising the steps of: (a) introducing into cells a nucleic acid comprising a sequence encoding a protein of interest; (b) selecting cells that harbor the nucleic acid; (c) culturing the selected cells in a cell culture medium of any of claims 1001 to 1018 or according to any of the methods of claims 1019 to 1037; and (d) expressing the protein of interest in the cells, wherein the protein of interest is secreted into the medium. [This invention 1039] The method of claim 1038, wherein the cell is a CHO cell, a 293 cell or a BHK cell. [The present invention 1040] 1039. The method of any one of claims 1038 to 1039, wherein said protein of interest is an antigen-binding protein. [The present invention 1041] 1040. The method of any of claims 1038 to 1040, wherein said protein of interest comprises an Fc domain. [The present invention 1042] 1042. The method of any of claims 1038 to 1041, wherein said protein of interest is selected from the group consisting of a receptor-Fc-fusion protein (TRAP), a soluble TCR-Fc fusion protein, an antibody, an Fc-fusion protein and an ScFv protein. [This invention 1043] 1042. The method of any of claims 1038 to 1042, wherein the protein of interest is produced at an average day 7 titer that is at least 7% greater than the average day 7 titer produced by similar cells in a cell culture medium containing less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine. [This invention 1044] 1043. The method of any of claims 1038 to 1043, wherein the protein of interest is produced at an average day 7 titer that is at least 14% greater than the average day 7 titer produced by similar cells in a cell culture medium comprising less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine. [This invention 1045] 1045. Any of the methods of claims 1038 to 1044, wherein the protein of interest is produced at an average day 7 titer that is at least 80% greater than the average day 7 titer produced by similar cells in a cell culture medium containing less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine. [The present invention 1046] 1046. The method of any of claims 1038 to 1045, wherein the protein of interest is produced at an average day 7 titer that is at least 2-fold greater than the average day 7 titer produced by similar cells in a cell culture medium containing less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine. [This invention 1047] 1046. The method of any of claims 1038 to 1046, wherein said protein of interest is produced at an average day 7 titer that is at least three times greater than the average day 7 titer produced by similar cells in a cell culture medium comprising less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine. [This invention 1048] 1048. The method of any of claims 1038 to 1047, wherein said protein of interest is a recombinant human antibody. DETAILED DESCRIPTION OF THE INVENTION

[0025] Applicants have surprisingly discovered that the addition of ornithine, or a combination of ornithine and putrescine ("OS medium"), improves viable cell density, cell doubling time, and protein production by cells in cell culture compared to serum-free medium containing little or no ornithine, or little or no combination of ornithine and putrescine ("non-OS medium").

[0026] Before describing the cell cultures and methods of the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0027] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, certain specific methods and materials are described herein. Units, prefixes, and symbols may be denoted in their SI-approved form. Numerical ranges cited herein are in open brackets, meaning that they are inclusive of the numbers defining the range. Unless otherwise specified, the terms "a" or "an" should be construed to mean "at least one of." Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. The methods and techniques described herein are generally performed according to conventional methods known in the art and described in various general and more specific references cited and discussed throughout this specification, unless otherwise specified.See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992); Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990); and Julio E. Celis, Cell Biology: A Laboratory Handbook, 2nd ed., Academic Press, New York, NY (1998); and Dieffenbach and Dveksler, PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1995). All publications mentioned throughout this disclosure are incorporated herein by reference in their entirety.

[0028] definition As used herein, "peptide," "polypeptide," and "protein" are used interchangeably throughout and refer to molecules comprising two or more amino acid residues connected to each other by peptide bonds. Peptides, polypeptides, and proteins may also include modifications such as glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, alkylation, hydroxylation, and ADP-ribosylation. Peptides, polypeptides, and proteins may be of scientific or commercial interest, including protein-based drugs. Peptides, polypeptides, and proteins include, inter alia, antibodies and chimeric or fusion proteins. Peptides, polypeptides, and proteins are produced by recombinant animal cell lines using cell culture methods.

[0029] The term "heterologous polynucleotide sequence," as used herein, refers to a nucleic acid polymer encoding a protein of interest, such as a chimeric protein (such as a trap molecule), antibody, or antibody portion (e.g., VH, VL, CDR3), to be produced as a biopharmaceutical drug substance. The heterologous polynucleotide sequence can be produced by genetic engineering techniques (e.g., a sequence encoding a chimeric protein, or a codon-optimized sequence, an intronless sequence, etc.) and introduced into a cell, where it can exist episomally or be integrated into the cell's genome. The heterologous polynucleotide sequence can be a naturally occurring sequence introduced into an ectopic site within the producing cell's genome. The heterologous polypeptide sequence can be a naturally occurring sequence from another organism, such as a sequence encoding a human ortholog.

[0030] An "antibody" refers to an immunoglobulin molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain has one heavy chain variable region (HCVR or VH) and one heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain has one light chain variable region and one light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody" includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass. The term "antibody" includes antibody molecules prepared, expressed, created, or isolated by recombinant means, e.g., antibodies isolated from host cells transfected to express the antibody. The term antibody also includes bispecific antibodies, including heterotetrameric immunoglobulins capable of binding to more than one different epitope. Bispecific antibodies are generally described in U.S. Patent Application Publication No. 2010 / 0331527, which is incorporated herein by reference.

[0031] The term "antigen-binding portion" of an antibody (or "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a monovalent fragment, a Fab fragment, consisting of the VL, VH, CL, and CH1 domains; (ii) a bivalent fragment, an F(ab')2 fragment, comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment, consisting of the VH and CH1 domains; (iv) an Fv fragment, consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment, consisting of the VH domain (Ward et al. (1989) Nature 241:544-546); (vi) an isolated CDR; and (vii) an scFv, consisting of the two domains, VL and VH, of an Fv fragment connected by a synthetic linker to form a single protein chain, wherein the VL and VH domains pair to form a monovalent molecule. Other forms of single-chain antibodies, such as diabodies, are also encompassed under the term "antibody" (see, e.g., Holliger et al. (1993) PNAS USA 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

[0032] Furthermore, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of streptavidin core regions to generate tetrameric scFv molecules (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to generate bivalent biotinylated scFv molecules (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as by papain or pepsin digestion of whole antibodies. Furthermore, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques generally known in the art (see Sambrook et al., 1989).

[0033] The term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include, for example, amino acid residues in the CDRs, and particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro by random or site-specific mutagenesis or in vivo by somatic mutation). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0034] The term "recombinant human antibody," as used herein, is intended to include all human antibodies prepared, expressed, created, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created, or isolated by any other means, including splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of these recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally occur within the human antibody germline repertoire in vivo.

[0035] An "Fc fusion protein" comprises part or all of two or more proteins, one of which is the Fc portion of an immunoglobulin molecule, that are not otherwise found together in nature. The preparation of fusion proteins containing particular heterologous polypeptides fused to various portions of antibody-derived polypeptides (including Fc domains) is described, for example, by Ashkenazi et al., Proc. Natl. Acad. ScL USA 88:10535 (1991); Byrn et al., Nature 344:677 (1990); and Hollenbaugh et al., "Construction of Immunoglobulin Fusion Proteins," Current Protocols in Immunology, Suppl. 4, pp. 10.19.1-10.19.11 (1992). A "receptor-Fc fusion protein" comprises one or more extracellular domain(s) of a receptor coupled to an Fc portion, and in some embodiments, comprises a hinge region followed by the CH2 and CH3 domains of an immunoglobulin. In some embodiments, the Fc-fusion protein comprises two or more separate receptor chains that bind to one or more ligand(s). For example, the Fc-fusion protein is a trap, such as an IL-1 trap (e.g., rilonacept, which comprises the IL-1RAcP ligand-binding region fused to the IL-1R1 extracellular region fused to the Fc of hIgG1; see U.S. Patent No. 6,927,004), or a VEGF trap (e.g., aflibercept, which comprises the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to the Fc of hIgG1; see U.S. Patent Nos. 7,087,411 and 7,279,159).

[0036] Culture medium The present invention provides serum-free media useful in culturing cells and producing biopharmaceutical drug substances. "Serum-free" refers to cell culture media that do not contain animal serum, such as fetal bovine serum. The serum-free media may contain ≦7.5 g / L of a hydrolysate, such as soy hydrolysate. The present invention also provides chemically defined media that are not only serum-free but also hydrolysate-free. "Hydrolysate-free" refers to cell culture media that do not contain exogenous protein hydrolysates, such as animal or plant protein hydrolysates, such as peptone, tryptone, etc.

[0037] While eliminating serum and reducing or eliminating hydrolysates from cell culture media reduces lot-to-lot variability and enhances downstream processing steps, unfortunately, it also reduces cell growth, viability, and protein expression. Therefore, chemically defined, serum-free, low-to-no hydrolysates media require additional components to improve cell growth and protein production. The cell culture media of the present invention can be supplemented with additional components such as polyamines, or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, etc., depending on the requirements of the cells being cultured or the desired cell culture parameters. Specifically, the cell culture media herein are supplemented with ornithine, putrescine, or both to improve cell growth, cell viability, and recombinant protein production ("OS medium").

[0038] In some embodiments, the OS medium comprises at least about 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 540, 545, 550, 555, 560, 565, 568, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, ornithine at a concentration of 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 620, 625, 630, 635, 640, 645, 650, 700, 750, 800, 850, or 900 μM (expressed in micromoles per liter).

[0039] In some embodiments, the medium comprises ornithine at a concentration of about 85, 90, 95, 100, 105, 110, 113, or 115 μM. In one embodiment, the medium comprises 100 μM±15 μM ornithine. In one embodiment, the medium comprises 15 mg / L±2.25 mg / L ornithine·HCl.

[0040] In some embodiments, the medium comprises ornithine at a concentration of about 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, or 345 μM. In one embodiment, the medium comprises 300 μM ± 45 μM ornithine. In one embodiment, the medium comprises 50 mg / L ± 7.5 mg / L ornithine·HCl.

[0041] In some embodiments, the medium comprises ornithine at a concentration of about 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, or 690 μM. In one embodiment, the medium comprises 600 μM ± 90 μM ornithine. In one embodiment, the medium comprises 100 mg / L ± 15 mg / L ornithine·HCl.

[0042] In some embodiments, the medium comprises 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 91 The medium contains ornithine at a concentration of 0, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, 1,000, 1,005, 1,010, 1,015, 1,020, 1,025, 1,030, or 1,035 μM. In one embodiment, the medium contains 900 μM ± 135 μM ornithine. In one embodiment, the medium contains 150 mg / L ± 22.5 mg / L ornithine·HCl.

[0043] Putrescine can optionally be added to ornithine-supplemented media. Putrescine has been included as a component in some cell culture media formulations at very low concentrations, e.g., WO2005 / 028626, which describes putrescine at 0.02-0.08 mg / L; U.S. Pat. No. 5,426,699 (0.08 mg / L); U.S. Pat. No. Re. 30,985 (0.16 mg / L); U.S. Pat. No. 5,811,299 (0.27 mg / L); U.S. Pat. No. 5,122, 469 (0.5635 mg / L); U.S. Pat. No. 5,063,157 (1 mg / L); WO 2008 / 154014 (about 100 μM to about 1000 μM); U.S. Patent Application Publication No. 2007 / 0212770 (0.5 to 30 mg / L polyamines; 2 mg / L putrescine; 2 mg / L putrescine + 2 mg / L ornithine; 2 mg / L putrescine + 10 mg / L ornithine).

[0044] In some embodiments, the medium comprises a combination of ornithine and putrescine, wherein the putrescine may be at a concentration of at least about 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 260, 365, 370, 375, 380, 385, 390, 395, 400, 405, or 410 μM.

[0045] In some embodiments, the medium comprises putrescine at a concentration of about 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, or 230 μM. In one embodiment, the medium comprises 200 μM±30 μM putrescine in addition to ≧90 μM±14 μM ornithine. In one embodiment, the medium comprises ≧15 mg / L±2.25 mg / L ornithine·HCl in addition to 30 mg / L±4.5 mg / L putrescine·2HCl.

[0046] In some embodiments, the medium contains putrescine at a concentration of about 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, or 405 μM. In one embodiment, the medium contains 350 μM ± 52.5 μM putrescine in addition to ≧90 μM ± 14 μM ornithine. In one embodiment, the medium contains 57 mg / L ± 8.55 mg / L putrescine·2HCl in addition to ≧15 mg / L ± 2.25 mg / L ornithine·HCl.

[0047] In some embodiments, the medium comprises putrescine at a concentration of about 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, or 805 μM. In one embodiment, the medium comprises 714 μM±105 μM putrescine in addition to ≧90 μM±14 μM ornithine. In one embodiment, the medium contains ≧15 mg / L±2.25 mg / L ornithine·HCl plus 115 mg / L±17.25 mg / L putrescine·2HCl.

[0048] In some embodiments, the medium comprises a paired combination of putrescine and ornithine at any of the concentrations listed above. In some embodiments, the medium comprises a concentration of about 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, or 810. 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685 or 690 μM ornithine. For example, in one embodiment, the medium contains about 700 μM putrescine plus any one of 510, 511, 512 μM ornithine, see below; or 701 μM putrescine plus any one of 510, 511, 512 μM ornithine, see below; etc. Also, for example, in one embodiment, the medium contains about 600 μM ornithine plus any one of 700, 701, 702 μM ornithine, see below; or 601 μM ornithine plus any one of 700, 701, 702 μM ornithine, see below; etc. In some embodiments, the medium contains 702 μM ± 106 μM purescine plus 593 μM ± 89 μM ornithine. In one particular embodiment, the medium contains about 714 μM putrescine and 593 μM ornithine. In one embodiment, the medium comprises 115 mg / L±17 mg / L putrescine·2HCl and 100 mg / L±15 mg / L ornithine·HCl. In a particular embodiment, the medium comprises 115 mg / L putrescine·2HCl and 100 mg / L ornithine·HCl.

[0049] In one embodiment, in addition to containing ornithine or putrescine, the medium comprises a mixture of nucleosides at a cumulative concentration of at least 50 μM, at least 60 μM, at least 70 μM, at least 80 μM, at least 90 μM, at least 100 μM, at least 110 μM, at least 115 μM, at least 120 μM, at least 125 μM, at least 130 μM, at least 135 μM, at least 140 μM, at least 145 μM, at least 150 μM, at least 155 μM, at least 160 μM, at least 165 μM, or at least 170 μM. In one embodiment, the medium comprises about 174 μM±26 μM of nucleosides. In one embodiment, the medium contains a purine derivative at a cumulative concentration of at least 40 μM, at least 45 μM, at least 50 μM, at least 55 μM, at least 60 μM, at least 65 μM, at least 70 μM, at least 75 μM, at least 80 μM, at least 85 μM, at least 90 μM, at least 95 μM, at least 100 μM, or at least 105 μM. In one embodiment, the medium contains about 106 μM±5 μM of a purine derivative. Purine derivatives include hypoxanthine and the nucleosides adenosine and guanosine. In one embodiment, the medium contains a pyrimidine derivative at a cumulative concentration of at least 30 μM, at least 35 μM, at least 40 μM, at least 45 μM, at least 50 μM, at least 55 μM, at least 60 μM, or at least 65 μM. In one embodiment, the medium contains about 68 μM±5 μM of a pyrimidine derivative. Pyrimidine derivatives include the nucleosides thymidine, uridine and cytidine. In one particular embodiment, the medium comprises adenosine, guanosine, cytidine, uridine, thymidine and hypoxanthine.

[0050] In addition to including ornithine or putrescine, in one embodiment, the medium also includes an amino acid at a cumulative concentration of at least 40 mM, where the amount of glutamine is not included in the cumulative calculation. In one embodiment, glutamine is not included in the medium, but may be provided as a "point-of-use additive" to the medium during cell culture, such as during protein production. Thus, in some embodiments, the medium may be supplemented with glutamine as a point-of-use additive, such as in a method for culturing cells or producing a protein of interest. In such an embodiment, glutamine is added in an amount of less than about 40 mM, less than about 35 mM, less than about 30 mM, less than about 25 mM, less than about 20 mM, less than about 15 mM, less than about 10 mM, less than about 8 mM, less than about 7 mM, less than about 6 mM, less than about 5 mM, less than about 4 mM, less than about 3 mM, or less than about 2.5 mM. In one embodiment, the amount of glutamine in the glutamine-supplemented medium is about 2 mM±0.5 mM.

[0051] In one embodiment, in addition to containing ornithine or a combination of both ornithine and putrescine, the medium also contains amino acids having nonpolar side groups at a concentration of at least 15 mM, at least 24 mM, at least 25 mM, at least 26 mM, at least 27 mM, at least 28 mM, at least 29 mM, or at least 30 mM. In one embodiment, the medium contains about 30 mM of amino acids having nonpolar side groups. In one embodiment, at least 32 mol%, at least 33 mol%, at least 34 mol%, at least 35 mol%, at least 36 mol%, at least 37 mol%, at least 38 mol%, at least 39 mol%, at least 40 mol%, or at least 41 mol% of the total amino acids contained in the medium are amino acids having nonpolar side groups. In one embodiment, about 42 mol% ± 1 mol% of the amino acids in the medium are amino acids having nonpolar side groups. Amino acids having nonpolar side groups include alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine.

[0052] In one embodiment, in addition to including ornithine or a combination of both ornithine and putrescine, the medium also includes an amino acid having an uncharged polar side group at a concentration of about 10 mM to 34 mM, about 11 mM to 33 mM, about 12 mM to 32 mM, about 13 mM to 31 mM, about 14 mM to 30 mM, about 15 mM to 29 mM, about 16 mM to 28 mM, about 17 mM to 27 mM, about 18 mM to 26 mM, about 19 mM to 25 mM, about 20 mM to 24 mM, about 21 mM to 23 mM, or about 22 mM. In one embodiment, the medium includes about 22 mM of an amino acid having an uncharged polar side group. In another embodiment, the medium includes about 12 mM of an amino acid having an uncharged polar side group. In one embodiment, about 14 mol% to 46 mol%, about 15 mol% to 45 mol%, about 16 mol% to 44 mol%, about 17 mol% to 43 mol%, about 18 mol% to 42 mol%, about 19 mol% to 41 mol%, about 20 mol% to 40 mol%, about 21 mol% to 39 mol%, about 22 mol% to 38 mol%, about 23 mol% to 37 mol%, about 24 mol% to 36 mol%, about 25 mol% to 35 mol%, about 26 mol% to 34 mol%, about 27 mol% to 33 mol%, about 28 mol% to 32 mol%, about 29 mol% to 31 mol%, or about 30 mol% of the total amino acids contained in the medium are amino acids having an uncharged polar side group. In one embodiment, about 30 mol% ± 3 mol% of the amino acids in the medium are amino acids having an uncharged polar side group. Amino acids having uncharged polar side groups include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.

[0053] In one embodiment, in addition to containing ornithine or a combination of both ornithine and putrescine, the medium also contains an amino acid having a negative charge at pH 6 (i.e., an acidic amino acid) at a concentration of about 4 mM to 14 mM, about 5 mM to 13 mM, about 6 mM to 12 mM, about 7 mM to 11 mM, about 8 mM to 10 mM, about 9 mM, or about 4 mM. In one embodiment, the medium contains about 9 mM of an acidic amino acid. In one embodiment, the medium contains 9 mM ± 1 mM of an acidic amino acid. In one embodiment, about 8 mol% to 18 mol%, about 9 mol% to 17 mol%, about 10 mol% to 16 mol%, about 11 mol% to 15 mol%, about 12 mol% to 14 mol%, or about 13 mol% of the total amount of amino acids contained in the medium are acidic amino acids. In one embodiment, about 12.6 mol% ± 1 mol% of the amino acids in the medium are acidic amino acids. Acidic amino acids include aspartic acid and glutamic acid.

[0054] In one embodiment, in addition to containing ornithine or a combination of both ornithine and putrescine, the medium also contains a positively charged amino acid at pH 6 (i.e., a basic amino acid) at a concentration of at least 3.5 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, or at least 11 mM. In one embodiment, the medium contains about 11 mM basic amino acids. In one embodiment, the medium contains about 11.42 mM±1 mM basic amino acids. In one embodiment, at least 5 mol%, at least 6 mol%, at least 7 mol%, at least 8 mol%, at least 9 mol%, at least 10 mol%, at least 11 mol%, at least 12 mol%, at least 13 mol%, at least 14 mol%, or at least 15 mol% of the total amount of amino acids contained in the medium are basic amino acids. In one embodiment, about 16 mol% of the amino acids in the medium are basic amino acids. In one embodiment, about 15.8 mol% ± 2.4 mol% of the amino acids in the medium are basic amino acids. In one embodiment, about 21 mol% ± 3.2 mol% of the amino acids in the medium are basic amino acids. Basic amino acids include lysine, arginine, and histidine.

[0055] In one embodiment, in addition to containing ornithine or a combination of both ornithine and putrescine, the medium also contains about 30 mM nonpolar amino acids, about 22 mM uncharged polar amino acids, about 9 mM acidic amino acids, and about 11 mM basic amino acids. In one embodiment, of the amino acids in the medium, about 42 mol % are nonpolar amino acids, about 30 mol % are uncharged polar amino acids, about 13 mol % are acidic amino acids, and about 16 mol % are basic amino acids.

[0056] In addition to containing ornithine or a combination of both ornithine and putrescine, in one embodiment, the medium contains micromolar amounts of fatty acids (or fatty acid derivatives) and tocopherol. In one embodiment, the fatty acids include any one or more of linoleic acid, linolenic acid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and octanoic acid. In one embodiment, the medium contains tocopherol, linoleic acid, and thioctic acid.

[0057] In one embodiment, the medium also contains a mixture of vitamins, including other nutrients and essential nutrients, at a cumulative concentration of at least about 700 μM or at least about 2 mM. In one embodiment, the mixture of vitamins includes one or more of D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, pyridoxine HCl, D-pantothenic acid (hemiCa), riboflavin, thiamine HCl, vitamin B12, etc. In one embodiment, the mixture of vitamins includes all of D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, pyridoxine HCl, D-pantothenic acid (hemiCa), riboflavin, thiamine HCl, and vitamin B12.

[0058] Various embodiments of the media of the present invention include any of the combinations of the above embodiments, including serum-free media that do not contain chemically defined hydrolysates, including the indicated amounts of ornithine or putrescine plus, inter alia, (a) amino acids; (b) optionally nucleosides; (c) salts of divalent cations; (d) fatty acids and tocopherols; and (e) vitamins. In some embodiments, small amounts of all hydrolysates may be added to the OS medium.

[0059] Applicants contemplate that any one or more of a variety of basal media or combinations thereof to which ornithine or a combination of both ornithine and putrescine is added may be used in the practice of the present invention. Basal media are commonly known in the art and include, among others, Eagle's MEME (Minimum Essential Medium) (Eagle, Science, 1955, Vol. 112(3168): pp. 501-504), Ham's F12 (Ham, Proc. Nat'l. Acad. Sci. USA, 1965, Vol. 53: pp. 288-293), F-12 K medium, Dulbecco's medium, Dulbecco's modified Eagle's medium (Proc. Natl. Acad. Sci. USA, August 1952; Vol. 38(8): pp. 747-752), DMEM / Ham's F12 1:1, Trowell's T8, A2 medium (Holmes and Wolf, Biophys. Biochem. Cytol., 1961, Vol. 10: pp. 389-401), Waymouth's medium (Davidson and Waymouth, Biochem. J., 1945, 39(2):188-199), Williams E medium (Williams et al., Exp. Cell Res., 1971, 69:105ff.), RPMI 1640 (Moore et al., J. Amer. Med. Assoc., 1967, 199:519-524), MCDB 104 / 110 medium (Bettger et al., Proc. Nat'l. Acad. Sci. USA, 1981, 78(9):5588-5592), Ventrex HL-1 medium, albumin-globulin medium (Orr et al., Appl. Microbiol.1973, 25(1):49-54), RPMI-1640 medium, RPMI-1641 medium, Iscove's modified Dulbecco's medium, McCoy's 5A medium, Leibovitz's L-15 medium, and serum-free medium, such as EX-CELL™ 300 Series (JRH Biosciences, Lenexa, Kansas), protamine-zinc-insulin medium (Weiss et al., 1974, U.S. Pat. No. 4,072,565), biotin-folate medium (Cartaya, 1978, U.S. Pat. No. Re. 30,985), transferrin-fatty acid medium (Baker, 1982, U.S. Pat. No. 4,560,655), transferrin-EGF medium (Hasegawa, 1982, U.S. Pat. No. 4,615,977; Chessebeuf, 1984, U.S. Pat. No. 4,786,599), as well as other media permutations (Inlow (See, e.g., U.S. Patent No. 6,048,728; Drapeau, U.S. Patent No. 7,294,484; Mather, U.S. Patent No. 5,122,469; Furukawa, U.S. Patent No. 5,976,833; Chen, U.S. Patent No. 6,180,401; Chen, U.S. Patent No. 5,856,179; Etcheverry, U.S. Patent No. 5,705,364; Etcheverry, U.S. Patent No. 7,666,416; Ryll, U.S. Patent No. 6,528,286; Singh, U.S. Patent No. 6,924,124; Luan, U.S. Patent No. 7,429,491; etc.).

[0060] In one particular embodiment, the medium is chemically defined and contains, in addition to ornithine or a combination of both ornithine and putrescine, the following: CaCl22H2O; HEPES buffer, KCl; MgSO4; NaCl; Na2HPO4 or other phosphate salts; pyruvate; L-alanine; L-arginine HCl; L-asparagine HO; L-aspartic acid; L-cysteine ​​HCl HO; L-glutamic acid; glycine; L-histidine HCl HO; L-isoleucine; L-leucine; L-lysine HCl; L-methionine; L-ornithine HCl; L-phenylalanine; L-proline; L-serine; L-threonine; L-tryptophan; L-tyrosine Disodium 2H2O; L-valine; D-biotin; choline chloride; folic acid; myo-inositol; niacinamide; pyridoxine HCl; D-pantothenic acid; riboflavin; thiamine HCl; vitamin B12; p-aminobenzoic acid; ethanolamine HCl; Pluronic F68; DL-α-tocopherol phosphate; linoleic acid; Na2SeO3; thioctic acid; and glucose; and optionally adenosine; guanosine; cytidine; uridine; thymidine; and hypoxanthine 2Na.

[0061] In one embodiment, the starting osmolality of the medium of the invention is 200-500, 250-400, 275-350, or about 300 mOsm. During growth of cells in the medium of the invention, particularly after any feeding according to a fed-batch protocol, the osmolality of the culture can increase up to as high as about 350, 400, 450, or 500 mOsm.

[0062] In some embodiments where the osmolality of the defined medium is less than about 300, the osmolality is brought to about 300 by the addition of one or more salts in excess of a specified amount. In one embodiment, the osmolality is increased to a desired level by adding one or more osmolytes selected from sodium chloride, potassium chloride, magnesium salts, calcium salts, amino acid salts, fatty acid salts, sodium bicarbonate, sodium carbonate, potassium carbonate, chelator salts, sugars (e.g., galactose, glucose, sucrose, fructose, fucose, etc.), and combinations thereof. In one embodiment, the osmolyte is added in excess of and above the concentration in the components already present in the defined medium (e.g., sugars are added in excess of and above the concentration specified for the sugar component).

[0063] Each and every embodiment of the above medium, as well as any other serum-free medium containing at least about 90 μM ornithine (or containing a combination of at least about 100 μM ornithine and at least about 200 μM putrescine), is hereinafter referred to as an ornithine-supplemented ("OS") medium. Conversely, a medium that does not contain ornithine (or does not contain an ornithine / putrescine combination), or a medium that contains less than 100 μM ornithine (or a medium that contains less than 100 μM ornithine and less than 200 μM putrescine), is hereinafter referred to as a non-ornithine-supplemented ("non-OS") medium.

[0064] cell culture The present invention provides a cell culture comprising a cell line expressing a protein of interest in the OS medium. In one embodiment, the cell culture comprises insulin, which can be added to the medium as a point-of-use component or included in the medium formulation. In one embodiment, the cell line comprises cells capable of producing a biotherapeutic protein. Examples of cell lines commonly used to produce protein biotherapeutics include primary cells, BSC cells, HeLa cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, CHO cells, CHO-K1 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, and chicken embryo cells, among others. In one embodiment, the cell line is a CHO cell line or one or more of several specific CHO cell variants optimized for large-scale protein production, for example, CHO-K1.

[0065] "Cell culture" or "culture" refers to the proliferation and growth of cells outside a multicellular organism or tissue. Culture conditions suitable for mammalian cells are known in the art. See, for example, Animal Cell Culture: A Practical Approach, edited by D. Rickwood, Oxford University Press, New York (1992). Mammalian cells can be cultured in suspension or attached to a solid substrate. Fluidized-bed bioreactors, hollow-fiber bioreactors, roller bottles, shake flasks, or stirred-tank bioreactors, operating in batch, fed-batch, continuous, semi-continuous, or perfusion modes, with or without microcarriers, are available for mammalian cell culture. Cell culture medium or concentrated feed medium can be added to the culture continuously or at intervals during cultivation. For example, the culture can be fed once daily, every other day, every three days, or when the concentration of a particular medium component being monitored is outside the desired range.

[0066] Animal cells, e.g., CHO cells, can be cultured in small-scale cultures, e.g., in 125 ml containers with about 25 ml of medium, in 250 ml containers with about 50-100 ml of medium, or in 500 ml containers with about 100-200 ml of medium. Alternatively, these cultures can be on a larger scale, e.g., in 1000 ml containers with about 300-1000 ml of medium, in 3000 ml containers with about 500-3000 ml of medium, in 8000 ml containers with about 2000-8000 ml of medium, and in 15000 ml containers with about 4000-15000 ml of medium. Cultures for manufacturing can contain 10,000 L or more of medium. Large-scale cell cultures, e.g., for clinical manufacturing of protein therapeutics, are typically maintained for several days or even weeks while the cells produce the desired protein(s). During this time, the culture can be supplemented with a concentrated feed medium containing components consumed during the course of the culture, such as nutrients and amino acids. The concentrated feed medium can be based on any cell culture medium formulation. Such concentrated feed medium can contain most of the components of a cell culture medium in their normally useful amounts, for example, about 5x, 6x, 7x, 8x, 9x, 10x, 12x, 14x, 16x, 20x, 30x, 50x, 100x, 200x, 400x, 600x, 800x, or even about 1000x. Concentrated feed medium is often used in fed-batch processes.

[0067] In some embodiments, the cell culture medium is supplemented with "point-of-use additives," also known as additives, point-of-use components, or point-of-use chemicals, during the process of cell growth or protein production. Point-of-use additives include any one or more of growth factors or other proteins, buffers, energy sources, salts, amino acids, metals, and chelators. Other proteins include transferrin and albumin. Growth factors, including cytokines and chemokines, are generally known in the art and are known to stimulate cell proliferation, or in some cases, cell differentiation. Growth factors are typically proteins (e.g., insulin), small peptides, or steroid hormones, such as estrogen, DHEA, and testosterone. In some cases, growth factors can be non-naturally occurring chemicals that promote cell proliferation or protein production, such as tetrahydrofolate (THF), methotrexate, and the like. Non-limiting examples of protein and peptide growth factors include angiopoietin, bone morphogenetic proteins (BMPs), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), glial cell line-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin, insulin-like growth factor (IGF), migration-stimulating factor, and myostatin (GDF-8). These include nerve growth factor (NGF) and other neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), wnt signaling pathway agonists, placental growth factor (PlGF), fetal bovine somatotrophin (FBS), interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, and IL-7.In one embodiment, the cell culture medium is supplemented with the point-of-use additive growth factor insulin. In one embodiment, the concentration of insulin in the medium, i.e., the amount of insulin in the cell culture medium after addition, is about 0.1 μM to 10 μM. One or more point-of-use additives may also be included in the medium formulation of some embodiments.

[0068] Buffers are generally known in the art. The present invention is not limited to any particular buffer(s), and one of skill in the art can select an appropriate buffer or buffer system for use with a particular cell line producing a particular protein. In one embodiment, the point-of-use additive buffer is NaHCO. In one embodiment, the point-of-use additive buffer comprises NaHCO. In another embodiment, the buffer is HEPES.

[0069] Energy sources for use as point-of-use additives in cell culture are also well known in the art. Without limitation, in one embodiment, the point-of-use additive energy source is glucose. Taking into account the specific and specific requirements of a particular cell line and the protein being produced, in one embodiment, the glucose may be added to a concentration of about 1 to 20 mM in the medium. In some cases, glucose may be added at a high level of up to 10 g / L.

[0070] Chelators are also well known in the fields of cell culture and protein production. Tetrasodium EDTA dehydrate and citrate are two common chelators used in the art, although other chelators can be used in the practice of the present invention. In one embodiment, the point-of-use additive chelator is tetrasodium EDTA dihydrate. In one embodiment, the point-of-use additive chelator is citrate, e.g., Na3C6H5O7.

[0071] In one embodiment, the cell culture may be supplemented with one or more point-of-use additive amino acids, such as glutamine, hi one embodiment, the cell culture medium is supplemented with the point-of-use additive glutamine at a final concentration of about 1 mM to 13 mM.

[0072] Other point-of-use additives include one or more of various metal salts, such as iron, nickel, zinc, and copper salts. In one embodiment, the cell culture medium is supplemented with any one or more of copper sulfate, zinc sulfate, ferric chloride; and nickel sulfate.

[0073] In one embodiment, the cell culture medium is supplemented with any one or more or all of the following point-of-use additives: about 29.8 mM NaHCO, about 2 mM glutamine, about 0.86 μM insulin, about 11.1 mM glucose, about 6.54 μM zinc sulfate, about 0.168 μM copper sulfate, about 75 μM ferric chloride, about 0.639 μM nickel sulfate, about 85 μM EDTA, and about 50 μM citrate.

[0074] In one embodiment, the medium is replenished at intervals during cell culture according to a fed-batch process, which is commonly known in the art and used for optimized protein production (see YM Huang et al., Biotechnol Prog. 2010 Sep-Oct;26(5):1400-10).

[0075] Cell viability, viable cell density, and cell doublings are improved compared to cells grown in cultures that do not contain ornithine or putrescine. With respect to cell viability, cells grown in OS medium exhibit at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, or at least 3-fold greater viability than similar or identical cells grown in non-OS medium.

[0076] In some embodiments, the doubling rate of surviving mammalian cells in OS medium is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or at least 3 times greater than the doubling rate of mammalian cells cultured in non-OS medium. In some embodiments, the doubling rate of viable mammalian cells in OS medium is about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% greater than the doubling rate of mammalian cells in non-OS medium.

[0077] In some embodiments, the doubling time of actively cycling mammalian cells is less than 30 hours, less than 29 hours, less than 28 hours, less than 27 hours, less than 26 hours, less than 25 hours, less than 24 hours, less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, or less than 18 hours in OS medium. In some embodiments, the doubling time of actively growing mammalian cells is less than 28 hours in OS medium. In some embodiments, the doubling time of mammalian cells is about 27±1 hours, about 26±1 hours, about 25±1 hours, about 24±1 hours, about 23±1 hours, about 22±1 hours, or about 21±1 hours in OS medium. In some embodiments, the doubling time of actively cycling mammalian cells is about 24±1 hours in OS medium. In some embodiments, the doubling time of actively dividing cells cultured in OS medium is at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, or at least 25% shorter than the doubling time of actively cycling cells cultured in non-OS medium.

[0078] Protein production In addition to chemically defined OS medium and methods for culturing cells in OS medium, the present invention provides methods for producing proteins, such as therapeutically effective antibodies or other biopharmaceutical drug substances, in cells cultured in OS medium.

[0079] In some embodiments, the rate of protein production by mammalian cells cultured in OS medium is at least 5%, 10%, 15%, or 20% greater than the rate of protein production by the same mammalian cells cultured in non-OS medium, hi some embodiments, the rate of protein production in cells cultured in OS medium is at least 1 pg / cell / day ("PCD"), at least 2 PCD, at least 3 PCD, at least 4 PCD, at least 5 PCD, at least 6 PCD, at least 7 PCD, at least 8 PCD, at least 9 PCD, at least 10 PCD, at least 15 PCD, at least 20 PCD, at least 25 PCD, at least 30 PCD, at least 35 PCD, at least 40 PCD, at least 45 PCD, at least 50 PCD, at least 75 PCD, or at least 100 PCD.

[0080] In some embodiments, the protein production yield or titer from cells cultured in OS medium, which may be expressed in grams of protein product per liter of culture medium, is at least 100 mg / L, at least 1 g / L, at least 1.2 g / L, at least 1.4 g / L, at least 1.6 g / L, at least 1.8 g / L, at least 2 g / L, at least 2.5 g / L, at least 3 g / L, at least 3.5 g / L, at least 4 g / L, at least 4.5 g / L, at least 5 g / L, at least 5.5 g / L, at least 6 g / L, at least 6.5 g / L, at least 7 g / L, at least 7.5 g / L, at least 8 g / L, at least 8.5 g / L, at least 9 g / L, at least 9.5 g / L, at least 10 g / L, or at least 20 g / L.

[0081] In some embodiments, the protein product (protein of interest) is an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antigen-binding antibody fragment, a single-chain antibody, a diabody, a triabody or a tetrabody, a Fab or F(ab')2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In one embodiment, the antibody is an IgG1 antibody. In one embodiment, the antibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4 antibody.

[0082] In some embodiments, the protein of interest is a recombinant protein (e.g., an Fc-fusion protein) comprising an Fc portion and another domain. In some embodiments, the Fc-fusion protein is a receptor Fc-fusion protein comprising one or more extracellular domain(s) of a receptor coupled to an Fc portion. In some embodiments, the Fc portion comprises a hinge region followed by the CH2 and CH3 domains of IgG. In some embodiments, the receptor Fc-fusion protein comprises two or more separate receptor chains that bind either a single ligand or multiple ligands. For example, the Fc-fusion protein is a trap, such as an IL-1 trap (e.g., rilonacept, which comprises the IL-1RAcP ligand-binding region fused to the extracellular region of IL-1R1 fused to the Fc of hIgG1; see U.S. Pat. No. 6,927,004, which is incorporated herein by reference in its entirety), or a VEGF trap (e.g., aflibercept, which comprises the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to the Fc of hIgG1; see U.S. Pat. Nos. 7,087,411 and 7,279,159).

[0083] The present invention is not limited to any particular type of cell for protein production. Examples of cell types suitable for protein production include mammalian cells, insect cells, avian cells, bacterial cells, and yeast cells. These cells can be stem cells or recombinant cells transformed with a vector for recombinant gene expression, or cells transfected with a virus to produce a viral product. These cells can contain a recombinant heterologous polynucleotide construct encoding a protein of interest. This construct can be episomal or an element physically integrated into the cell's genome. These cells can also produce a protein of interest without the protein being encoded on a heterologous polypeptide construct. In other words, the cell can naturally encode a protein of interest, such as a B cell that produces an antibody. These cells can also be primary cells, such as chicken embryo cells, or primary cell lines. Examples of useful cells include BSC cells, LLC-MK cells, CV-1 cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK-21 cells, chicken embryo cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 293 cells, RK cells, Per.C6 cells, and CHO cells. In various embodiments, the cell line is a CHO cell derivative, such as CHO-K1, CHO DUX B-11, CHO DG-44, Veggie-CHO, GS-CHO, S-CHO, or a CHO lec mutant line.

[0084] In one embodiment, the cell, which is a CHO cell, ectopically expresses a protein. In one embodiment, the protein comprises an immunoglobulin heavy chain region, such as a CH1, CH2, or CH3 region. In one embodiment, the protein comprises human or rodent immunoglobulin CH2 and CH3 regions. In one embodiment, the protein comprises human or rodent immunoglobulin CH1, CH2, and CH3 regions. In one embodiment, the protein comprises a hinge region and CH1, CH2, and CH3 regions. In a specific embodiment, the protein comprises an immunoglobulin heavy chain variable domain. In a specific embodiment, the protein comprises an immunoglobulin light chain variable domain. In a specific embodiment, the protein comprises an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain. In a specific embodiment, the protein is an antibody, such as a human antibody, a rodent antibody, or a chimeric human / rodent antibody (e.g., human / mouse, human / rat, or human hamster).

[0085] The production phase can be carried out in cultures of any scale, from individual flasks and shaker flasks or wave bags to 1-liter bioreactors and large-scale individual bioreactors. Large-scale processes can be carried out in volumes of about 100 liters to 20,000 liters or more. One or more of several means, such as temperature shift or chemical induction, can be used to control protein production. The growth phase can occur at a higher temperature than the production phase. For example, the growth phase can occur at a first temperature of about 35°C to 38°C, and the production phase can occur at a second temperature of about 29°C to 37°C, optionally about 30°C to 36°C or about 30°C to 34°C. Additionally, chemical inducers of protein production, such as caffeine, butyrate, tamoxifen, estrogen, tetracycline, doxycycline, and hexamethylene bisacetamide (HMBA), can be added simultaneously with, before, or after the temperature shift. If inducers are added after the temperature shift, they can be added 1 hour to 5 days after the temperature shift, for example, 1 to 2 days after the temperature shift. The production cell culture can be run as a continuous-feed culture system, such as in a chemostat (see C. Altamirano et al., Biotechnol Prog. 2001 Nov-Dec;17(6):1032-41), or according to a fed-batch process (Huang, 2010).

[0086] The present invention is useful for improving protein production via cell culture processes. The cell lines used in the present invention can be genetically engineered to express polypeptides of commercial or scientific interest. Genetically engineering cell lines involves transfecting, transforming, or transducing cells with recombinant polynucleotide molecules, or otherwise modifying them (e.g., by homologous recombination and gene activation, or by fusion of recombinant cells with non-recombinant cells), so that the host cells express the desired recombinant polypeptide. Methods and vectors for genetically engineering cells or cell lines to express a polypeptide of interest are well known to those skilled in the art; for example, various techniques are set forth in Current Protocols in Molecular Biology, edited by Ausubel et al. (Wiley & Sons, New York, 1988 and quarterly revisions); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); Kaufman, RJ, Large Scale Mammalian Cell Culture, 1990, pp. 15-69. A wide variety of cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and suppliers. Examples of cell lines commonly used in industry include VERO, BHK, HeLa, CV1 (including Cos), MDCK, 293, 3T3, myeloma cell lines (e.g., NSO, NS1), PC12, WI38 cells, and Chinese hamster ovary (CHO) cells. CHO cells are widely used for the production of complex recombinant proteins, such as cytokines, clotting factors, and antibodies (Brasel et al. (1996), Blood 88:2004-2012; Kaufman et al. (1988), J. Biol. Chem 263:6352-6362; McKinnon et al. (1991), J. MoI. Endocrinol 6:231-239; Wood et al. (1990), J. Immunol. 145:3011-3016).The dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al. (1980) Proc Natl Acad Sci USA 77:4216-4220), DXB1 1, and DG-44, are desirable CHO host cell lines because an efficient DHFR-selectable, amplifiable gene expression system allows high-level recombinant protein expression in these cells (Kaufman RJ. (1990) Meth Enzymol 185:537-566). Furthermore, these cells are easy to manipulate as adherent or suspension cultures and exhibit relatively good genetic stability. CHO cells and the proteins recombinantly expressed by them have been extensively characterized and approved by regulatory agencies for use in clinical and commercial manufacturing. In some embodiments, the CHO cell line is a cell line as described in U.S. Patent Application Publication Nos. 2010 / 0304436, 2009 / 0162901, and 2009 / 0137416, as well as U.S. Patent Nos. 7,455,988, 7,435,553, and 7,105,348.

[0087] The present invention is not limited in scope by the specific embodiments described herein, which are to be construed as illustrative of individual aspects or embodiments of the invention. Functionally equivalent methods and components are within the scope of the invention. Various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications fall within the scope of the invention.

[0088] The present invention is based, in part, on the discovery that the addition of ornithine or a combination of ornithine and putrescine to serum-free cell culture media results in increased cell growth, viability, and polypeptide production from recombinantly engineered animal cell lines (or native cells) expressing a protein of interest, thereby enhancing culture robustness and improving yield of the polypeptide of interest. [Example]

[0089] Example 1 Improved viable cell culture density A 250 mL shake flask was inoculated with a seed culture of a recombinant antibody-producing cell line derived from CHO K1. The inoculated cells were grown at 36.5°C for 7 days, with glucose feeding on days 3 and 5. Cells were grown in each of two separate chemically defined (hydrolysate-free, serum-free) media. The first medium contained approximately 75 mM amino acids (Medium 1), the second medium contained approximately 40 mM amino acids (Medium 2), and both formulations contained up to 2.5 μM (0.4 mg / L) putrescine. Another group of media conditions was generated by adding soy hydrolysate to Medium 2 at a concentration of 7.5 g / L. Each of the three control cultures was supplemented with approximately 593 μM ornithine (as 100 mg / L L-ornithine·HCl) or a combination of approximately 593 μM ornithine (as 100 mg / L L-ornithine·HCl) and approximately 714 μM putrescine (as 115 mg / L putrescine·2HCl). Aliquots of 3 mL cultures were removed on days 3, 5, and 7, and viable cell counts were performed using trypan blue exclusion on a BioProfile FLEX™ instrument (Nova Biomedical). On day 0, all cultures had a viable cell count of 0.8 × 10 per mL. 6 For a given medium (Medium 1, Medium 2, or Medium 2 + soy), viable cell counts over a 7-day period revealed that CHO cells grown in medium supplemented with ornithine or ornithine + putrescine had increased viable cell densities. This effect was particularly pronounced in the hydrolysate-free medium over the 7-day period (i.e., a 2- to 4-fold or greater increase in viable cell density). Hydrolysate-free OS Medium 2 performed comparably to soy-containing non-OS Medium 2, indicating that the cell growth benefits of soy hydrolysate can be replicated by ornithine replacement. Increased cell densities were also observed with the addition of ornithine or ornithine and putrescine to Medium 2 + soy. The results are shown in Table 1.

[0090] [Table 1]

[0091] We also tested the effect of varying amounts of ornithine·HCl (i.e., 50 mg / mL, 100 mg / mL, and 150 mg / mL) on viable cell density in Medium 3 ("Medium 3") containing approximately 75 mM amino acids and 0.4 mg / L putrescine HCl. Using a single seed training culture of a recombinant antibody-producing cell line derived from CHO K1, we cultured 0.4 × 10 cells in a 15 mL working volume. 6 50 mL TubeSpin® Bioreactors (TPP) were inoculated with 1000 cells / mL. Cells were grown in a 37°C incubator for 3 days. An aliquot of 3 mL culture was removed on day 3, and viable cell counts were performed using trypan blue exclusion on a BioProfile FLEX™ instrument (Nova Biomedical). All three levels of ornithine improved cell density on average by slightly more than two-fold (N=3). The results are shown in Table 2.

[0092] [Table 2]

[0093] Example 2 Improved cell culture doubling times The doubling time of a recombinant antibody-producing cell line derived from CHO K1 cells in logarithmic growth phase was determined under various cell culture medium conditions. Seed training cultures were passaged in each of three separate media: Medium 1, Medium 2, and Medium 2 containing soy hydrolysate (Medium 2 + soy) in 250 mL shaker flasks at 36.5°C over a 14-day period. One-mL aliquots were removed from each condition on day 0 and at the time of the seed training passage (every 2 or 3 days), and viable cell counts were performed using trypan blue exclusion on a CDV™ instrument (Nova Biomedical). Medium 1 was tested unsupplemented or supplemented with 100 mg / L ornithine·HCl or both 115 mg / L putrescine·2HCl and 100 mg / L ornithine·HCl. Medium 2, containing low putrescine·2HCl (0.4 mg / L), was tested unsupplemented or supplemented with 100 mg / L ornithine·HCl or both 115 mg / L putrescine·2HCl and 100 mg / L ornithine·HCl. The results are shown in Tables 3 and 4. Ornithine supplementation to Medium 1, either with or without putrescine, was necessary to achieve significant growth. Supplementing hydrolysate-free Medium 2 with ornithine or ornithine plus putrescine reduced cell doubling time by approximately 25%–30%. Doubling time was also reduced to a lesser extent upon addition of ornithine or ornithine plus putrescine to hydrolysate-containing Medium 2.

[0094] [Table 3]

[0095] [Table 4]

[0096] Example 3 Improved antibody titers Having established that the inclusion of ornithine or ornithine plus putrescine improved cell proliferation and viable cell density in culture, we further investigated the effect of these conditions on recombinant protein production titers. We tested the expression and secretion of recombinant IgG by a CHO-K1-derived cell line. In this experiment, mean antibody titers were determined on day 7 in culture under various media formats. As described above, Medium 1 containing low putrescine (0.4 mg / L putrescine·2HCl), ornithine (100 mg / L ornithine·HCl), and both ornithine and putrescine (100 mg / L ornithine·HCl / 115 mg / L putrescine·2HCl) was tested. Medium 2 and Medium 2 + soy containing low putrescine (0.4 mg / L putrescine 2HCl), ornithine (100 mg / L ornithine HCl), and both ornithine and putrescine (100 mg / L ornithine HCl / 115 mg / L putrescine 2HCl) were also tested. In all cases, the inclusion of ornithine or ornithine and putrescine at levels above 0.4 mg / L resulted in significantly higher protein titers, i.e., titers at least approximately two-fold higher. The results are shown in Table 5.

[0097] [Table 5]

[0098] We also tested the effect of various amounts of ornithine·HCl (i.e., 50 mg / mL, 100 mg / mL, and 150 mg / mL) in Medium 3 on antibody production. Using a single seed training culture of a recombinant antibody-producing cell line derived from CHO K1, 0.4 × 10 cells were cultured in a 15 mL working volume. 6 Cells / mL were seeded into 50 mL TubeSpin® Bioreactors (TPP). Cells were grown in a 37°C incubator for 3 days. All three levels of ornithine supplementation improved antibody titers, on average, by slightly more than 50% (N=3). The results are shown in Table 6.

[0099] Table 6

Claims

1. A cell culture medium that is serum-free and contains ≧0.09 mM±0.014 mM ornithine.

2. 2. The cell culture medium of claim 1, comprising ≧0.20±0.03 mM putrescine.

3. 3. The cell culture medium of claim 1, comprising 0.09±0.014 mM to 0.9±0.14 mM ornithine.

4. 4. The cell culture medium of claim 1, comprising ornithine at 0.09±0.014 mM, 0.3±0.05 mM, 0.6±0.09 mM or 0.9±0.14 mM.

5. 5. The cell culture medium of claim 1, comprising 0.20±0.03 mM to 0.714±0.11 mM putrescine.

6. 5. The cell culture medium of claim 1, comprising putrescine at 0.20±0.03 mM, 0.35±0.06 or 0.714±0.11 mM.

7. 7. The cell culture medium of claim 1, which is hydrolysate-free.

8. 7. The cell culture medium of claim 1, which is chemically defined.

9. 9. The cell culture medium of claim 1, comprising ≧40±6 mM of a mixture of amino acids or salts thereof.

10. 10. The cell culture medium of claim 9, wherein the mixture of amino acids consists of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

11. 11. The cell culture medium of claim 1, comprising one or more fatty acids.

12. 12. The cell culture medium of claim 11, wherein the one or more fatty acids are selected from the group consisting of linoleic acid, linolenic acid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and octanoic acid.

13. 13. The cell culture medium of claim 1, comprising a mixture of nucleosides.

14. 14. The cell culture medium of claim 13, wherein the mixture of nucleosides comprises one or more of adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

15. 15. The cell culture medium of claim 1, comprising adenosine, guanosine, cytidine, uridine, thymidine and hypoxanthine.

16. 16. The cell culture medium of claim 1, comprising one or more divalent cations.

17. 17. The cell culture medium of claim 16, wherein the divalent cation is magnesium, calcium, or both.

18. Ca ２＋ and Mg ２＋ 18. The cell culture medium of claim 1 , comprising:

19. 20. A method for culturing cells, comprising: (a) providing a cell culture medium according to any one of claims 1 to 18; and (b) growing or maintaining cells in the cell culture medium to form a cell culture.

20. 20. The method of claim 19, wherein the cell is selected from the group consisting of a mammalian cell, an avian cell, an insect cell, a bacterial cell, and a yeast cell.

21. 21. The method of claim 19 or claim 20, wherein the cells are CHO cells.

22. 22. The method of any one of claims 19 to 21, wherein the cells express a protein of interest.

23. 23. The method of claim 22, wherein the protein of interest is an antigen-binding protein.

24. 24. The method of claim 22 or 23, wherein the protein of interest comprises an Fc domain.

25. The method of any one of claims 22 to 24, wherein the protein of interest is a receptor-Fc-fusion protein.

26. The method of claim 25, wherein the receptor-Fc-fusion protein is a trap protein.

27. 27. The method of claim 26, wherein the trap protein is an IL-1 antagonist or a VEGF antagonist.

28. 24. The method of claim 22 or 23, wherein the protein of interest is an antibody or antibody fragment.

29. 29. The method of claim 28, wherein the antibody or antibody fragment is a recombinant human antibody or fragment thereof.

30. 30. The method of any one of claims 19 to 29, wherein the cells have an average doubling time of ≦30 hours.

31. 31. The method of any one of claims 19 to 30, wherein the cells have a mean doubling time of ≦24 hours.

32. 32. The method of any one of claims 19 to 31, wherein the cells have an average doubling time that is at least one-third of the average doubling time of cells grown in a cell culture medium comprising <0.3±0.045 mM ornithine and <0.2±0.03 mM putrescine.

33. 33. The method of any one of claims 19 to 32, wherein the cell culture is able to reach a viable cell count density that is at least 15% greater than a similar cell culture in a medium containing <0.09±0.014 mM ornithine and <0.2±0.03 mM putrescine.

34. 34. The method of any one of claims 19 to 33, wherein the cell culture is capable of reaching a viable cell count density that is at least three times greater than a similar cell culture in a similar cell culture medium comprising <0.09±0.014 mM ornithine and <0.2±0.03 mM putrescine.

35. 35. The method of any one of claims 19 to 34, comprising adding one or more point-of-use additives to the cell culture medium.

36. The point-of-use additive is NaHCO ３ , glutamine, insulin, glucose, CuSO ４ , ZnSO ４ , FeCl ３ , NiSO ４ , Na ４ EDTA and sodium citrate ３ 36. The method of claim 35, comprising one or more of:

37. NaHCO ３ , glutamine, insulin, glucose, CuSO ４ , ZnSO ４ , FeCl ３ , NiSO ４ , Na ４ EDTA and sodium citrate ３ 37. The method of claim 35 or 36, wherein each of is added to the medium as a point-of-use addition.

38. 38. A method for producing a protein, the method comprising: (a) introducing into cells a nucleic acid comprising a sequence encoding a protein of interest; (b) selecting cells that harbor the nucleic acid; (c) culturing the selected cells in a cell culture medium according to any one of claims 1 to 18 or according to the method according to any one of claims 19 to 37; and (d) expressing the protein of interest in the cells, wherein the protein of interest is secreted into the medium.

39. 39. The method of claim 38, wherein the cell is a CHO cell, a 293 cell, or a BHK cell.

40. 40. The method of claim 38 or 39, wherein the protein of interest is an antigen-binding protein.

41. 41. The method of any one of claims 38 to 40, wherein the protein of interest comprises an Fc domain.

42. 42. The method of any one of claims 38 to 41, wherein the protein of interest is selected from the group consisting of receptor-Fc-fusion proteins (TRAP), soluble TCR-Fc fusion proteins, antibodies, Fc-fusion proteins and ScFv proteins.

43. 43. The method of any one of claims 38 to 42, wherein the protein of interest is produced at an average day 7 titer that is at least 7% greater than the average day 7 titer produced by similar cells in a cell culture medium comprising less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine.

44. 44. The method of any one of claims 38 to 43, wherein the protein of interest is produced at an average day 7 titer that is at least 14% greater than the average day 7 titer produced by similar cells in a cell culture medium comprising less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine.

45. 45. The method of any one of claims 38 to 44, wherein the protein of interest is produced at an average day 7 titer that is at least 80% greater than the average day 7 titer produced by similar cells in a cell culture medium comprising less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine.

46. 46. ​​The method of any one of claims 38 to 45, wherein the protein of interest is produced at an average day 7 titer that is at least 2-fold greater than the average day 7 titer produced by similar cells in a cell culture medium comprising less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine.

47. 47. The method of any one of claims 38 to 46, wherein the protein of interest is produced at an average day 7 titer that is at least three times greater than the average day 7 titer produced by similar cells in a cell culture medium comprising less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine.

48. 48. The method of any one of claims 38 to 47, wherein the protein of interest is a recombinant human antibody.

Images