WO2008009641A1

WO2008009641A1 - Cell culture media

Info

Publication number: WO2008009641A1
Application number: PCT/EP2007/057262
Authority: WO
Inventors: Caspar Foghsgaard; Tanja Maria Rosenkilde Kjaer; Nana Kristensen Soni
Original assignee: Novozymes A/S
Priority date: 2006-07-17
Filing date: 2007-07-13
Publication date: 2008-01-24
Also published as: WO2008009642A1

Abstract

The invention is a cell culture medium comprising recombinant albumin and which preferably is free of animal derived components. The medium supports the growth of cells for both small scale and large scale propagation of cells. The invention also includes a method of cultivating cells using the cell culture medium of the present invention.

Description

TITLE: CELL CULTURE MEDIA

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. Biotechnology drugs are medicines, such as therapeutic proteins (monoclonal antibodies, blood proteins and enzymes) that are produced by living organisms to fight disease. Unlike other medicines, biotech drugs are generally not produced synthetically, but are usually produced through microbial fermentation and in mammalian cell culture. They can be more difficult, time-consuming and expensive (at least $250 million in production facility costs alone) to produce than synthetic drugs.

It is estimated that there are more than 370 new biotechnology medicines in the pipeline. Producing biotech drugs is a complicated and time-consuming process. Cells must be grown in large stainless-steel fermentation vats under strictly maintained and regulated conditions. In some cases the proteins are secreted by the cells; in other cases the cells must be broken open so the protein can be extracted and purified. Once the method is tested, devised and scaled up, the biotech medicines can be produced in large batches. This is done by growing host cells that have been transformed to contain the gene or antibody of interest in carefully controlled conditions in large stainless-steel tanks. The cells are kept alive and stimulated to produce the target proteins through precise culture conditions that include a balance of temperature (which can often vary by no more than one degree Celsius), oxygen, acidity (if pH levels change by even a small fraction, cells can easily die), media components and other variables. After careful culture in the appropriate media or serum (the duration varies depending on the protein produced and the nature of the organism), the proteins are isolated from the cultures, stringently tested at every step of purification, and formulated into pharmaceutically-active products. All of these procedures are in strict compliance with Food and Drug Administration (FDA) regulations, (http://www.bio.org/pmp/factsheet1.asp, "A Brief Primer on Manufacturing Therapeutic Proteins"). There are many varied types of cell culture media that can be used to support cell viability, for example DMEM medium (H. J. Morton, In Vitro, 6, 89/1970), F12 medium (R. G. Ham, Proc. Natl. Acad. Sci. USA, 53, 288/1965) and RPMI 1640 medium (J. W. Goding, J. Immunol. Methods, 39, 285/1980; JAMA 199, 519/1957). Such media (often called "basal media"), however, are usually seriously deficient in the nutritional content required by most animal cells. Typically, serum must be added to the basal media to overcome these deficiencies. Generally, fetal bovine serum (FBS, harvested from the fetuses of cows), human serum, porcine serum and horse serum is used in significant concentrations. While the use of serum is desirable, and often necessary, for proper cell growth, it has several disadvantages. It is difficult to obtain serum with consistent growth characteristics. Further, the biochemical complexity of the serum can complicate the downstream processing of the proteins of interest, therefore raising the production costs. In an attempt to solve this problem the serum has been removed and specific components have been added instead. One of these components is albumin, any animal or mammalian albumin, such as bovine serum albumin (BSA) or human serum albumin (HSA).

However, there is an increased focus on the risk of possible contamination with pathogens when using animal derived components such as albumins. In the case of BSA, the presence of prions thought to be responsible for mad-cow disease (BSE) is in particular a problem associated with the production of BSA by blood fractionation. For HSA there is a risk of possible contamination by hepatitis and immunodeficiency viruses like HIV, when HSA is produced by blood fractionation.

A recombinant albumin medium is an excellent alternative to standard serum-containing media for the cultivation of cells. It has several advantages, which include better definition of the composition, and very importantly no risk for contamination with pathogens originating from animals.

Due to the increased focus on blood transferred diseases from humans and animals, there has been an increased focus on finding a medium free from animal derived albumin/serum having cultivation ability comparable to that of the conventional serum-containing medium. There is a continuing need in the art for cell culture media that include no risk of using with regard to transferal of diseases but provide all of the necessary nutrients and growth factors, at suitable concentrations, to optimize the growth of the cells.

Most of the effort in recent years has been to develop serum free media by supplementing the basal media with appropriate nutrients to avoid the addition of serum, without sacrificing cell viability and/or cell growth and/or protein production. Examples of such components include bovine albumin and human albumin; certain growth factors derived from natural (animal) or recombinant sources, including epidermal growth factor (EGF) or fibroblast growth factor (FGF); lipids such as fatty acids, sterols and phospholipids; lipid derivatives and complexes such as phosphoethanolamine, ethanolamine and lipoproteins; protein and steroid hormones such as insulin, insulin like growth factor (IGF), hydrocortisone and progesterone; nucleotide precursors; and certain trace elements (reviewed by Waymouth, C, in: Cell Culture Methods for Molecular and Cell Biology, Vol. 1 : Methods for Preparation of Media, Supplements, and Substrata for Serum-Free Animal Cell Culture, Barnes, D. W., et al., eds., New York: Alan R. Liss, Inc., pp. 23-68 (1984), and by Gospodarowicz, D., Id., at pp 69-86 (1984)).

However, most of the prior art listed below are still describing medias comprising animal derived components. It is known that cholesterol and cholesterol-containing fractions obtained from bovine serum are useful to promote the growth of various organisms. J. Bacterid., Vol. 135, pp. 818-827 (1978) describes the use of a cholesterol-containing bovine serum fraction in the growth of Mycoplasma pneumoniae and Mycoplasma arthritidis. J. Gen. Microbiology, Vol. 116, pp. 539-543 (1980) describes the use of USP cholesterol in the growth of Treponema hyodysenteriae. In Vitro, Vol. 17, No. 5, pp. 519-530 (1981 ) discloses that mixtures of high density lipoproteins and transferrin can be used to grow certain mammalian cells in the absence of serum.

U.S. Patent No. 4,290,774 describes the production of a specific cholesterol-rich fraction from mammalian plasma or serum by a process that involves the step of treatment with an alkaline carbonate and an alkaline earth salt. Zeit. Klin. Chem. 6(3), pp. 186-190 (1968) describes the removal of certain lipoproteins from human serum by use of colloidal silicic acid.

U.S. Patent No. 4,762,792 and European Patent No. EP0201800 disclose a process for isolating a cholesterol-rich fraction from mammalian blood plasma or serum using a silica adsorbant followed by several alkaline steps, which is useful as a growth medium ingredient, especially in cell culture.

U.S. Patent No. 5,409,840 describes an improved process for the recovery of cholesterol rich fractions from mammalian serum or plasma. The process involves adsorbing the fraction on precipitated silica gel agglomerates which are then separated from the serum or plasma whereupon the adsorbed cholesterol rich fraction is eluted from the silica and recovered. EX-CYTE® is a concentrated aqueous mixture of cholesterol, lipoproteins and fatty acids that is manufactured by Serologicals, Inc. using the process described in U.S. Patent No. 4,762,792. EX-CYTE® is typically made from bovine serum.

In 1989, Hewlett et al. (Miles Science Journal 1989,11: 9-14) described the effects of the addition of EX-CYTE® to serum-free or low serum containing culture media on the growth of several cell types, including L929 cells, CHO-K1 cells, BHK-21 cells, AHT-107 hybridoma cells, mouse myeloma cells and monkey-fibroblast cells. The cells were grown in media containing several of the following components (the components varied depending on cell type): insulin, transferrin, selenite, bovine EX-CYTE®, human EX-CYTE®, freeze-dried EX- CYTE®, 1% FBS, selenite, human serum albumin (HSA) and/or trace elements. In 1991 , Guy Hewlett (Prod. Biol. Anim. Cells Cult. (1991 ) ESACT 10 Meet., 67-69) further described the effect of the addition of EX-CYTE®, to serum-free or low serum containing culture media on the growth of several cell types, including A431 human keratinocytes, L929 mouse fibroblast cells, 3T3 cells, X63 myeloma cells, AHT hybridoma cells and HeLa cells. The cells were grown in media containing 50/50 mix of Dulbecco's modification of Eagle's medium (DMEM) and Ham's nutrient solution F12 (F12 ) and several of the following additional components (the components varied depending on cell type): bovine insulin (10 mg/L), transfrerrin (10 mg/L), EX-CYTE®, lipoprotein/lipid (30 mg cholesterol/mL), albumin (200 mg/L), selenium (100 nmol/L) and/or 0.5% Fetal Calf Serum.

Savonniere et al. (Journal of Biotechnology 48 (1996) 161-173) described the effect of lipid supplementation of culture media on cell growth, antibody production, membrane structure and dynamics in two hybridoma cell lines (B9 cells (fusion of SP2/OAg14 cells with mouse Balb/C spleen cells) and A49 cells (fusion of SP2/O myeloma cells with mouse Balb/B lymphocytes)). B9 cells were grown in RPMI 1640 medium (Seromed. Strasbourg, France) supplemented with 100 Ul/ml penicillin, 10 μg/ml streptomycin, 5% (v/v) fetal calf serum, 50 μM 2-mercaptoethanol, 100 Ul/ml of recombinant human IL-6, and Ex-Cyte. A49 cells were grown in RPMI 1640 medium supplemented with antibiotics, 0.5% (v/v) FCS, 2% Ultroser HY and EX-CYTE®. They reported that the response to the two cell lines to EX-CYTE® was different, the addition of EX-CYTE® was without effect on the B9 cells, while A49 cells showed an increased growth rate.

PCT Publication No. WO 90/07007 filed b y the United States of America discloses a serum free media for culturing animal epithelial cells, including human epithelial cells. The patent discloses a media with the following components: L-glutamine, 2mM, Insulin, 10 ug/ml, Hydrocortisone, 0.2 uM, epidermal growth factor, 5.0 ng/ml, transferrin, 10 ug/ml, phosphoethanolamine, 0.5 uM, cholera toxin, 25 ng/ml, triiodothyronine, 10 nM, retinoic acid, 10 nM, ornithine, 2 mM, CaCI₂, 0.4 mM, Glucose, 2.0 mg/ml, bovine pituitary extract, 7.5 ug/ml, EX-CYTE® V, 312 ug/ml, FeS0₄x7H₂O, 2.7 uM, ZnSO₄x7H₂O, 0.5 uM, Na₂SeO₃, 3.0 x 10^"8 M, MnCI₂x4H₂O, 1.0 nM, Na₂Si0₃x9H₂0, 5.0 x 10^"7 M, (NH₄)6 Mo₇O₂₄x4H₂0, 1.0 nM, NH₄VO₃, 5.0 nM, NiSO₄x6H₂0, 0.5 nM, SnCI₂x2H₂0,0.5 nM, and Gentamicin, 50 ug/ml. U.S. Patent No. 6,733,746 to Daley et al. and U.S. Publication No. 2004/0072349 filed by Daley et al. disclose a hematopoetic cell culture nutrient supplement. The supplement disclosed contains one or more antioxidants, one or more albumins or albumin substitutes, one or more lipid agents, one or more insulins or insulin substitutes, one or more transferrins or transferrin substitutes, one or more trace elements, and one or more glucocorticoids. The patent application specifically discloses formulations for culturing hematopoetic stem cells that contain, for example N-acetyl-L-cysteine, human serum albumin, Human EX-CYTE®, ethanolamine HC1 , zinc insulin, human iron saturated transferrin, a Se⁴⁺ salt, hydrocortisone, D,L-tocopherol acetate, 2-mercaptoefhanol and/or glutamine.

US. Patent No. 5,932,703 to ICOS Corporation describes purified and isolated nucleotide sequences encoding a human macrophage-derived chemokine (MDC) and methods for the recombinant production of the same. Transfected CHO cells were used to express MDC. The media used to culture the CHO cells contained P5 medium (which consists of various components including glutamine) containing 0.2% to 1.0% FBS, 3 g/l sodium bicarbonate, 2 ug/l sodium selenite, 1% soy bean hydrolysate, ferrous sulfate/EDTA solution, 1.45 ml/L EX- CYTE VLE solution, 10 ug/ml recombinant insulin, 0.1% pluronic F-68, 30 ug/ml glycine, 50 uM ethanolamine and 1 mM sodium pyruvate.

Gorfien et al. (Biotechnol. Prog. 2000, 16, 682-687) describe the growth of NSO hybridoma cells in a protein free, chemically defined media. The media contains CD Hybridoma Medium and lipoprotein supplements, including EX-CYTE VLE (Bayer, catalog number 81-129) at 1 :300, 1 :500, and 1 :1000 dilutions. This reference teaches that Ex-Cyte did not support the long term growth of NSO cells.

U.S. Patent Publication No. 2003/0166146 to Lee et al. describes a myeloma line usefid for manufacturing recombinant proteins in chemically defined media. The chemically defined media used to culture the myeloma cell line (C463A myeloma cell line, a spontaneous mutant cloned from a Sp2/0-Ag14 cell bank) contained IMDM, Primatone, Albumin, and Ex- Cyte.

U.S. Patent No. 5,240,848 to Monsanto Company describes a cDNA sequence for human vascular permeability factor and methods to recombinantly produce the same. U-937 cells (a human cell line established from a diffuse histiocytic lymphoma, ATCC CRL 1593) were used to produce the vascular permeability factor protein. The cells were cultured in media that contained the following components: RPMI 1640, DME (high glucose), Ham's F12 in a 1 :1 :1 ratio, HEPES (25 mM, pH 7.10-7.15) glutathione (1 mM), ethanolamine (20 uM), selenium (30 nM) or 5200 ug/ml, NaHCO₃ (2 mM), CuSO₄ (5 nM), NH₄ VOs (5 nM), ZnSO₄ (0.5 uM), MnSO₄ (0.5 nM), FeSO₄ (4 uM), bovine serum albumin, Miles "Pentex" (100 ug/ml), iron rich transferrin, Miles (5 ug/ml), bovine insulin (10 ug/ml), F-68 Pluracol(0.05% w/v) and 0.1 % Ex- Cyte.

In 2002, Serologicals, Inc. published a Technical Bulletin disclosing increased protein yield in an antihuman IgGG monoclonal antibody-producing cell line. The publication disclosed the use of Ex-Cyte (30 ug cholesterol), human serum albumin (10 mg/ml), human APO- transferrin (2.5 ug/ml), insulin (5 ug/ml) and sodium selenite (10^"7M). In accordance with the present invention, the medium includes recombinant albumin.Albumin binds and transports nutrients, metabolites and other substances. It is known that albumin has a positive effect on the culture of cells for research, diagnostic or therapeutic purposes; the culture of genetically engineered or non-genetically engineered mammalian cells, including but not limited to CHO, Sp2/0, NSO, BHK, HEK 293, Vero, PER.C6, MDCK, Namalwa and PERC.6, A431 , for the production of biopharmaceuticals, diagnostic reagents or native or recombinant proteins, or adenoviruses to be used for medical or cosmetic purposes, and culture media for the same; the culture of normal primary human cells, for example those offered commercially from Clonetics, Cascade or Cell Applications, and culture media for the same; the culture of stem cells, for example cells available from Stem Cell Technologies or patient derived samples for bone marrow transplants or myocardial infarct repair, and culture media for the same; the culture of mammalian fibroblasts with and without kerotinocytes, for example Dermagraft © from Smith+Nephew, and the culture media for the same; the culture and expansion of mammalian tissue for implant and lesion repair, for example autologous chondrocyte implantation or myocyte implantation, and culture media for the same; (Yamane I. 1978. Development and application of a serum-free culture medium for primary culture. In H. Katsuta (ed), Nutritional Requirements of Cultured Cells. Baltimore, University Park Press, pp 1-21 ; US patent 5,021 ,349; Iscove NN, Melchers F (1978). Complete replacement of serum by albumin, transferrin and soybean lipid in cultures of lipopolysaccharide-reactive B lymphocytes. J Exp Med 147: 928-33; US patent 5,198,349; US patent 5,250,421 ; Berntorp E (1997). Thrombosis Haemostasis 78: 256-60; McGrew JT, Richards CL, Smidt P, Dell B and Price V. 1998. Lipid requirements of a recombinant Chineese Hamster Ovary Cell Line (CHO), ibidem, pp 205-207; Yamane I. 1978. Role of bovine albumin in a serum-free culture medium and its application. Natl Cancer Inst Monogr 48:131-133; Sato JD, Kawamoto T, McClure DB and Sato GH. 1984. Cholesterol requirement of NS-1 mouse myeloma cells for growth in serum-free medium. MoI Biol Med 2(2):121-134; Kovar J. Hybridoma cultivation in defined serum-free media: growth-supporting substances. IV. Lipids and serum albumin. Folia Biol (Praha). 1987, 33(6):377-84; Jaeger, V, Lehmann J, Friedl P. Serum-free growth medium for the cultivation of a wide spectrum of mammalian cells in stirred bioreactors. Cytotechnology 1988, 1 :319-29; Glassy CM, Tharakan JP, Chau, PC. Serum-free media in hybridoma culture and monoclonal antibody production. Biotech Bioeng 1988, 32:1015-28). The use of insulin and insulin-like growth factor in cell culture media has been described. (Yamaguchi et al, 1992, Biosci. Biotechnol. Biochem. 56: 600-604); (Morris & Schmid, 2000, Biotechnol. Prog, 16, 693-697); (Yandall et al., 2004, An Analogue of IGF-I: A Potent Substitute for Insulin in Serum-Free Manufacture of Biologies by CHO Cells BioProcess International, 2(3), 56-58, 60, 62, 64, 2004); WO Patent 2006/047380. It is an object of this invention to provide culture media that supports the growth of various cell types.

Another object of this invention is to provide culture media that are free of animal-derived serum or animal derived components to support the growth of various cell types. A further object of the present invention is to provide cell culture media for use in either small-scale culture or large-scale commercial production of cells.

A still further object of this invention is to provide a cell culture medium that increases the yield of biological materials, for example, peptides, produced by the cells cultured in such media.

Another object of this invention is to provide a method of culturing cells in a suitable medium to allow cell growth.

A further object of this invention is to provide a method of culturing cells in a suitable medium for the production of biological materials of interest. A still further object of the present invention is to provide methods to make cell culture media.

SUMMARY OF THE INVENTION

The invention provides novel cell culture media compositions that comprise recombinant albumin that eliminates the use of animal derived serum. The invention also includes methods of culturing cells using the cell culture media compositions. The compositions and or methods are useful in the culture of a variety of cell types, including, for example, hybridoma cells and/or cancer cells.

In a first embodiment, the cell culture composition comprises (i) basal media; and (ii) recombinant albumin.

The production of recombinant albumin is known in the art and numerous hosts such as

Escherichia coli (EP 73,646), yeast has been reported in WO 00/44772, EP 0683233 A2, and

US 5,612,196, and Bacillus subtillis (Saunders et al, 1987, J. Bacterid, 169, 2917-2925),

Aspergillus. Production of albumin has been demonstrated in transgenic plants such as but not limited to tobacco, rice, and maize and in transgenic animals such as but not limited to chicken and bovine.

In a second embodiment of the present invention the composition comprises (i) basal media;

(ii) recombinant albumin; and one or more components selected from the group consisting of insulin, sodium selenite, glutamine, transferrin, peptone, ethanolamine, fetuin, vitamins, lipoprotein, fatty acids and amino acids.

In one embodiment of the present invention the composition comprises between 0.0001-

10%, 0.0005-7.5%, 0.001-5.0%, most particularly between 0.05-3.0% recombinant albumin.

In one embodiment of the present invention the composition comprises between 0.01-1000 mg/L, more particular between 0.01-500 mg/L, even more particular between 0.1-100 mg/L, such as 1-50 mg/L and most particular between 4-20 mg/L insulin. In one embodiment of the present invention the composition comprises between 0.0001-10 mg/L, more particular between 0.005-7.5 mg/L, even more particular between 0.1-5.0 mg/L and most particular between 0.75-3.5 mg/L lipoprotein.

In another embodiment, the composition comprises (i) basal media; (ii) recombinant albumin; (iii) insulin; (iv) sodium selenite; and/or (v) transferrin.

In one embodiment, the composition comprises at least 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 or 9 mg/ml albumin; at least 3.0, 3.5,4.0,4.5, 5.0, 5.5, 6.0, 7.0, 10, 15 or 20 ug/ml transferrin; approximately 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5, 11 , 11.5, 12, 15 or 20 ug/ml insulin; at least 1 , 2, 3,2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5, 6.7, 7.0, 7.5, 8.0, 9.0, 10, 15 or 20 ug/L sodium selenite.

In one specific embodiment, the cell culture media comprises approximately 4 mg/ml recombinant albumin; approximately 5.5 ug/ml transferrin; approximately 10 ug/ml insulin; approximately 6.7, ug/L sodium selenite in basal media. In another embodiment, the composition comprises (i) basal media; (ii) recombinant albumin (iii) glutamine; (iv) insulin; (v) transferrin; and/or (vi) ethanolamine. In one embodiment, the composition comprises approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM glutamine; approximately 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 ,2, 3,4, 5, 6,7, or 8% recombinant albumin; approximately 1 , 2, 3,4, 5, 6, 7, 8, 9, 9.5, 10, 10.5, 11 , 1 1.5, 12, 13, 14, 15, 16, 17, 18, 19,20 mg/L insulin; approximately 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 1 , 1.5, 2, 2.5, 3,4, 5, 6, or 7 mg/L transferrin; approximately 1 ,2, 3, 4, 5, 6, 7, 8, 8.5, 9, 9.5, 10, 10.5, 1 1 , 1 1.5, 12, 13, 14, 15, 16, 17, 18, 19 or 20 μM. ethanolamine in basal media. In one specific embodiment, the invention comprises approximately 4mM glutamine; approximately 0.5% recombinant albumin; approximately 10 mg/L insulin; approximately 1 mg/L transferrin; and/or approximately 10 molar ethanolamine, in basal media. In another embodiment, the composition may include (i) basal media; (ii) recombinant albumin; and one or more of the following components selected from the group consisting of (iii) glutamine; (iv) insulin; and (v) transferrin. In one embodiment, the composition comprises approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM glutamine; approximately 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1 , 1.5,2,2.5, 3, 3.5, 3.5 to 5, 5 to 10, 10 to 20% albumin; approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, 9.5, 10, 10.5, 1 1 , 1 1.5, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/L insulin; and or approximately 0.1 , 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 0.95, 1 , 1.5, 2, 2.5, 3, 4, 5, 6, or 7 mg/L transferrin. In one specific embodiment, the composition comprises approximately 4mM glutamine, approximately approximately 1 % albumin, approximately 10 mg/L insulin, and/or approximately 1 mg/L transferrin in basal media. In another embodiment, the composition may comprise (i) basal media; (ii) glutamine; (iii) recombinant albumin; (iv) insulin; and/or (v) transferrin; and/or (vii) peptone. In one embodiment, the composition comprises approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM glutamine; approximately 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.5, 2, 2.5, 3, 3.5, 3.5 to 5, 5 to 10, 10 to 20% albumin; approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, 9.5, 10, 10.5, 1 1 , 11.5, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/L insulin; approximately 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 1 , 1.5, 2, 2.5, 3, 4, 5, 6, or 7 mg/L transferrin; and/or approximately 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 , 0.2, 0.3, 0.4, 0.5, 1 , 2 or 3 % peptone. In one specific embodiment, the composition may comprise approximately 4mM glutamine, approximately 1% recombinant albumin, approximately 10 mg/L insulin, approximately 1 mg/L transferrin, and/or approximately 0.1 % peptone in basal media. In embodiments of the present invention, the peptone or peptone mixture is a protein hydrolysate, which is obtained from hydrolyzed animal or plant protein. The peptones can be derived from animal by-products from slaughter houses, purified gelatin, or plant material. The protein from the animal or plant sources can be hydrolyzed using acid, heat or various enzyme preparations. Peptone mixtures that can be used include spy peptone, "Primatone RL" and/or "Primatone HS", both of which are commercially available (Sheffield, England or; Quest International (IPL5X59051 ), PRIMATONE® RL). In another embodiment, the composition may comprise (i) basal media; (ii) glutamine; (iii) albumin; (iv) insulin; (v) transferrin; and/or (vi) fetuin (such as Pedersen). In one embodiment, the composition may comprise approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM glutamine; approximately 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.5, 2, 2.5, 3, 3.5, 3.5 to 5, 5 to 10, 10 to 20% albumin; approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, 9.5, 10, 10.5, 11 , 11.5, 12, 13, 14, 15,16, 17, 18, 19, or 20 mg/L insulin; approximately 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 1 , 1.5, 2, 2.5, 3, 4, 5, 6, or 7 mg/L transferrin; and/or approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 10.5, 1 1 , 1 1.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 16, 17, 18, 19, 20 μg/ml of fetuin. In one specific embodiment, the composition of the present invention may comprise approximately 4mM glutamine, approximately 1% albumin, approximately 10 mg/L insulin, approximately 1 mg/L transferrin, and/or approximately 12.5 μg/ml fetuin (such as Pedersens) in basal media. In another embodiment, the composition may comprise (i) basal media; (ii) albumin (iii) glutamine; (vi) insulin; (v) transferrin; and/or (vi) vitamin E. In one embodiment, the composition may comprise approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM glutamine; approximately 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.5, 2, 2.5, 3, 3.5, 3.5 to 5, 5 to 10, 10 to 20% albumin; approximately 1 , 2, 3,4, 5, 6, 7, 8, 9, 9.5, 10, 10.5, 1 1 , 1 1.5, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/L insulin; approximately 0.1 , 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 0.95, 1 , 1.5, 2, 2.5, 3, 4, 5, 6, or 7 mg/L transferrin; and/or approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 micromolar vitamin E. In one specific embodiment, the composition of the present invention may comprise approximately 4mM glutamine, approximately 1 % recombinant albumin, approximately 10 mg/L insulin, approximately 1 mg/L transferrin, and/or approximately 5 μM vitamin E in basal media. In a preferred embodiment of the invention, the composition may comprise approximately

4mM glutamine, approximately 1 % recombinant albumin, approximately 10 mg/L insulin, approximately 1 mg/L transferrin, approximately 0.1 % peptone, approximately 12.5 μg/mL fetuin (such as Pederson), and/or approximately 5 μM vitamin E.

In a further embodiment, the composition may comprise any media listed in table 1 in WO 2005/070120 hereby incorporated by reference. In another embodiment, the serum free media is either Hybridoma Media, animal component free or Ex-Cell (JRH Biosceinces, Inc.). In another aspect of the present invention compositions are provided that are useful as a cell culture medium that serves to increase the yield of biological products, such as proteins, produced by the cells cultured in the media. In one embodiment, compositions can increase the yield of biological products at least 25%, 30%, 50%, 100%, 200% or 300%. In another embodiment, the biological products produced can be a peptide, such as a therapeutic or diagnostic peptide, polypeptide, protein, monoclonal antibody, immunoglobulin, cytokine (such as interferon), integrin, antigen, growth factor, cell cycle protein, hormone, neurotransmitter, receptor, fusion peptide, blood protein and/or chimeric protein. In a further aspect of the present invention, compositions are provided that are useful as a cell culture medium for a variety of cells. In one embodiment, the cell culture media of the present invention can be used for adherent cell culture. In another embodiment, the cell culture media described herein can be used for suspension cell culture. In other embodiments, the cell culture media described herein can. be used as culture media for hybridoma cells, monoclonal antibody producing cells, virus-producing cells, transfected cells, cancer cells and/or recombinant peptide producing cells- In one embodiment, the compositions may be used to culture eukaryotic cells, such as plant and/or animal cells. The cells may be mammalian cells, fish cells, insect cells, amphibian cells or avian cells. Other types of cells can be selected from the group consisting of MK2.7 cells (ATCC Catalogue No. CRL1909, an anti-murine-VCAM IgGI expressing hybridoma cell), HEK 293 cells, PER-C6 cells, CHO cells, COS cells, 5L8 hybridoma cells, Daudi cells, EL4 cells, HeLa cells, HL-60 cells, K562 cells, Jurkat cells, THP-1 cells, Sp2/0 cells; and/or the hybridoma cells listed in WO 2005/070120, table Il hereby incorporated by reference or any other cell type disclosed herein or known to one skilled in the art. Basal media may comprise, but are not limited to Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, .alpha-modified Minimal Essential Medium (.alpha. MEM), Glasgow's Minimal Essential Medium (G-MEM), and/or Iscove's Modified Dulbecco's Medium. The present invention also provides a method of cultivating eukaryotic cells including contacting the cells with the compositions that are useful as cell culture medium of the present invention and/or maintaining the cells under conditions suitable to support cultivation of the cells in culture. In a particular embodiment, the cells are cancer cells or hybridoma cells. In other embodiments, methods of cultivating tissue explants are cultures are provided including contacting the tissues with the cell culture media compositions described herein. In one embodiment, the method includes contacting hybridoma cells with a composition including: (i) basal media; (ii) recombinant albumin; (iii) glutamine; (iv) insulin; (v) transferrin; and/or (vi) ethanolamine, and/or maintaining the hybridoma cells under conditions suitable to support cultivation of the hybridoma cells in culture. In a specific embodiment, the method includes contacting hybridoma cells with a composition including (i) basal media; (ii) approximately 0.5% recombinant albumin; (iii) approximately 4mM glutamine; (iv) approximately 10 mg/L insulin; (v) approximately 1 mg/L transferrin; (vi) approximately 1 0 μM ethanolamine. In another embodiment, the present invention is a method of cultivating cancer cells by contacting the cells with compositions that are useful as cell culture medium of the present invention and/or maintaining the cancer cells under conditions suitable to support cultivation of the cancer cells in culture. In a specific embodiment, the method includes contacting cancer cells with a composition including (i) basal media; (ii) recombinant albumin; and one or more of the following components selected from the group consisting of (iii) glutamine; (iv) insulin; and (v) transferrin.

In a specific embodiment, the method includes contacting cancer cells with a composition including (i) basal media; (ii) recombinant albumin; and one or more of the following components selected from the group consisting of (iii) glutamine; (iv) insulin; (v) transferrin and (vi) peptone. In a further embodiment, the method includes contacting cancer cells with a composition including (i) basal media; (ii) recombinant albumin; and one or more of the following components selected from the group consisting of (iii) glutamine; (iv) insulin; (v) transferrin and (vi) fetuin. In a specific embodiment, fetuin protein can be Pedersen's fetuin. In another embodiment, the method involves contacting cancer cells with a composition including (i) basal media; (ii) recombinant albumin; (iii) glutamine; (vi) insulin; (v) transferrin; and/or (vi) vitamin E.

In a specific embodiment, the method of the present invention involves contacting cancer cells with a composition including (i) basal media; (ii) recombinant albumin and one or more of the following components selected from the group consisting of (iii) glutamine; (iv) insulin; (v) transferrin; (vi) peptone and (vii) fetuin (such as Pedersens).

In yet another embodiment, the method of the present invention involves contacting cancer cells with a composition including (i) basal media; (ii) recombinant albumin; (iii) glutamine; (iv) insulin; (v) transferrin; (vi) fetuin; and/or (vii) vitamin E. In a specific embodiment, the method of the present invention involves contacting cancer cells with a composition including (i) basal media; (ii) recombinant albumin; (iii) glutamine; (iv) insulin; (v) transferrin; (vi) peptone; (vii) fetuin (such as Pedersens); and/or (vii) vitamin E. In a particular embodiment, the method of the present invention involves contacting cancer cells with a composition including (i) basal media; (ii) recombinant albumin; (iii) approximately 4mM glutamine; (iv) approximately 10 mg/L insulin; (v) approximately 1 mg/L transferrin; (vi) approximately 0.1 % peptone; (vii) approximately 12.5 μg/ml fetuin (such as Pedersens); and/or (vii) approximately 5 μM vitamin E.

The present invention also provides a kit for the cultivation of cells in vitro, the kit comprising the compositions of the present invention. In another embodiment, the kit can contain compositions of the present invention in combination with specific cell lines.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Shows the effect of recombinant albumin (rHSA) on the growth performance of the CHO cell line by adding the following concentrations of recombinant albumin: 0.00mg/ml, 0.03mg/ml, 0.3mg/ml, 1 mg/ml, 3mg/ml and 5mg/ml.

Figure 2: Shows the effect of recombinant albumin (rHSA) on percent dead cells of CHO cells by adding the following concentrations of recombinant albumin: Oug/ml, 20ug/ml, 200ug/ml and 2mg/ml.

Figure 3: Shows the effect of recombinant albumin (rHSA) on cell growth of the CHO-K1 cell line in a feed batch process by adding the following concentrations of recombinant albumin: Oug/ml, 20ug/ml, 100ug/ml, 500ug/ml, 1 mg/ml and 5mg/ml.

Figure 4: Shows the effect of recombinant albumin (rHSA) on cell growth of the SP2/0 cell line. No addition of albumin was compared to addition of bovine serum albumin (Probumin) and recombinant albumin at a concentration of 0.5g/L.

Figure 5: Shows the effect of recombinant albumin (rHSA) on cell growth of the SP2/0 cell line. No addition of albumin was compared to addition of bovine serum albumin (Probumin) and recombinant albumin at a concentration of 2g/L.

Figure 6: Compares the ability of recombinant albumin (rHSA) at stimulating CHO-K1 cell growth and viability with bovine serum albumin (Probumin).

DETAILED DESCRIPTION

Cultured cells are widely used in the biopharmaceutical industry for the production of biopharmaceuticals, such as vaccines, proteins, peptides and monoclonal antibodies. The invention is a serum free recombinant albumin comprising composition which is useful as a cell culture medium.

The invention provides novel cell culture media compositions that include recombinant albumin to eliminate the use of animal derived albumin. The invention also includes methods of culturing cells using the cell culture media compositions. The compositions and/or methods are useful in the culture of a variety of cell types, including, for example, hybridoma cells and/or cancer cells.

Besides recombinant albumin and basal media, the composition may comprise but are not limited to:

• Water.

• Osmolality regulators such as salts including NaCI, KCI, KNO3. It is advantageous to have the osmolality range in the 150-450 milli-Osmols (mOsm) range, particular in the 200-400 mOsm range and more particular 290-350 mOsm. • Buffers to maintain pH such as carbonates such as NAHCO3; also chlorides, sulphates and phosphates such as CaCI₂x2H₂O, MgSO₄x7H₂O, NaHPO₄x2H₂O, or sodium pyruvate or HEPES, or MOPS. Buffers maintain the pH of the medium at pH 6-8, particular pH 6.5-7.5 and most particular 7.

• Energy sources including glucose, mannose, fructose, galactose and maltose. The energy source is present in an amount 250-20.000 mg/liter, particular 500-15.000 mg/liter, and most particular, 1000-10.000 mg/liter.

• Non-ferrous metal ions such as magnesium, copper and zink, sodium, potassium and selenium. These are often added as salts such as chlorides and sulphates. Selenium particularly in the form of sodium selenite, Na₂SeOs and in a range between 0.004- 0.08 mg/liter, particularly 0.009-0.02 mg/liter.

• Vitamins and enzyme co-factor vitamins (co-factors) include Vitamin B6 (pyridoxine), Vitamin B12 (cyanocobalamin), Vitamin K (biotin) present in an amount 0.005-0.75 mg/liter, particular 0.01 -0.5 mg/liter. Vitamin C (ascorbic acid) present in an amount 5- 45 mg/liter, particular 10-30 mg/liter. Vitamin B2 (riboflavin) present in amount 0.05-2 mg/liter, particularly 0.1-1 mg/liter. Vitamin B1 (thiamine), nicotinamide, Vitamin B5 (D calcium pentothenate, folic acid, i-insositol present in an amount 0.05-16 mg/liter, particular 0.1-12 mg/liter, more particular 0.2-8 mg/liter. Vitamin E present in an amount 0.1-20 μM, particular 1-10 μM, more particular 2.5-7.5 μM.

• Lipids and fatty acids, and lipid factors such as choline chloride, lipoic acid, oleic acid, myrisic acid, palmitic acid, stearic acid, Tween® 80, Pluronic® F-68, phosphatidylcholine or methyl lineoleate or arachadonic acid. The amount present in the range 0.00075-30 mg/liter, particular 0.02-20 mg/lier, more particular 0.05-10 mg /liter.

• Lipoproteins such as any lipophilic compound that can be, for example, carried through the plasma by apolipoproteins, including but not limited to cholesteryl esters, unesterified cholesterol, cholesterol, triglycerides, fatty acids and/or phospholipids,

EX-CYTE® . The amount present in the range 0.0001-10%, particular 0.01-7.5% more particular, 0.1-5%, even more particular 1-3%.

• Compounds involved in lipid production for example alcoholamines such as ethanolamine. These may be present in an amount 1-30 μM, particular 3-20 μM, more particular 5-15 μM. Alternatively, the ethanolamine may be present in an amount of 0.01-1000 mg/L.

• Amino acids, such as but not limited to glutamine. They may be present in an amount in the range of 0.01-1000mg/L or 1-300 mg/liter, in particular 10-200 mg/liter. L- glutamine is often present in higher concentrations in the range 250-750 mg/ml, particular 400-600 mg/ml.

• It may be advantageous to include a pH indicator for example Phenol red sodium salt at a range present 2.5-75 mg/liter.

• Antibiotics such as polymyxin, neomycin, penicillin or streptomycin in an amount at a range from 2500-150,000 ul/liter. • Growth factors, hormones including peptide hormones such as but not limited to insulin in an amount 0.1-50 mg/liter, in particular 1-25 mg/ml, more in particular 4-20 mg/ml. Epidermal growth factor (EGF) in a range from 0.01-200 μg/liter, more particular 0.1-100 μg/liter. Platelet-derived growth factor (PDGF), thyroxtne T₃, thrombin, interleukins such as IL2 and IL6, progesterone, testosterone, hydrocortisone.

• Chelating agents, ion transporter and iron sources, radical scavengers such as EDTA, L-Ascorbic acid, α-Tocopherol, transferrin present in a range of 0.001-1000 mg/L, in particular 0.1-30 mg/liter, more in particular 0.25-15 mg/liter, even more in particular 0.5-10 mg/liter. Lactoferrin, chelated iron salts such as ferric citrate or ferric ammonium citrate.

• Protein or peptide digests, hydrolysates or extracts.

• Glycoproteins including fetuin.

• Putresscine, including as a salt such as HCI, which is known to play a role in maintaining the structure of the endoplasmic reticulum. • Cell protectants such as polyethylene glycol, polyvinyl alcohol or pluronic polyols

• Hypoxanthine, thymidine, methotrexate for selection pressure regulation.

• Antioxidants The components of the medium are mostly inorganic, synthetic, recombinant and as such not obtained from any animal or human source. Some components may be obtained from a plant, algae, fungal, bacterial or a yeast source. Recombinant components are prepared under highly pure conditions to minimize the risk of contamination from the parent tissue passing to the cells used to produce the components. Further purification steps may be employed to remove cell proteins. Thus, a medium which is essentially free from all protein, lipid and carbohydrate isolated from an animal source, can be achieved. The preferred culture medium of the invention contains no protein, lipid and carbohydrate isolated from an animal or human source.

In another embodiment, the composition comprises (i) basal media; (ii) 0.0001-10% recombinant albumin; and one or more components selected from the group consisting of (iii) insulin, (iv) glutamine, (v) transferrin, (vi) ethanolamine, (vii) fetuin, (viii) vitamins, (ix) lipoprotein, (x) fatty acids, (xi) amino acids, (xii) sodium selenite, (xiii) peptone. In a further embodiment, the composition comprises (i) basal media; (ii) recombinant albumin (iii) glutamine; (iv) insulin; (v) transferrin; and/or (vi) ethanolamine. In another embodiment, the composition comprises (i) basal media; (ii) recombinant albumin; (iii) glutamine; (iv) insulin; and/or (v)transferrin. In a further embodiment, the composition comprises (i) basal media; (ii) recombinant albumin; (iii) glutamine; (iv) insulin; and/or (v) transferrin; and/or (vi) peptone. In another embodiment, the composition comprises (i) basal media; (ii) recombinant albumin; (iii) glutamine; (iv) insulin; (v) transferrin; and/or (vi) fetuin (such as Pedersen). In another embodiment, the composition comprises (i) basal media; (ii) recombinant albumin; and one or more of the following components selected from the group consisting of (iii) glutamine; (iv) insulin; (v) transferrin and (vi) vitamin E.

In another aspect of the present invention compositions are provided that are useful as a cell culture medium that serves to increase the yield of biological products, such as proteins, produced by the cells cultured in the media. In a further aspect of the present invention, compositions are provided that are useful as a cell culture medium for a variety of cells. In one embodiment, the cell culture media of the present invention can be used for adherent cell culture. In another embodiment, the cell culture media described herein can be used for suspension cell culture. In one embodiment, the method includes contacting hybridoma cells with a composition including: (i) basal media; (ii) 0.0001-10% recombinant albumin; and one or more components selected from the group consisting of (iii) insulin, (iv) glutamine, (v) transferrin, (vi) ethanolamine, (vii) fetuin, (viii) vitamins, (ix) lipoprotein, (x) fatty acids, (xi) amino acids, (xii) sodium selenite, (xiii) peptone and/or maintaining the hybridoma cells under conditions suitable to support cultivation of the hybridoma cells in culture.

In a further embodiment, the method includes contacting hybridoma cells with a composition including: (i) basal media; (ii) recombinant albumin; (iii) glutamine; (iv) insulin; (v) transferrin; and/or (vi) ethanolamine, and/or maintaining the hybridoma cells under conditions suitable to support cultivation of the hybridoma cells in culture.

In another embodiment, the present invention is a method of cultivating cancer cells by contacting the cells with compositions that are useful as cell culture medium of the present invention and/or maintaining the cancer cells under conditions suitable to support cultivation of the cancer cells in culture. In a specific embodiment, the method includes contacting cancer cells with a composition including (i) basal media; (ii) 0.0001-10% recombinant albumin; and one or more components selected from the group consisting of (iii) insulin, (iv) glutamine, (v) transferrin, (vi) ethanolamine, (vii) fetuin, (viii) vitamins, (ix) lipoprotein, (x) fatty acids, (xi) amino acids, (xii) sodium selenite, (xiii) peptone.

In a further embodiment, the method includes contacting cancer cells with a composition including (i) basal media; (ii) recombinant albumin; and one or more of the following components selected from the group consisting of (iii) glutamine; (iv) insulin; and (v) transferrin.

Definitions

The terms "cell culture medium," "culture medium" and "medium formulation" refer to a nutritive solution for culturing or growing cells.

A "serum-free" medium is a medium that contains no serum (e.g., fetal bovine serum (FBS), horse serum, goat serum, or any other animal-derived serum known to one skilled in the art). The term "basal medium" refers to any medium which is capable of supporting growth of cells. The basal medium supplies standard inorganic salts, such as zinc, iron, magnesium, calcium and potassium, as well as trace elements, vitamins, an energy source, a buffer system, and essential amino acids. Suitable basal media include, but are not limited to Dulbecco's Modified Eagle's Medium (DMEM), DME/F12, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, .alpha. Minimal Essential Medium (.alpha. MEM), Glasgow's Minimal Essential Medium (G-MEM), and Iscove's Modified Dulbecco's Medium. As used herein the term "albumin" means a polypeptide compound having the biologic function of albumin.

The term "protein yield" refers to the amount of protein expressed by cultured cells, and can be measured, for example, in terms of grams of protein produced ml medium. If the protein is not secreted by the cells, the protein can be isolated from the interior of the cells by methods known to those of ordinary skill in the art. If the protein is secreted by the cells, the protein can be isolated from the culture medium by methods known to those of ordinary skill in the art. The amount of protein expressed by the cell can readily be determined by those of ordinary skill in the art. The protein can be a recombinant protein. The term "suspension culture" refers to cells in culture in which the majority or all of cells in culture are present in suspension, and the minority or none of the cells in the culture vessel are attached to the vessel surface or to another surface within the vessel (adherent cells). The "suspension culture" can have greater than about 50%, 60%, 65%, 75%, 85%, or 95% of the cells in suspension, not attached to a surface on or in the culture vessel. The term "adherent culture" refers to cells in culture in which the majority or all of cells in culture are present attached to the vessel surface or to another surface within the vessel, and the minority or none of the cells in the culture vessel are in suspension. The "adherent culture" can have greater than 50%, 60%, 65%, 75%, 85%, or 95% of the cells adherent.

As used herein, the term "mammal" is meant to include any human or non-human mammal, including but not limited to porcine, ovine, bovine, rodents, ungulates, pigs, sheep, lambs, goats, cattle, deer, mules, horses, monkeys, dogs, cats, rats, and mice.

I. Cell Culture Media The invention provides novel cell culture media compositions and methods to cultivate cells with the novel media. In general, cell culture media contains a base solution or "basal media" into which all of the desired components are added.

Basal media which can be used in the present invention include but are not limited to Iscove's Modified Dulbecco's Medium, RPMI 1640, Minimal Essential Medium-alpha. (MEM- alpha), Dulbecco's Modification of Eagle's Medium (DMEM), DME/F12, alpha MEM, Basal Medium Eagle with Earle's BSS, DMEM high Glucose, with L-Glutamine, DMEM high glucose, without L-Glutamine, DMEM low Glucose, without L-Glutamine, DMEM:FI2 1 :1 , with L-Glutamine, GMEM (Glasgow's MEM). GMEM with L-glutamine, Grace's Complete Insect Medium, Grace's Insect Medium, without FBS, Ham's F-10, with L-Glutamine, Ham's F-12, with L-Glutamine, IMDM with HEPES and L-Glutamine, IMDM with HEPES and without L- Glutamine, IPL-41 Insect Medium, L-15 (Leibovitz)(2X), without L-Glutamine or Phenol Red, L-15 (Leibovitz), without L-Glutamine, McCoy's 5A Modified Medium, Medium 199, MEM Eagle, without L- Glutamine or Phenol Red (2X), MEM Eagle-Earle's BSS, with L-glutamine, MEM Eagle-Earle's BSS, without L-Glutamine, MEM Eagle-Hanks BSS, without L-Glutamine, NCTC-109, with L-Glutamine, Richter's CM Medium, with L-Glutamine, RPMI 1640 with HEPES, L-Glutamine and/or Penicillin-Streptomycin, RPMI 1640, with L-Glutamine, RPMI 1640, without L-Glutamine, Schneider's Insect Medium or any other media known to one skilled in the art. The compositions of the present invention may be used to culture a variety of cells. In one embodiment, the medium is used to culture eukaryotic cells such as plant and/or animal cells. The cells can be mammalian cells, fish cells, insect cells, amphibian cells or avian cells. The medium can be used to culture cells selected from the group consisting of MK2.7 cells, PER-C6 cells, NSO, GS-NSO, CHO cells, HEK 293 cells, COS cells and Sp2/0 cells. MK2.7 (ATCC Catalogue Number CRL 1909) is an anti-murine VCAM IgGI expressing Hybridoma cell line derived from the fusion of a rat splenocyte and a mouse Sp2/0 myeloma. MK2.7 is a non-adherent cell line that can be grown in serum-free media. Other types of cells can be selected from the group consisting of 5L8 hybridoma cells, Daudi cells, EL4 cells, HeLa cells, HL-60 cells, K562 cells, Jurkat cells, THP-1 cells, Sp2/0 cells; and/or the hybridoma cells listed in table 2, WO 2005/070120 which is hereby incorporated by reference or any other cell type disclosed herein or known to one skilled in the art. Additional mammalian cell types can include, but are not limited to, including primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, kidney epithelial cells and retinal epithelial cells) and established cell lines and their strains (e.g., 293 embryonic kidney cells, BHK cells, HeLa cervical epithelial cells and PER-C6 retinal cells, MDBK (NBL-1 ) cells, 91 1 cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS 180 cells, LS 174T cells, NCI-H-548 cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-I cells, LLC-PK.sub.2 cells, Clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK.sub.1 cells, PK(15) cells, GH. _λ cells, GH₃ cells, L2 cells, LLC-RC 256 cells, MH.sub.ICi cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, or derivatives thereof), fibroblast cells from any tissue or organ (including but not limited to heart, liver, kidney, colon, intestines, esophagus, stomach, neural tissue (brain, spinal cord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow, and blood), spleen, and fibroblast and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-2 1 cells, citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl.sub.1 cells, CHO cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB cells, BLO-1 1 cells, NOR-10 cells, C₃H/IOTI/2 cells, HSDM.sub.IC3 cells, KLN205 cells, McCoy cells, Mouse L cells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L) cells, L-MTK (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3 cells, Indian muntjac cells, SIRC cells, C_N cells, and Jensen cells, or derivatives thereof). The medium disclosed herein can be used to culture cells in suspension or adherent cells.

The compositions of the present invention are suitable for either adherent, monolayer or suspension culture, transfection, and cultivation of cells, and for expression of proteins or antibodies in cells in monolayer or suspension culture.

Cells supported by the medium of the present invention can be derived from any animal, such as a mouse or a human. The cells cultivated in the present media can be normal cells or abnormal cells (i.e., transformed cells, established cells, or cells derived from diseased tissue samples).

Cell culture can be performed using various culture devices, for example, a fermentor type tank culture device, an air lift type culture device, a culture flask type culture device, a spinner flask type culture device, a microcarrier type culture device, a fluidized bed type culture device, a hollow fiber type culture device, a roller bottle type culture device, a packed bed type culture device or any other suitable devise known to one skilled in the art.

In another aspect of the present invention compositions are provided that are useful as a cell culture medium that serves to increase the yield of biological products, such as proteins, produced by the cells cultured in the media. In one embodiment, compositions can increase the yield of biological products at least 25%, 30%, 50%, 100%, 200% or 300%. In another embodiment, the biological products produced can be a peptide, such as a therapeutic or diagnostic peptide, polypeptide, protein, monoclonal antibody, immunoglobulin, cytokine (such as interferon, for example, interferon alpha, beta or gamma), integrin, antigen, growth factor, cell cycle protein, hormone, neurotransmitter, receptor, fusion peptide, blood protein, blood clotting factor and/ or chimeric protein. The biological product can also be an IgG, IgM, IgE, IgA immunoglobulin, a single chain antibody or fragment thereof, such as a sFv fragment, a linked antibody fragment, and/or a humanized antibody.

II. Compositions to Replace Serum

The biochemical complexity of serum, such as FBS, can potentially complicate the downstream processing of the proteins of interests. The present invention also provides a unique formulation of complete media without the use of serum.

According to this aspect of the invention, the composition comprises (i) basal media; (ii) 0.0001-10% recombinant albumin; and one or more components selected from the group consisting of (iii) insulin, (iv) glutamine, (v) transferrin, (vi) ethanolamine, (vii) fetuin, (viii) vitamins, (ix) lipoprotein, (x) fatty acids, (xi) amino acids, (xii) sodium selenite, (xiii) peptone.

The composition may comprise any amount of recombinant albumin that achieves the desired effect, including but not limited to approximately 2, 2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 or 9 mg/ml recombinant albumin. In one embodiment, the composition may comprise 1 to 3, 1 to 5, 2 to 4, 2 to 7, 3 to 6, 5 to 9, 5 to 8, or 2 to 8 mg/ml recombinant albumin. In accordance with the present invention, the medium includes recombinant albumin. The production of recombinant albumin is known in the art and numerous hosts such as Escherichia coli (EP 73,646), yeast has been reported in WO 00/44772, EP 0683233 A2, and US 5,612,196, and Bacillus subtillis (Saunders et al, 1987, J. Bacterid, 169, 2917-2925), Aspergillus.

Production of albumin has been demonstrated in transgenic plants such as but not limited to tobacco, rice, and maize and in transgenic animals such as but not limited to chicken and bovine

In a specific embodiment, the composition can include 3.5 to 5.0 mg/ml, specifically, 4 mg/ml, recombinant albumin.

The composition may also comprise any amount of transferrin that achieves the desired effect, including but not limited to approximately 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0,10, 15 or 20 ug/ml transferrin. In one embodiment, the composition may comprise 2.5 to 3.0, 3.0 to 4.0, 3.0 to 5.0, 3.0 to 6.0, 4.0 to 8.0, or 6.0 to 10.0 ug/ml transferrin. In a specific embodiment, the composition may comprise 2 to 4 mg/ml, specifically 2.5 mg/ml transferrin. A transferrin substitute can also be used. A "transferrin substitute" refers to any compound which can replace transferrin and provides substantially similar results as transferrin. Examples of transferrin substitutes include but are not limited to any iron chelate compound, such as including, but not limited to, iron chelates of ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(beta-aminoethyl ether)- N,N,N',N'-tetraacetic acid (EGTA), desferoxamine mesylate, dimercaptopropanol, diethylenetriamine-pentaacetic acid (DPTA), and trans- 1 ,2- diaminocyclohexane-N,N,N',N'-tetraacetic adic (CDTA), as well as a ferric citrate chelate and a ferrous sulfate chelate. The transferrin can be iron saturated transferring, such as human transferrin. The transferrin can be a cell culture grade transferrin, such as that available from Serologicals, Inc.

The composition may also comprise any amount of insulin that achieves the desired effect, including but not limited to approximately 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5, 1 1 , 11.5, 12, 15 or 20 ug/ml insulin. In one embodiment, the composition may comprise 5 to 7, 5.5 to 6,7 to 10,9 to 1 1 ,8 to 12 or 10 to 15 ug/ml insulin.

An insulin substitute can also be used. The term "insulin substitute" may be any zinc containing compound which may be used in place of insulin that provides substantially similar results as insulin. Examples of insulin substitutes include but are not limited to zinc chloride, zinc nitrate, zinc bromide, and zinc sulfate. Additional insulins are known to those of ordinary skill in the art, see, for example, Gilman, A. G. et al., Eds., The Pharmacological Basis of Therapeutics, Pergamon Press, New York, 1990, pp. 1463-1495. The insulin can be zinc insulin or human zinc insulin. The insulin can be cell culture grade insulin, such as available from Serologicals, Inc.

The substitute may also be an insulin like growth factor such as IGF1. The composition may also comprise any amount of sodium selenite that achieves the desired result, including but not limited to approximately 1 , 2, 3, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 6.7, 7.0, 7.5, 8.0, 9.0, 10, 15 or 20 ug/L sodium selenite. In one embodiment, the composition may comprise 1 to 5, 5.5 to 15, 6.0 to 7.0 or 6.0 to 10 ug/L sodium selenite. The composition may comprise any amount of purified lipoprotein material that achieves the desired result, including but not limited to 0.2, 0.3. , 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0, 1.5 or 2% purified lipoprotein material in basal media. In one embodiment, 0.1 to 1.0%, 0.1 to 0.5, 0.5 to 1.0%, 0.5 to 1.5%, 0.1 to 2%, 1 to 2%, 1 to 5%, 5 to 10% or 10 to 20% purified lipoprotein material may be used in the composition.

In one specific embodiment, approximately 4 mg/ml recombinant albumin; approximately 5.5 u/lml transferrin; approximately 10 ug/ml insulin; approximately 6.7 ug/L sodium selenite in basal media.

In another specific embodiment, the composition can include : approximately 3 to 5 mg/ml recombinant albumin, approximately 4.5 to 6.5 ug/ml transferrin, approximately 9 to 1 1 ug/ml insulin, approximately 6.0 to 7.0 ug/L sodium selenite in basal media.

III. Compositions to Boost the Performance of Serum-free Media

Other types of serum-free media have been developed to substitute for the use of serum in cell culture. Compositions of the present invention, such as those that contain a recombinant albumin, can be used as a supplemented to further boost the growth of cells and increase the yield of products produced. In one aspect, the composition can include (i) serum free media and (ii) recombinant albumin. In one embodiment, the serum free media may be selected from one of the media listed in Table 1 , WO 2005/070120 hereby incorporated by reference. In another embodiment, the serum free media is either Hybridoma Media, animal component free or Ex- Cell (SAFC). In another embodiment, approximately 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, or 10% recombinant albumin is used. In a further embodiment, 0.1 to 1.0%, 0.1 to 0.5, 0.5 to 1.0%, 0.5 to 1.5%, 0.1 to 2%, 1 to 2%, 1 to 5%, 5 to 10% or 10 to 20% recombinant albumin may be used in the composition.

In a specific embodiment, the composition comprises Ex-Cell and approximately 0.5% recombinant albumin. In another specific embodiment, the composition comprises Hybridoma Medium, Animal Component-free, approximately 0.2% recombinant albumin.

IV. Cell Culture Compositions and Methods to Enhance the Growth and Performance of Cells The compositions and methods of the present invention can also be used to enhance the growth and product yield of hybridomas in cell culture.

In one aspect of the invention, the composition may comprise (i) basal media; (ii) recombinant albumin; and one or more components selected from the group consisting of insulin, sodium selenite, glutamine, transferrin, peptone, ethanolamine, fetuin, vitamins, lipoprotein, fatty acids and amino acids. In one embodiment, the basal media can be DMEM.

The composition may comprise any amount of recombinant albumin that achieves the desired effect, including but not limited to up to approximately 0.1 , 0.2, 0.3,0.4, 0.5,0.6,0.7, 0.8, 0.9, 1 ,2,3,4,5, 6,7, 8,9, 10, 1 1 , 12, 13, 14, 15, 20, 25%; 3 to 5, 5 to 8 % albumin. In one embodiment, the composition contains from approximately 0.2 to 0.4, 0.4 to 0.6, 0.6 to 0.9%, particularly approximately 0.5% albumin. The production of recombinant albumin is known in the art and numerous hosts such as Escherichia coli (EP 73,646), yeast has been reported in WO 00/44772, EP 0683233 A2, and US 5,612,196, and Bacillus subtillis (Saunders et al, 1987, J. Bacterid, 169, 2917-2925), Aspergillus.

The composition may comprise any amount of glutamine that achieves the desired effect, including but not limited to approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,12, 13, 14, 15, 20, 25 or 30 mM glutamine. In one embodiment, the composition may comprise 2 to 3,3 to 4,4 to 5, or 5 to 6 mM glutamine, particularly approximately 4mM glutamine. The composition may also comprise any amount of insulin that achieves the desired effect, including but not limited to approximately 1 , 2, 3, 4, 5 , 6 , 7, 8, 9, 9.5, 10, 10.5, 1 1 , 1 1.5, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40% or 16 to 18, 18 to 20 mg/L insulin. In one embodiment, the composition may comprise 7 to 9, 9 to 11 , 1 1 to 13, specifically about or approximately 10 mg/L insulin. An insulin substitute can also be used. The term "insulin substitute" may be any zinc containing compound which may be used in place of insulin that provides substantially similar results as insulin it may also be insulin like growth factor (IGF). Examples of insulin substitutes include but are not limited to zinc chloride, zinc nitrate, zinc bromide, and zinc sulfate. Additional insulins are known to those of ordinary skill in the art, see, for example, Gilman, A. G. et al., Eds., The Pharmacological Basis of Therapeutics, Pergamon Press, New York, 1990, pp. 1463-1495. In one embodiment, the insulin can be zinc insulin or human zinc insulin. The insulin can be cell culture grade insulin, such as available from Serologicals, Inc. In a specific embodiment, the insulin is human recombinant insulin such as that available from Serologicals.

The composition may also comprise any amount of transferrin that achieves the desired effect, including but not limited to approximately 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 1 , 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1 , 2.2, 2.3, 2.4, 2.5, 3, 4, 5, 10, or 15 mg/L transferrin; 3 to 5, or 5 to 7 mg/L transferrin. In one embodiment, the composition may comprise 0.5 to 1.0, 1.0 to 1.5, 1.5 to 2.0 mg/L transferrin. In a specific embodiment, the composition can include approximately 1.0 mg/L transferring. A transferrin substitute can also be used. A "transferrin substitute" refers to any compound which can replace transferrin and provides substantially similar results as transferrin. Examples of transferrin substitutes include but are not limited to any iron chelate compound, such as including, but not limited to, iron chelates of ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), deferoxamine mesylate, dimercaptopropanol, diethylenetriamine-pentaacetic acid (DPTA), and trans-l,2-diaminocyclohexane-N,N,N',N'- tetraacetic adic (CDTA), as well as a ferric citrate chelate and a ferrous sulfate chelate. The transferrin may be iron saturated transferrin, such as human transferrin. The transferrin may be a cell culture grade transferrin, such as that available from Serologicals, Inc. In a specific embodiment, the transferrin is human holo-transferrin, such as that available from Serologicals, Inc. The composition may comprise any amount of ethanolamine that exerts the desired effect, including but not limited to approximately 1 , 2, 3,4, 5, 6, 7, 8, 8.5, 9, 9.1 , 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1 , 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11 , 1 1.5, 12, 13 to 15, or 15 to 20 μM ethanolamine. In one embodiment, the composition can include 9.0 to 9.5, 9.5 to 10, 10 to 10.5, or 10.5 to 11 μM ethanolamine. In a specific embodiment, the composition can include approximately 10 μM ethanolamine. In other embodiments, the cell culture media may comprise basal media, approximately between 1 and 4,2 and 4, 1 and 3, 1 and 5,0.5 and 5,0.5 and 4.5,3 and 5, 3.5 and 4.5 mM glutamine, approximately between 0.2 and 1.0%, 0.1 and 1.0%, 0.5 and 1.0%, 0.3 and 1.5%; 0.2 and 5%; 0.2 and 3% and 0.3 and 2% recombinant albumin; approximately between 1 and 10 mg/L, 8 and 12 mg/L, 5 and 15 mg/L or greater than 25 mg/L insulin; approximately between 0.5 and 9.5,0.5 and 9.7,0.5 and 1.5,0.5 and 5,0.5 and 7, and 0.5-9 mg/L transferrin; approximately 1 and 10 uM, 8 and 12 uM, 5 and 15 uM or greater than 25 uM ethanolamine. In one specific embodiment, the composition of the present invention comprises approximately 4mM glutamine, approximately 0.5% recombinant albumin, approximately 10 mg/L insulin, approximately 1 mg/L transferrin and approximately 10 micromolar ethanolamine.

The present invention also includes a method of culturing cells using a involving contacting the cells with a composition described herein, including, but not limited to: (i) basal media; (ii) recombinant albumin; and one or more components selected from the group consisting of insulin, sodium selenite, glutamine, transferrin, peptone, ethanolamine, fetuin, vitamins, lipoprotein, fatty acids and amino acids, and maintaining the cells under conditions suitable to support cultivation of the cells in culture. In one embodiment of the present invention, the cells are eukaryotic cells, such as plant or animal cells or any other cell described herein. In a particular embodiment, the cells are MK2.7 cells, HEK 293 cells, CHO cells, PER-c6 cells, 5L8 cells, COS cells and Sp2/0 cells. In one embodiment, the preset invention provides a method of culturing hybridoma cells involving contacting the cells with a composition including (i) basal media; (ii) recombinant albumin; and one or more components selected from the group consisting of insulin, sodium selenite, glutamine, transferrin, peptone, ethanolamine, fetuin, vitamins, lipoprotein, fatty acids and amino acids, and maintaining the cells under conditions suitable to support cultivation of the hybridoma cells in culture. In one embodiment, the hybridoma is one of the hybridomas listed in table II, WO 2005/070120 hereby incorporated by reference. In a particular embodiment, the hybridoma is MK2.7.4. In a further embodiment, the hybridoma is 5L8. In a specific embodiment, the present invention is a method of culturing hybridoma cells involving contacting the cells with a composition including (i) basal media; (ii) approximately 0.5 % recombinant albumin; (iii) approximately 4mM glutamine; (iv)approximately 10 mg/L insulin; (v) approximately 1 mg/L transferrin; and (vi) approximately 10 micromolar ethanolamine, and maintaining the cells under conditions suitable to support cultivation of the hybridoma cells in culture.

The composition may comprise any amount of glutamine that achieves the desired effect, including but not limited to approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM glutamine. In one embodiment, the composition may comprise 2 to 3,3 to 4,4 to 5,2 to 10, 1 to 20,3 to 10,3 to 6, or 5 to 6 mM glutamine, specifically approximately 4mM glutamine. The composition may comprise any amount of albumin that achieves the desired effect, including but not limited to up to approximately 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1 , 1.5, 2, 2.5, 3, 3.5, 3.5 to 5, 5 to 10, 10 to 20% albumin. In one embodiment, the composition contains from 0.5 to 1 , 1 to 1.5, 1.5 to 2, specifically approximately 0.5% recombinant albumin. The production of recombinant albumin is known in the art and numerous hosts such as Escherichia coli (EP 73,646), yeast has been reported in WO 00/44772, EP 0683233 A2, and US 5,612,196, and Bacillus subtillis (Saunders et al, 1987, J. Bacterid, 169, 2917- 2925), Aspergillus.

The composition may also comprise any amount of insulin that achieves the desired effect, including but not limited to approximately 1 ,2, 3,4, 5 , 6, 7, 8 , 9 , 9.5, 10, 10.5, 11 , 1 1.5, 12, 13, 14, 15, 16 to 18, 18 to 20 mg/L insulin. In one embodiment, the composition can include 7 to 9, 9 to 1 1 , 1 1 to 13, including approximately 10 mg/L insulin. An insulin substitute can also be used. The term "insulin substitute" may be any zinc containing compound which may be used in place of insulin that provides substantially similar results as insulin or it may be insulin like growth factor (IGF),. Examples of insulin substitutes include but are not limited to zinc chloride, zinc nitrate, zinc bromide, and zinc sulfate. Additional insulins are known to those of ordinary skill in the art, see, for example, Gilman, A. G. et al., Eds., The Pharmacological Basis of Therapeutics, Pergamon Press, New York, 1990, pp. 1463-1495.

The insulin can be zinc insulin or human zinc insulin. The insulin can be cell culture grade insulin, such as available from Serologicals, Inc. In a specific embodiment, the insulin is human recombinant insulin such as that available from Serologicals.

The composition may also comprise any amount of transferrin that achieves the desired effect, including but not limited to approximately 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 1 , 1.5, 2, 2.5, 3 to 5, or 5 to 7 mg/L transferrin. In one embodiment, the composition can include 0.5 to 1.0, 1.0 to 1.5, 1.5 to 2.0 mg/L transferrin. In a specific embodiment, the composition can include approximately 1.0 mg/L transferrin. A transferrin substitute can also be used. A "transferrin substitute" refers to any compound which can replace transferrin and provides substantially similar results as transferrin. Examples of transferrin substitutes include but are not limited to any iron chelate compound, such as including, but not limited to, iron chelates of ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), deferoxamine mesylate, dimercaptopropanol, diethylenetriamine-pentaacetic acid (DPTA), and trans-1 ,2- diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), as well as a ferric citrate chelate and a ferrous sulfate chelate. The transferrin can be iron saturated transferrin, such as human transferrin. The transferrin can be a cell culture grade transferrin, such as that available from Serologicals, Inc. In a specific embodiment, the transferring is human holo-transferrin, such as that available from Serologicals, Inc. In one specific embodiment, the composition of the present invention comprises approximately 1 % recombinant albumin, approximately 4mM glutamine, approximately 10 mg/L insulin, and approximately 1 mg/L transferrin.

In another embodiment, the composition comprises (i) basal media; (ii) recombinant albumin; (iii) glutamine; (iv) insulin; (v) transferrin; and (vi) peptone. The composition can include any amount of peptone that exerts the desired effect, including but not limited to approximately 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 , 0.2, 0.3, 0.4, 0.5, 1 ,2 or 3 % peptone. In one embodiment, the composition can include approximately 0.05 to 0.1 , 0.1 to 0.2, 0.2 to 0.3, 0.3 to 0.5 % peptone. In one specific embodiment, the composition of the present invention can include approximately 4mM glutamine, approximately 1 % recombinant, approximately 10 mg/L insulin, approximately 1 mg/L transferrin, and approximately 0.5% peptone.

In further embodiment, the composition can include (i) basal media; (ii) recombinant albumin; (iii) glutamine; (iv) insulin; (v) transferrin; and (vi) fetuin. The composition may include any amount of fetuin that exerts the desired effect, including but not limited to approximately 2,3,4, 5, 6,7, 8, 9, 10, 10.5, 11 , 1 1.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 16 to 18, 18 to 20 micrograms/ml of fetuin. In one embodiment of the invention, fetuin is Pedersen's fetuin. In a particular embodiment, the composition contains approximately 8 to 10, 10 to 12, 12 to 14 micrograms/mL Pedersen's fetuin, specifically approximately 12.5 μg/mL Pedersen's fetuin, such as that available from Serologicals, Inc. In a specific embodiment of the present invention, the composition can include approximately 4mM glutamine, approximately 1 % recombinant albumin approximately 10 mg/L insulin, approximately 1 mg/L transferrin, and approximately 12.5 μg/mL of Pedersen's fetuin.

In yet another embodiment, the composition comprises (i) basal media; (ii) recombinant albumin; (iii) glutamine; (iv) insulin; (v) transferrin; and (vi) vitamin E. The composition can include any amount of vitamin E that exerts the desired effect, including but not limited to approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM vitamin E. In one embodiment, the composition can include approximately 3 to 5, 5 to 8 or 8 to 10 μM vitamin E, specifically approximately 5 μM vitamin E. In a specific embodiment of the present invention, the composition can include approximately 4mM glutamine, approximately 1 % recombinant albumin, approximately 10 mg/L insulin, approximately 1 mg/L transferrin, and approximately 5 μM vitamin E.

In a preferred embodiment, the composition of the present invention comprises (i) basal media; (ii) recombinant albumin (iii) glutamine; (iv) insulin; (v) transferrin; and (vi) peptone; (viii) fetuin; and (ix) vitamin E.

In one specific embodiment, the composition comprises approximately 1% recombinant albumin, approximately 4mM glutamine, approximately 10 mg/L insulin, approximately 1 mg/L transferrin, approximately 0.1 % peptone, approximately 12.5 micrograms/ml fetuin, and approximately 5 micromolar vitamin E. The composition may be used to boost the growth and production of cells in culture, including eukaryotic cells. Thus, one aspect of the present invention is a method of cultivating cells comprising contacting the cells with a composition including: (i) basal media; (ii) recombinant albumin; and one or more components selected from the group consisting of insulin, sodium selenite, glutamine, transferrin, peptone, ethanolamine, fetuin, vitamins, lipoprotein, fatty acids and amino acids.

A further aspect of the present invention is a method of cultivating cancer cells comprising contacting the cells with a composition of the present invention, including, but not limited to: (i) basal media; (ii) recombinant albumin (iii) glutamine; (iv) insulin; and/ or (v) transferrin. In one embodiment, the present invention provides a method of cultivating cancer cells comprising contacting the cells with a composition described herein, such as including: (i) basal media; (ii) recombinant albumin; (iii) glutamine; (iv) insulin; (v) transferrin; and/ or (vi) peptone.

In another embodiment, present invention is a method of cultivating cancer cells comprising contacting the cells with a composition including: (i) basal media; (ii) recombinant albumin; (iii) glutamine; (iv) insulin; (v) transferrin; and (vi) fetuin. In a specific embodiment, the fetuin is Pedersens fetuin. In one embodiment, present invention is a method of cultivating cancer cells comprising contacting the cells with a composition including (i) basal media; (ii) recombinant albumin; (iii) glutamine; (iv) insulin; (v) transferrin; and (vi) vitamin E.

In a further embodiment, the present invention is a method of cultivating cancer cells comprising contacting the cells with a composition including(i) basal media; (ii) recombinant albumin; (iii) glutamine; (iv) insulin; (v) transferrin; (vi) peptone; and/ or (vii) fetuin. In yet another embodiment, the present invention is a method of cultivating cancer cells comprising contacting the cells with a composition including: (i) basal media; (ii) recombinant albumin; (iii) glutamine; (iv) insulin; (v) transferrin; (vi) peptone; and/ or (vii) vitamin E. In another embodiment, the present invention is a method of cultivating cancer cells comprising contacting the cells with a composition including: (i) basal media; (ii) recombinant albumin; (iii) glutamine; (iv) insulin; (v) transferrin; (vi) vitamin E; and/ or (vii) fetuin. In a specific embodiment, the method of the present invention is a method of cultivating cancer cells comprising contacting the cells with a composition including: (i) basal media; (ii) recombinant albumin; (iii) glutamine; (iv) insulin; (v) transferrin; (vi) peptone; (vii) Pedersen's fetuin; and/ or (viii) vitamin E.

In a preferred embodiment, the method of the present invention is a method of cultivating cancer cells comprising contacting the cells with a composition including: (i) basal media; (ii) approximately 1 % recombinant albumin; (iii) approximately 4mM glutamine; (iv) approximately 10 mg/L insulin; (v) approximately 1 mg/L transferrin; (vi) approximately 0.1 % peptone; (vii) approximately 12.5 μg/ml Pedersen's fetuin; and or (viii) approximately 5 μM vitamin E.

Any number of cancer cell lines is familiar to those skilled in the art. Representative examples of cancer cell lines that can be cultivated by the method of the present invention include but are not limited to the following cancer cell lines: human myeloma (e.g., KMM-1 , KMS-1 1 , KMS-12-PE, KMS-12-BM, KMS-18, KMS-20, KMS-21-PE, U266, RPMI8226); human breast cancer (e.g., KPL-1 , KPL-4, MDA-MB-231 , MCF-7, KPL-3C, T47D, SkBr3, HS578T, MDA4355, Hs 606 (CRL-7368), Hs 605.T (CRL-7365) HS 742.T (CRL-7482), BT- 474, HBL-100, HCC202, HCC1419, HCC1954, MCF7, MDA-361 MDA-436, MDA-453, SK- BR-3, ZR-75-30, UACC-732, UACC-812, UACC-893, UACC-3133, MX-1 and EFM-192A); ductal (breast) carcinoma (e.g., HS 57HT (HTB-126), HCC1008 (CRL-2320), HCC1954 (CRL-2338; HCC38 (CRL-2314), HCC1 143 (CRL-2321 ), HCC1 187 (CRL-2322), HCC1295 (CRL-2324), HCC1599 (CRL-2331 ), HCC1937 (CRL-2336), HCC2157 (CRL-2340), HCC2218 (CRL-2343), Hs574.T (CRL-7345), Hs 742.T (CRL-7482); skin cancer (e.g., COLO 829 (CRL-1974), TE 354.T (CRL-7762), Hs 925.T (CIU-7677)); human prostate cancer (e.g., MDA PCa 2a and MDA PCa 2b); bone cancer (e.g., Hs 919.T (CRL-7672), Hs 821. T (CRL- 7554), Hs 820.T (CRL-7552)y HS 704.T (CRL-7444), HS 707(A)T (CRL-7448), HS 735.T

(CRL-7471 ), HS 860.T (CRL-7595)y HS 888.T.(CRL-7622); HS 889.T (CRL-7626); HS 890.T (CRL-7628), Hs 709.T (CRL-7453)); human lymphoma (e.g., K562); human cervical carcinoma (e.g., HeLA); lung carcinoma cell lines (e.g., H125, H522, H1299, NCI-H2126 (ATCC CCL-256), NCI-H1672 (ATCC CRL-5886), NCI- 2171 (CRL-5929); NCI-H2195 (CRL05931 ); lung adenocarcinoma (e.g., NCI-H1395 (CRL-5856), NCI-H1437 (CRL-5872), NCI-H2009 (CRL-591 1 ), NCI-H2122 (CRL-5985), NCI-H2087 (CRL-5922); metastatic lung cancer (e.g., bone) (e.g., NCI-H209 (HTB-172); colon carcinoma cell lines (e.g., LN235, DLD2, Colon A, LIM2537, LIM1215, LIM1863 , LIM1899 , LIM2405 , LIM2412 , SK-CO1 (ATCC HTB-77), HT29 (ATCC HTB38), LoVo (ATCC CCL-229), SW1222 (ATCC HB-11028), and SW480 (ATCC CCL-228); ovarian cancer (e.g., OVCAR-3 (ATCC HTB-161 ) and SKOV- 3 (ATCC HTB-77); mesothelioma (e.g., NCI-h2052 (CRL-5915); neuroendocrine carcinoma (e.g., HCI-H1770 (e.g., CRL-5893); gastric cancer (e.g., LIM1839); glioma (e.g., T98, U251 , LN235); head and neck squamous cell carcinoma cell lines (e.g., SCC4, SCC9 and SCC25); medulloblastoma (e.g., Daoy, D283 Med and D341 Med); testicular non-seminoma (e.g., TERA1 ); prostate cancer (e.g., 178-2BMA, Du145, LNCaP, and PC-3). Other cancer cell lines are well known in the art.

The present invention will now be exemplified with reference to the following non-limiting examples and figures.

EXAMPLE 1 : recombinant albumin has a beneficial effect on growth performance and viability of CHO cells and SP2/0 cells

The effect of recombinant albumin on the growth performance of a CHO cell line was measured using a standard cell growth assay with cell growth measured using cell doubling time. Cells were grown on cell culture plates (cluster trays) and incubated at 37C° and 5% CO₂.

Media was not changed during the period of the experiment. Recombinant albumin was added to the CHO cells in the following concentrations: 0.00mg/ml, 0.03mg/ml, 0.3mg/ml,

1 mg/ml, 3mg/ml and 5mg/ml. Cell number was determined using a hemocytometer.

As indicated by Figure 1 , the cell doubling time (time taken for the cells to double in number) decreased with increasing concentrations of recombinant albumin. At the highest concentration of recombinant albumin, the cell doubling time was decreased by 10 hours compared to no addition of albumin.

The ability of the recombinant albumin to increase cell viability was determined by measuring the percentage of dead cells over a set period of time. Cells were grown on cell culture plates (cluster trays) and incubated at 37C° and 5% CO₂.

Media was not changed during the period of the experiment. Recombinant albumin was added to the CHO-K1 cells in the following concentrations: Oug/ml, 20ug/ml, 200ug/ml and 2mg/ml. Cell number was determined using a hemocytometer.

As indicated by Figure 2, recombinant albumin provided a protective effect on the cells grown over an extended time without medium change. The results indicated that cell survival increases with increasing concentrations of recombinant albumin.

The ability of the recombinant albumin to increase growth rate and cell viability in a feed batch process was determined by measuring the level of viable cells over a set period of time. Cells were grown on cell culture plates (cluster trays) and incubated at 37C° and 5% CO₂.

Recombinant albumin was added to the CHO-K1 cells in the following concentrations: Oug/ml, 20ug/ml, 100ug/ml, 500ug/ml, 1 mg/ml and 5mg/ml. Cell number was determined using a hemocytometer. As indicated by Figure 3, recombinant albumin provided a protective effect on the cells grown over an extended time without medium change. The results indicated that cell survival and cell viability increases with rising concentrations of recombinant albumin. In a further aspect of this experiment the effect of recombinant albumin on the growth performance of the SP2/0 cell line was measured using a standard cell growth assay. Cells were grown in DMEM/F12 supplemented with 0.5 g/l probumin and 1 % ITS before initiation of the experiment. Upon initiation of the experiment, probumin was withdrawn and either no albumin (control), probumin or recombinant albumin was added at a concentration of 0.5g/L or 2g/L. Cells were counted by a hemocytometer at the following intervals: 0, 1 , 4, 5, 6, 7 days. The results of the experiment are presented in Figures 4 and 5. The results show that the growth performance of viable SP2/0 cells was superior when recombinant albumin was added to the cell culture medium.

EXAMPLE 2: Recombinant albumin is equivalent to bovine serum albumin when measuring the growth performance and viability of the CHO-K1 cell line

The inventors compared the performance of recombinant albumin at stimulating CHO-K1 cell growth and viability with bovine serum albumin.

CHO-K1 cells where thawed in DMEM/F12 (1 :1 ), w/Hepes cat. no. 31330038 (Invitrogen) with 10% FCS, trypsinated in Trypsin-EDTA (GIBCO), 0.05%, washed in Hanks balanced salt solution and and dissolved in medium with 0.5 g/l of bovine serum albumin (with 1 % Insulin Transferrin Selenium). The culture medium used for at least 3 passages before the survival assay is DMEM/F12 (1 :1 ) with 1 % ITS and 0.5 g/l Probumin. The cells are counted using a hemocytometer and the cell concentration adjusted to 5000 cells/ml. The following concentrations of recombinant albumin or bovine serum albumin (Probumin (81-068-02, Celliance) is added to the medium: 4, 2, 1 , 0.5, 0.25, 0.125, 0.0.0625, 0.03125 g/l and 100 μl of each concentration is added to triplicate wells in a 96 well Round Bottom plates (NUNC, cat. no. 163320). Following this step, 100ul of the cell suspension with 5000 cells/ml is added to all wells. After 6 days of incubation, 0.5 uCi ³H-Thymidine is added to each well. The plates are incubated for 20 h at 37°C and 5% CO₂ followed by harvesting of DNA from cells. The proliferation of the cells (cell division) is determined by measuring incorporated radioactivity by liquid scintillation counting and the results are presented in Figure 6. The results show that bovine serum albumin in CHO-K1 cell culture can be substituted with recombinant albumin without loss of cell survival.

Claims

1. A cell culture media comprising the following components: basal media, 0.0001-10% recombinant albumin, and one or more components selected from the group consisting of; glutamine, insulin, IGF, transferrin, ethanolamine, fetuin, vitamins, lipoprotein, fatty acids, amino acids, sodium selenite, peptone and antioxidants.

2. The cell culture media of claim 1 , comprising the following components: basal media, recombinant albumin, glutamine, transferrin, insulin and/or IGF.

3. The cell culture media of claim 1 or 2, wherein the transferrin is present in an amount of 0.001-1000 mg/L.

4. The cell culture media of claim 1 comprising the following components: basal media, recombinant albumin, glutamine, ethanolamine, insulin and/or IGF.

5. The cell culture media of claim 1 , 2 or 4, wherein the insulin is present in an amount of 0.01-1000 mg/L.

6. The cell culture media of claim 1 , comprising the following components: basal media, recombinant albumin, glutamine and ethanolamine.

7. The cell culture media of claim 1 , 2, 4 or 6, wherein the glutamine is present in an amount of 0.01 -1000 mg/L.

8. The cell culture media of claim 1 , 4 or 6, wherein the ethanolamine is present in an amount of 0.01-1000 mg/L.

9. The cell culture media of claim 1 , comprising the following components: basal media, recombinant albumin, ethanolamine, insulin and/or IGF.

10. A method to culture cells comprising incubating the cells in a culture media consisting essentially of the following components: basal media, 0.0001-10% recombinant albumin, and one or more components selected from the group consisting of; glutamine, insulin, transferrin, ethanolamine, fetuin, vitamins, lipoprotein, fatty acids, amino acids, sodium selenite, peptone and antioxidants.

1 1. A method to culture cells comprising incubating the cells in a culture media comprising the following components: basal media, sodium selenite, recombinant albumin, insulin, transferrin, peptone, fetuin, and vitamin E.

12. The method of claim 10, wherein the cells produce a peptide.

13. The method of claim 12, wherein the peptide is an antibody.

14. The method of claim 12, wherein the peptide is an immunoglobulin.

15. The method of claim 12, wherein the peptide is a blood clotting factor.

16. The method of claim 10, wherein the cells are hybridoma cells.

17. The method of claim 1 1 , wherein the cells are cancer cells.

18. The method of claim 1 1 , wherein the cells are in suspension.

19. The method of claim 1 1 , wherein the cells are adherent.

20. The method of claim 1 1 , wherein the cells are selected from the group consisting of K562 cells, HL-60 cells, Daudi cells, HeLA cells, THP-1 cells, Jurkat cells, CHO, BHK, HEK 293, Vero, MDCK, fibroblast, A431 , Sp2/0, GS-NSO, NSO and EL4 cells.

21. The method of claim 10, wherein the cells are human cells.

22. A method for culturing hybridoma cells in vitro comprising incubating the cells in a cell culture media comprising the following components: basal media, 0.0001-10% recombinant albumin, and one or more components selected from the group consisting of: glutamine, insulin, transferrin, ethanolamine, fetuin, vitamins, lipoprotein, fatty acids, amino acids, sodium selenite, peptone and antioxidants.

23. A cell culture media consisting essentially of the following components: basal media, 0.0001-10% recombinant albumin, and one or more components selected from the group consisting of: glutamine, insulin, transferrin, ethanolamine, fetuin, vitamins, lipoprotein, fatty acids, amino acids, sodium selenite, peptone and antioxidants.