WO2008013809A1 - Cell culture methods - Google Patents

Abstract

The invention relates to concentrated feed media, methods of preparing the concentrated feeds and methods of using these feeds in culturing mammalian cells.

Description

CELL CULTURE METHODS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of United States provisional application serial number 60/833,126, filed July 24, 2006, the entire disclosure of which is relied upon and incorporated by reference herein.

FIELD OF THE INVENTION

The invention is in the field of cell culture and particularly in the field of recombinant cell culture. More specifically, the invention relates to concentrated feed media, methods of preparing the concentrated feeds and methods of using these feeds in culturing mammalian cells.

BACKGROUND Mammalian cells are commonly used for production of recombinant proteins.

One example of a mammalian cell line suited for recombinant production of therapeutic proteins is CHO (Chinese hamster ovary) cell lines. CHO cell lines efficiently produce proteins that are correctly folded and have desired post- translational modifications. Further, CHO cells have gained acceptance for use in manufacturing of recombinant protein therapeutics by regulatory agencies.

Optimization of recombinant protein expression and purification is important in order to obtain the greatest possible recovery of product, thereby reducing costs associated with the manufacture of protein therapeutics. Some examples of methods to increase production include monitoring and altering osmolarity during production, decreasing temperatures during specific phases of a cell culture, using high concentration growth media, and/or the addition of sodium butyrate to induce expression during the production phase (e.g., U.S. Pat. No. 5,705,364).

Additional methods include periodic feedings of batch culture grown cells with nutrients to increase production of recombinant proteins (e.g., U.S. Pat. No. 5,672,502). However, the use of a periodic feed has limitations because in order to deliver a sufficient amount of nutrients a high volume must be used resulting in a dilution effect in fed-batch cell culture process, which limits the ability to deliver more nutrients to cells, limits support for higher cell density and higher titer. Furthermore, oftentimes the feeds include undefined components such as animal serum or peptones. Since serum and peptones are derived from animals or plants, each of these have potential issues such as plant or animal derived contaminants and problems with consistency from suppliers.

Accordingly, there remains a need to continue improvement in yields of recombinant protein from each cell culture run.

SUMMARY OF THE INVENTION

The present invention provides, in part, a method of producing recombinant proteins comprising culturing a mammalian cell culture genetically engineered to produce the protein in a culture medium, and adding a feed solution to the cell culture, wherein the feed solution comprises a highly concentrated, chemically defined media. This media is advantageous in part because the feed media is highly concentrated and thus can be added to a batch fed culture at lower amounts, limiting the dilution effect, delivery of sufficient nutrients to cells for maximal growth, without limiting support for higher cell density higher titer without supplemental, undefined serum or peptones. Furthermore, as the media of the invention is defined, it does not carry the same level of risk of contaminants or have problems with lot consistency.

In addition, the invention comprises a new method to prepare the concentrated defined feed media called ,a two-powder method, that can be up to 100 fold (100x) concentrated, in contrast to the more standard 10 fold (10x) starting concentration. The present two-powder method has advantages over standard methods including it can be efficiently prepared without pH titration step and without a heating step and further, and it improves the manufacturability and scalability for media compounding. Generally, it has been found that the high concentration feed media and feed strategy described herein can improve the yield of recombinant proteins by more than 5% over a feed with chemically defined media that are not at high concentration. It is contemplated that the feed media of the invention can improve recombinant protein production by 10%, 15%, 20%, or 25% or more when compared to a defined feed media that is not at the higher concentration.

The invention finds particular use when the cells are grown in production conditions when the feed solution is added, namely, the cell culture is in the batch reactor and the protein of interest is being produced for later harvest. Accordingly, in this embodiment the feed is added repeatedly, such as, for example, about every two days for 4 to 10 days. The methods of the invention are particularly useful for large scale culturϊng of mammalian cell cultures.

It is also contemplated that the recombinant proteins expressed using the methods and compositions of the invention will be used as therapeutic molecules and are dosed, formulated and administered as appropriate. DETAILED DESCRIPTION OF THE INVENTION

During batch culture of recombinant cells, nutrients can become limiting leading to a reduction in cell performance (as measured by cell viability, viable cell density, and recombinant protein production). To overcome these effects, batch cultures can be fed with a solution of medium and/or amino acids. In order for enough nutrients to be added, the feed is often undesirably diluting to the production culture. In addition, the feed media typically contains serum or other complex mixtures of nutrients such as peptones that can be less predictable and in the case of bovine serum can present a risk of presenting a contaminant such as found in mad cow disease. Accordingly, the present invention is directed toward concentrated chemically defined feed media and methods of making them, that provide nutrients to cells in batch culture and help maintain titers of cells such that protein production and recovery is maintained.

Cells in batch culture are typical grown from a frozen stock of engineered clonal cell lines. They are initially thawed from the frozen storage vessel and grown in media for scale up. After the scale up the cells fed into a batch media during a production run, where the recombinant protein of interest is to be expressed and later harvested. During the production run, various techniques are employed to increase the expression of the protein of interest including adding feeds to the media to restore nutrient depletion and inducing agents, for example, butyrate or temperature changes to assist in 'inducing' greater expression of the protein of interest.

Thus, the invention finds particular use when the cells are grown in production conditions when the feed solution is added, namely, the cell culture is in the batch reactor and the protein of interest is being produced for harvest. Accordingly, in this embodiment the feed is added repeatedly, such as, for example, about every two days for 4 to 10 days. The methods of the invention are particularly useful for large scale culturing of mammalian cell cultures.

The present invention provides, in part, a method of improved production of recombinant proteins comprising culturing a mammalian cell culture genetically engineered to produce the protein in a medium, and adding a feed solution to the cell culture, wherein the feed solution comprises a highly concentrated, chemically defined media. This media is advantageous in part because the feed media is highly concentrated and thus can be added to a batch fed culture at lower amounts, limiting the dilution effect, delivery of sufficient nutrients to cells for maximal growth, support for higher cell density, and eventually achieve higher titer without supplemental, undefined serum or peptones. Furthermore, as the media of the invention is defined, it does not carry the same level of risk of contaminants or have problems with lot consistency. (n addition, the invention comprises a method to prepare the concentrated defined feed media called a two-powder method, that can be up to 100 fold (100x) concentrated, in contrast to the more standard 10 fold (1Ox) starting concentration. The present method has advantages over standard methods including it can be efficiently prepared without pH titration step and without a heating step and further, and it improves the manufacturability and scalability for media compounding.

In the examples described herein, the solubility of individual cell culture feed media components was investigated based on their pH values, solubility, concentration levels, and interactions with other components. These components were then grouped into two powders soluble at difference pH, e.g., acidic (low pH) and basic (high pH). The two groups were then mixed at different points to guarantee solubility for each.

During the mixing, or compounding of the media, the basic powder is added first into room temperature water at 80-90% of final volume, achieving a high pH of 9-11. After the basic powder is in solution, the second acidic powder is added driving the pH down to 3.5-6.5, depending on the concentration of the acidic powder.

The final pH can then be manipulated to the desired, final pH of 5.4-6.0 by adding additional components such as glucose, glutamine, NaHCO₃, NaOH or HCl. With this method it is shown that concentrated media can be prepared in a practical way that can be scaled for manufacturing purposes, e.g., mixing in a single vessel, in room temperature water (20+5⁰C), without acid/base titration, and without heating/cooling steps.

While high temperature mixing can be detrimental to some components of media during mixing and that the current invention can be performed at room temperature, it is contemplated that high temperatures can be used. Accordingly, it is understood that room temperature can vary between about 18 to 24 degrees

Celsius. However, the invention contemplates uses above or even below room temperature as is necessary.

In another embodiment, the two groups of compounds, basic soluble and acidic soluble are mixed in two separate respective solutions. The basic soluble compounds are dissolved first in a solution followed by dissolving the acid compounds. The two solutions are then combined and the pH adjusted as needed for suitable use as a feed media.

Furthermore, feed solutions can be added repeatedly. More frequent feeds will call for the addition of lower amounts of feed media each time; conversely, less frequent feeds will call for the addition of higher amounts of feed media. However, very high concentrations of feed media should be avoided as such can be toxic to CHO cells.

It has been found that the high concentration feed media and feed strategy described herein can improve the yield of recombinant proteins by 5% or more over feed chemically defined media that are not high concentration. It is contemplated that the feed media of the invention can improve recombinant protein production by 10%, 15%, 20%, or 25% or more when compared to a defined feed media that is not at the higher concentration. As used herein, a high concentration chemically defined media is understood to be more than 10x concentrated, in one embodiment, more than 15x, in another embodiment, more than 2Ox, in another embodiment, more than 25x, in another embodiment, more than 3Ox, in another embodiment, more than 35x, in another embodiment, more than 40x, in another embodiment, more than 45x, in another embodiment more than 5Ox, in another embodiment, more than 55x, in another embodiment, more than 60x, in another embodiment, more than 65x, in another embodiment, more than 70x, in another embodiment, more than 75x, in another embodiment, more than 80x, in another embodiment, more than 85X, in another embodiment, more than 90x, in another embodiment, more than 95X, in another embodiment, approximately 100x. It is understood that the typical reference concentration is at or around 1x, and this is only loosely based on the growth media for the cells and the feed media concentrations of individual components can vary. Accordingly, in one example, if a feed media is 4Ox concentrated and a reference media has 50 compounds, a chemically defined feed media does not require all 50 compounds to be present. The non-essential compounds may be omitted. The term animal cell is meant to encompass a cell whose progenitors were derived from a multicellular animal. Preferably, the animal cell lines are mammalian cell lines. A wide variety of animal cell lines suitable for growth in culture are available from, for example, the American Type Culture Collection (ATCC, Manassas, Va.) and NRRL (Peoria, III.). Some of the more established cell lines used in industrial or academic laboratories and which are preferred are CHO, NSO, VERO, BHK, HeLa, Cos, CV1 , MDCK, 293, 3T3, PC12, hybridoma, myeloma, and WI38 cell lines, to name but a few examples. The dihydrofolate reductase (DHFR)- deficient mutant cell lines (Urlaub et al., 1980, Proc Natl Acad Sci USA 77:4216 4220), DXB11 and DG-44, are the CHO host cell lines of choice because the efficient DHFR selectable and amplifiable gene expression system allows high level recombinant polypeptide expression in these cells (Kaufman R. J., 1990, Meth Enzymol 185:527 566). In addition, these cells are easy to manipulate as adherent or suspension cultures and exhibit relatively good genetic stability. In addition, new animal cell lines can be established using methods well known by those skilled in the art (e.g., by transformation, viral infection, and/or selection, etc.).

By in vitro cell culture is meant the growth and propagation of cells outside of a multicellular organism or tissue. Typically, in vitro cell culture is performed under sterile, controlled temperature and atmospheric conditions in culture plates (e.g., 10 cm plates, 96 well plates, etc.), or other adherent culture (e.g., on microcarrier beads) or in suspension culture in a reactor and/or in roller bottles. Cultures can be grown in shake flasks, small scale bioreactors, and/or large-scale bioreactors. A bioreactor is a device used to culture animal cells in which environmental conditions such as temperature, atmosphere, agitation, and/or pH can be monitored and adjusted. A number of companies (e.g., ABS Inc., Wilmington, Del.; Cell Trends, Inc., Middletown, Md.) as well as university and/or government-sponsored organizations (e.g., The Cell Culture Center, Minneapolis, Minn.) offer cell culture services on a contract basis.

Further, the methods and cell cultures of the invention (adherent or non- adherent and growing or growth arrested), can be small scale cultures, such as for example in 100 ml containers having about 30 ml of media, 250 ml containers having about 80 to 90 ml of media, 250 ml containers having about 150 to 200 ml of media. Alternatively, the cultures can be large scale such as for example 1000 ml containers having about 300 to 1000 ml of media, 3000 ml containers having about 500 to 3000 ml of media, 8000 ml containers having about 2000 to about 8000 ml of media, and 15000 ml containers having about 4000 ml to about 15000 ml of media. Both small scale and large scale culturing can be performed in bioreactors. In preferred embodiments, the size of the culture is at least about 100 liters, more preferably at least about 1000 liters, still more preferably at least about 5000 liters, even more preferably at least about 7000 liters.

Cell culture medium is defined, for purposes of the invention, as a medium suitable for growth of animal cells, and preferably mammalian cells, in in vitro cell culture. Typically, culture media contains a buffer, salts, energy source, amino acids, vitamins and trace essential elements. Any medium capable of supporting growth of the appropriate cell in culture can be used; as shown below by way of example, variations in a serum-free medium composition did not affect the superior results obtained when the high concentration media was fed to the cell culture.

Cell culture media suitable for use in the invention are commercially available. For example, any one or combination of the following media can be used: RPMl- 1640 Medium, Dulbecco's Modified Eagle's Medium, Minimum Essential Medium Eagle, F-12K Medium, Iscove's Modified Dulbecco's Medium. When defined medium that is serum-free and/or peptone-free is used, the medium is usually enriched for certain amino acids and trace elements (see, for example, U.S. Pat. No. 5,122,469 to Mather et al., and U.S. Pat. No. 5,633,162 to Keen et al.). However, these enriched feeds are typically not more than 10x above the growth media.

When defined medium that is serum-free and/or peptone-free is used, the medium is usually enriched for particular amino acids, vitamins andJor trace elements (see, for example, U.S. Pat. No. 5,122,469 to Mather et al., and U.S. Pat. No. 5,633,162 to Keen et al.). Depending upon the requirements of the particular cell line used, medium also contains a serum additive such as Fetal Bovine Serum, or a serum replacement. Examples of serum-replacements (for serum-free growth of cells) are TCH.TM., TM-235.TM., and TCH.TM.; these products are available commercially from Celox (St. Paul, Minn.).

In the methods and compositions of the invention, cells can be grown in serum-free, protein-free, growth factor-free, and/or peptone-free media. The term "serum-free" as applied to media includes any mammalian cell culture medium that does not contain serum, such as fetal bovine serum. The term "insulin-free" as applied to media includes any medium to which no exogenous insulin has been added. By exogenous is meant, in this context, other than that produced by the culturing of the cells themselves. The term "growth-factor free" as applied to media includes any medium to which no exogenous growth factor (e.g., insulin, IGF-1) has been added. The term "peptone-free" as applied to media includes any medium to which no exogenous protein hydrolysates have been added such as, for example, animal and/or plant protein hydrolysates. Preferably, the medium used is serum-free, or essentially serum-free. By "essentially serum-free" is meant that less than about 2% serum is present, more preferably less than about 1% serum is present, still more preferably less than about 0.5% serum is present, yet still more preferably less than about 0.1% serum is present. By "serum free", it is understood that the medium is preferably less than 0.1% serum and more preferably less than 0.01% serum.

The methods of the invention can be used in combination with other types of cell culture. For example, cell cultures can be serial subcultured in larger and larger volumes of culture medium to as to maintain the cells in exponential phase, and then converted to a batch culture system when a desired volume or cell density is achieved. Then, the batch cell culture can be fed using the methods of the invention. For example, a CHO cell culture can be grown and progressively transferred from a small scale culture to a large scale culture, and then seeded at a desired cell density into a batch cell culture. Once in the batch cell culture, the cells can be fed using the methods of the invention. CHO cells can be maintained in batch culture for as long as recombinant protein production occurs. Preferably, the batch culture is maintained in a production phase for about 2 to about 16 days, more preferably for about 6 to about 12 days.

Further, the methods of the invention can be used in combination with known or yet to be discovered methods of inducing the production of recombinant proteins. By "inducing conditions" is meant a technique to increase the relative production per cell of a desired recombinant protein during the production phase of the culture. Often, other cell processes (such as growth and division) are inhibited so as to direct most of the cells' energy into recombinant protein production. Such techniques include cold temperature shift, and additions of chemicals such as sodium butyrate (as described in U.S. Pat No. 5,705,364 to Etcheverry et al., incorporated herein by reference), DMSO, DMF, DMA, TNF-alpha, phorbol 12-myristate 13-acetate, PMA₁ propionate, forskolin, dibutyryl cAMP, 2-aminopurine, adenine, adenosine, okadaic acid, and combinations of any of these techniques, to name just a few examples, as well as any yet to be described and/or discovered induction techniques. Typically, a batch culture of cells at high density is induced to produce the recombinant protein. The invention can be used in the culture of cells that produce just about any protein, especially recombinant proteins. Examples of useful expression vectors that can be used to produce proteins are disclosed in WO 01/27299, and the pDC409 vector described in McMahan et al., 1991, Embo J. 10:2821. A protein is generally understood to be a polypeptide of at least about 10 amino acids, more preferably at least about 25 amino acids, even more preferably at least about 75 amino acids, and most preferably at least about 100 amino acids.

Generally, the methods of the invention are useful for the production of recombinant proteins. Recombinant proteins are proteins produced by the process of genetic engineering. The term "genetic engineering" refers to a recombinant DNA or RNA method used to create a host cell that expresses a gene at elevated levels, at lowered levels, or a mutant form of the gene. In other words, the cell has been transfected, transformed or transduced with a recombinant polynucleotide molecule, and thereby altered so as to cause the cell to alter expression of a desired protein. Methods and vectors for genetically engineering cells and/or cell lines to express a protein of interest are well known to those skilled in the art; for example, various techniques are illustrated in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates) and Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989). Genetic engineering techniques include but are not limited to expression vectors, targeted homologous recombination and gene activation (see, for example, U.S. Pat. No. 5,272,071 to Chappel) and trans activation by engineered transcription factors (see, for example, Segal et al., 1999, Proc. Natl. Acad. Sci. USA 96(6):2758-63). Proteins can be expressed under the control of a heterologous control element such as, for example, a promoter that does not in nature direct the production of that protein. For example, the promoter can be a strong viral promoter (e.g., CMV, SV40) that directs the expression of a mammalian protein. The host cell may or may not normally produce the protein. For example, the host cell can be a CHO cell that has been genetically engineered to produce a human protein.

Alternatively, the host cell can be a human cell that has been genetically engineered to produce increased levels of a human protein normally present only at very low levels (e.g., by replacing the endogenous promoter with a strong viral promoter).

Particularly preferred proteins for expression are protein-based therapeutics, also known as biologies. Preferably, the proteins are secreted as extracellular products. Proteins that can be produced using the invention include but are not limited to Flt3 ligand, CD40 ligand, erythropoeitin, thrombopoeitin, calcitonin, Fas ligand, ligand for receptor activator of NF-kappa B (RANKL), TNF-related apoptosis- inducing ligand (TRAIL), ORK/Tek, thymic stroma-derived lymphopoietin, granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor, mast cell growth factor, stem cell growth factor, epidermal growth factor, RANTES₁ growth hormone, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, interferons, nerve growth factors, glucagon, interleukins 1 through 18, colony stimulating factors, lymphotoxin-.beta., tumor necrosis factor, leukemia inhibitory factor, oncostatin-M, and various ligands for cell surface molecules Elk and Hek (such as the ligands for eph-related kinases, or LERKS). Descriptions of proteins that can be produced according to the invention may be found in, for example, Human Cytokines: Handbook for Basic and Clinical Research, Vol. Il (Aggarwal and Gutterman, eds. Blackwell Sciences, Cambridge Mass., 1998); Growth Factors: A Practical Approach (McKay and Leigh, eds., Oxford University Press Inc., New York, 1993) and The Cytokine Handbook (AW Thompson, ed.; Academic Press, San Diego Calif.; 1991).

Production of the receptors for any of the aforementioned proteins can also be improved using the invention, including the receptors for both forms of tumor necrosis factor receptor (referred to as p55 and p75), lnterleukin-1 receptors (type 1 and 2), lnterleukin-4 receptor, lnterleukin-15 receptor, lnterleukin-17 receptor, lnterleukin-18 receptor, granulocyte-macrophage colony stimulating factor receptor, granulocyte colony stimulating factor receptor, receptors for oncostatin-M and leukemia inhibitory factor, receptor activator of NF-kappa B (RANK), receptors for TRAIL, and receptors that comprise death domains, such as Fas or Apoptosis- lnducing Receptor (AIR). A particularly preferred receptor is a soluble form of the IL- 1 receptor type II; such proteins are described in U.S. Pat. No. 5,767,064, incorporated herein by reference in its entirety.

Other proteins that can be produced using the invention include cluster of differentiation antigens (referred to as CD proteins), for example, those disclosed in Leukocyte Typing Vl (Proceedings of the VUh International Workshop and Conference; Kishimoto, Kikutani et al., eds.; Kobe, Japan, 1996), or CD molecules disclosed in subsequent workshops. Examples of such molecules include CD27, CD30, CD39, CD40; and ligands thereto (CD27 ligand, CD30 ligand and CD40 ligand). Several of these are members of the TNF receptor family, which also includes 41BB and OX40; the ligands are,often members of the TNF family (as are 41BB ligand and OX40 ligand); accordingly, members of the TNF and TNFR families can also be produced using the present invention.

Proteins that are enzymatically active can also be produced according to the instant invention. Examples include metalloproteinase-disintegrin family members, various kinases, glucocerebrosidase, alpha-galactosidase A, superoxide dismutase, tissue plasminogen activator, Factor VIII, Factor IX, apolipoprotein E₁ apolipoprotein A-I, globins, an IL-2 antagonist, alpha-1 antitrypsin, TNF-alpha Converting Enzyme, and numerous other enzymes. Ligands for enzymatically active proteins can also be produced by applying the instant invention.

The inventive compositions and methods are also useful for production of other types of recombinant proteins, including immunoglobulin molecules or portions thereof, and chimeric antibodies (i.e., an antibody having a human constant region couples to a murine antigen binding region) or fragments thereof. Numerous techniques are known by which DNA encoding immunoglobulin molecules can be manipulated to yield DNAs capable of encoding recombinant proteins such as single chain antibodies, antibodies with enhanced affinity, or other antibody-based polypeptides (see, for example, Larrick et al., 1989, Biotechnology 7:934-938; Reichmann et al., 1988, Nature 332:323-327; Roberts et al., 1987, Nature 328:731- 734; Verhoeyen et al., 1988, Science 239:1534-1536; Chaudhary et al., 1989,

Nature 339:394-397). Recombinant cells producing fully human antibodies (such as are prepared using transgenic animals, and optionally further modified in vitro), as well as humanized antibodies, can also be used in the invention. The term humanized antibody also encompasses single chain antibodies. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., U.S. Pat No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. etal., European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Queen et a!., European Patent No. 0451 216 B1; and Padlan, E. A. et al., EP O 519 596 A1. For example, the invention can be used in the production of human and/or humanized antibodies that immunospecifically recognize specific cellular targets, e.g., any of the aforementioned proteins, the human EGF receptor, the her-2/neu antigen, the CEA antigen, Prostate Specific Membrane Antigen (PSMA), CD5, CD11a, CD18, NGF, CD20, CD45, CD52, Ep-cam, other cancer cell surface molecules, TNF-alpha, TGF-b1 , VEGF, other cytokines, alpha 4 beta 7 integrin, IgEs, viral proteins (for example, cytomegalovirus), etc., to name just a few.

Various fusion proteins can also be produced using the invention. A fusion protein is a protein, or domain or a protein (e.g. a soluble extracellular domain) fused to a heterologous protein or peptide. Examples of such fusion proteins include proteins expressed as a fusion with a portion of an immunoglobulin molecule, proteins expressed as fusion proteins with a zipper moiety, and novel polyfunctional proteins such as a fusion proteins of a cytokine and a growth factor (i.e., GM-CSF and IL-3, MGF and IL-3). WO 93/08207 and WO 96/40918 describe the preparation of various soluble oligomeric forms of a molecule referred to as CD40L, including an immunoglobulin fusion protein and a zipper fusion protein, respectively; the techniques discussed therein are applicable to other proteins. Another fusion protein is a recombinant TNFR:Fc, also known as "entanercept." Entanercept is a dimer of two molecules of the extracellular portion of the p75 TNF alpha receptor, each molecule consisting of a 235 amino acid TNFR-derived polypeptide that is fused to a 232 amino acid Fc portion of human IgGI . In fact, any of the previously described molecules can be expressed as a fusion protein including but not limited to the extracellular domain of a cellular receptor molecule, an enzyme, a hormone, a cytokine, a portion of an immunoglobulin molecule, a zipper domain, and an epitope.

After culturing using the methods of the invention, the resulting expressed protein can then be collected. In addition the protein can purified, or partially purified, from such culture or component (e.g., from culture medium or cell extracts or bodily fluid) using known processes. By "partially purified" means that some fractionation procedure, or procedures, have been carried out, but that more polypeptide species (at least 10%) than the desired protein is present. By "purified" is meant that the protein is essentially homogeneous, i.e., less than 1% contaminating proteins are present. Fractionation procedures can include but are not limited to one or more steps of filtration, centrifugation, precipitation, phase separation, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction chromatography (HIC; using such resins as phenyl ether, butyl ether, or propyl ether), HPLC, or some combination of above.

For example, the purification of the polypeptide can include an affinity column containing agents which will bind to the polypeptide; one or more column steps over such affinity resins as concanavalin A-agarose, HEPARIN-TOYOPEARL (chromatography medium) or Cibacrom blue 3GA SEPHAROSE (agarose beads); one or more steps involving elution; and/or immunoaffinity chromatography. The polypeptide can be expressed in a form that facilitates purification. For example, it may be expressed as a fusion polypeptide, such as those of maltose binding polypeptide (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion polypeptides are commercially available from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway, N.J.) and InVitrogen, respectively. The polypeptide can be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope FLAG (epitope tag) is commercially available from Kodak (New Haven, Conn.). It is also possible to utilize an affinity column comprising a polypeptide- binding polypeptide, such as a monoclonal antibody to the recombinant protein, to affinity-purify expressed polypeptides. Other types of affinity purification steps can be a Protein A or a Protein G column, which affinity agents bind to proteins that contain Fc domains. Polypeptides can be removed from an affinity column using conventional techniques, e.g., in a high salt elution buffer and then dialyzed into a lower salt buffer for use or by changing pH or other components depending on the affinity matrix utilized, or can be competitively removed using the naturally occurring substrate of the affinity moiety.

The desired degree of final purity depends on the intended use of the polypeptide. A relatively high degree of purity is desired when the polypeptide is to be administered in vivo, for example. In such a case, the polypeptides are purified such that no polypeptide bands corresponding to other polypeptides are detectable upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognized by one skilled in the pertinent field that multiple bands corresponding to the polypeptide can be visualized by SDS-PAGE, due to differential glycosylation, differential post-translational processing, and the like. Most preferably, the polypeptide of the invention is purified to substantial homogeneity, as indicated by a single polypeptide band upon analysis by SDS-PAGE. The polypeptide band can be visualized by silver staining, Coomassie blue staining, or (if the polypeptide is radiolabeled) by autoradiography. The invention also optionally encompasses further formulating the proteins. By the term "formulating" is meant that the proteins can be buffer exchanged, sterilized, bulk-packaged and/or packaged for a final user. For purposes of the invention, the term "sterile bulk form" means that a formulation is free, or essentially free, of microbial contamination (to such an extent as is acceptable for food and/or drug purposes), and is of defined composition and concentration. The term "sterile unit dose form" means a form that is appropriate for the customer and/or patient administration or consumption. Such compositions can comprise an effective amount of the protein, in combination with other components such as a physiologically acceptable diluent, carrier, or excipient. The term "physiologically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s).

Formulations suitable for administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The polypeptides can be formulated according to known methods used to prepare pharmaceutically useful compositions. They can be combined in admixture, either as the sole active material or with other known active materials suitable for a given indication, with pharmaceutically acceptable diluents (e.g., saline, Tris-HCI, acetate, and phosphate buffered solutions), preservatives (e.g., thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/or carriers.

Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Company, Easton, Pa. In addition, such compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No. 4,737,323. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application, so that the characteristics of the carrier will depend on the selected route of administration.

Sustained-release forms suitable for use include, but are not limited to, polypeptides that are encapsulated in a slowly-dissolving biocompatible polymer (such as the alginate microparticles described in U.S. Pat. No. 6,036,978), admixed with such a polymer (including topically applied hydrogels), and or encased in a biocompatible semi-permeable implant.

The invention having been described, the following examples are offered by way of illustration, and not limitation.

EXAMPLES MEDIA COMPOUNDING

All the milled powders were supplied by SAFCs Biosciences™ Immediate Advantage Lab. Individual media components were purchased from SAFC or Sigma. Small-scale media compounding was carried out in glass beakers with magnetic stir bar. Large-scale (20-100L) media preparation was carried out in stainless steel-tanks with top-mounted agitators.

10x to 100x concentrated feed media were made using the media formulation of DMEM mixed with F12 with additional supplements (Sigma).

CELL CULTURE

The cell culture process performance evaluations were carried out in duplicate 250 mL shaker flasks or 2 Liter Applikon bioreactors containing CHO cell lines producing recombinant proteins. Feed schedules and volumes were based on 50 mL (flasks) or 1400 mL (bioreactors) working volume and fed 10% each on day 3, 6, and 8. The following conditions were used: Forma 5% CO2, humidified incubator, agitation speed of 160 rpm, and grown in standard serum-free production cell media at standard temperatures. Each shaker flask was sampled on the feed day for viable cell density and % viability (Cedex), pH, gases (BGA Analyzer), cell metabolites, (Nova Bioanalyzer), and osmolality (Fiske Osmometer).

At the completion of the production runs, 1-50 mL of clarified, conditioned media were saved and frozen at -30⁰C for future analysis. Daily samples from the media from all growth conditions were submitted for titer analysis by the Poros Protein A method. Multiple projects were investigated for cell culture performance using concentrated chemically defined feed media.

AMINO ACID SOLUBILITY v

Twenty different amino acids were investigated for their solubility in water. Based on the Merck Index, amino acids were grouped per their R group properties, i.e. positively charged or negatively charged, polar or nonpolar. Individual amino acid stock solutions (> 10g/L) were then prepared and investigated in culture since different forms of amino acids were utilized in cell culture media rather than standard textbook version, e.g. L-Arginine.HCI was used instead of L-Arginine. Solubility of an amino acid was found to be highly dependent on its concentration and unique properties. The concentrations of amino acids tested ranged from 1-10 g/L, which equals to 10x to 100x of standard cell culture media. All the amino acids in "Neutral" and "Acidic" columns can be dissolved in room temperature water in less than 20 minutes. L-Tyrosine and Cystine can only be dissolved at pH >10-11. L-Tyrosine is soluble (>20 g/L) at pH >11 , but maximum solubility is ~2.3 g/L when the pH is at 6-7, which is the typical pH of most of cell culture media. The basic form L-Tyrosine.2Na was used for these studies.

Amino acids with low pH solubility include L-Aspartic acid and L-Glutamic acid (free acid), while high pH soluble amino acids include cystine.2HCI and L- Tyrosine.2Na are soluble in basic conditions and the remaining, L-Arginine.HCI, L- Histidine .HCI.H2O, L-Lysine HCI, L-Valine, L-Asparagine.H2O, Glycine, L- Isoleucine, L-Proline, L-Threonine, L-Serine, L-Cysteine HCI. H2O, L-Glutamine, L- Leucine, L-Methionine, L-Phenylalanine and L-Tryptophan are neutral soluble.

It should be noted that while this experiment utilized a particular grouping of these amino acids, depending on the amino acid and the pH of the solution, some amino acids can change groups from acidic to basic.

SALT SOLUBILITY

Salts typically have high solubility when dissolved in water separately, but will precipitate when dissolved together at certain pH values. Ca++, Mg++, and Zn++ can form precipitates with sodium phosphate dibasic/sodium phosphate monobasic (Na2HPO4/NaH2HPO4) at pH greater than 5.8 and concentrations of 10x-100x. CaCI2 was found to be particularly prone to precipitation at the concentrations tested. Since precipitations are reversible reactions, they will disappear when pH is titrated down. However, most cell culture media are kept at pH 6-7. Since manufacturing media typically contains CaCI2 at a sufficient level for cells during 11- day manufacturing run, it has been found that there is sufficient Ca++ in the starting media for an entire production run (data not shown). Accordingly the feed media is prepared without Ca++. If If Ca++ is eliminated from the media, final pH can be adjusted to a desirable final pH between 5.4 and 7.4. If the Ca++ is not eliminated from the media, the final pH can be manipulated into a desirable final pH between 5.4 and 5.8

VITAMINS/NUCLEOSIDES Initially, 15Ox to 150Ox concentration levels of vitamins and nucleosides were investigated since it was possible to accurately measure a small amount of powders. It was previously observed that solubility of certain vitamins (e.g., riboflavin, folic acid) was dependent on both concentration level and pH value.

Most vitamins and nucleosides have excellent solubility at room temperature water within the concentrations tested, but four components are hard to dissolve and therefore required further investigation. Biotin and Na Hypoxanthine needed more rigorous mixing to get into solution. Riboflavin needed the pH adjusted to 10.8 for a concentration of 150Ox, but had good solubility at 5Ox. Folic acid required a pH of 8.6 for solubilization and maintained above pH 5.4 at this stage to avoid precipitation. Finally, in order to dissolve the mixture of vitamins and nucleosides in water, an adjustment to pH 8.6-9 was required, and the resulting, clear solution formed precipitate when the pH was adjusted back to 5.4. Thus, dissolving mixtures of vitamins/nucleosides requires adjusting pH to over 8.6, and maintaining a fully dissolved solution requires maintaining the pH over 5.4.

TWO-POWDER COMPOUNDING METHOD

From solubility studies of amino acids, salts, vitamins, and nucleosides, it is particularly notable that L-Tyrosine has excellent solubility (>20 g/L) when the pH is greater than 10-11. In addition, the maximum solubility is only around 2.3 g/L when the pH is around 6-7; Ca++, Mg++, and Zn++ can form precipitation with sodium phosphate dibasic/sodium phosphate monobasic (Na2HPO4/NaH2HPO4) at pH greater than 5.8. CaCI2 precipitation has the highest turbidity within the concentration level tested, and to dissolve the mixture of vitamins/nucleosides, requires adjusting pH over 8.6; to maintain the stability of clear solution, requires keeping pH above 5.4.

All the media components are then grouped into two powders, namely acidic (low pH) powder and basic (high pH) powder. Take advantages of already existed very low pH value components (e.g., NaH2PO4, acidic amino acids) and very high pH value components (e.g., L-Tyrosine.2Na.2H2O, Na2HPO4, etc.) so that no extra base or acid are added to these two powders. The acidic powder has a good solubility at pH values between 3 to 6 that help to prevent salts, such as CaCI2, from forming precipitation. Basic powder has a good solubility at pH values between 8 to 11 that help to dissolve vitamins, such as folic acid. During two-powder compounding, the basic powder is added first into room temperature water at 80-90% of final volume, achieving a high pH of about 9-11. After the basic powder is in solution, the second acidic powder is added driving the pH down to between 3.5 to 6.5 depending on the concentration of acidic powder. The final pH can be manipulated into a desirable final pH between 5.4 and 5.8 by adding additional components such as glucose, glutamine, NaHCO3, NaOH or HCI. If CaCI2 is eliminated from the media, final pH can be adjusted to 5.4-7.4.

With this method it is shown that standard (1Ox) and concentrated (10Ox) media can be prepared in a single vessel with two-powder system, further improving the manufacturability. The two-powder compounding method is performed in room temperature water (20+5⁰C), without acid/base titration, without heating/cooling step, and without multiple powders/stock solutions.

Acidic powder

NaH2PO4.H2O monobasic MW 138, CaCI2.2H2O, L-Arginine HCI, L- Histidine.HCI.H2O, L-Lysine HCI, L-Valine, L-Asparagine.H2O, Glycine, L-lsoleucine, L-Proline, L-Threonine, L-Serine, L-Cysteine (or HCI, H2O), L-Glutamic acid (free acid), L-Leucine, L-Methionine, L-Phenylalanine, L-Tryptophan, L-Aspartic acid, CuSO4.5H2O, KCI (MW 74.55), MgCI2 (anhyd.) MW 95.21 , MgSO4 (anhyd.), ZnSO4.7H20 Basic powder

Sodium Pyruvate, Putrescine.2HCI, Thymidine, Na Hypoxanthine, Cyanocobalamin (B12), Thiamine HCI, Riboflavin, Pyridoxine HCI, Pyridoxal HCI, Niacinamide, i-lnositol, Folic Acid, Choline Chloride, D-Ca Pantothenate, Biotin, L- Tyrosine.2Na.2H2O, Na2HPO4 dibasic (anhydr.) MW 142. Additional Components L-Glutamine, Glucose, NaHCO3

FEED MEDIA PERFORMANCE The two powder method described herein concentrates each media component to approximately 5Ox except a few components (e.g., glucose, glutamine), and three scheduled feed volumes drop to 5%, 5%, 7% of the culture media compared to three feeds of 10% using the standard feed media. Out of five cell lines tested, four outperform the control feed (feed media with peptone) with titers within a range of +/- 15%. In contrast, the titers produced by the lower concentration defined feeds are on average only 65% of the high concentration feeds. The harvest viability is comparable between control feed and 5Ox feed.

Next, rather than increasing concentration of most feed components to 5Ox as illustrated above, all major medium components were categorized into three subgroups based on the function, namely, amino acids, salts and vitamins/nucleosides. The three groups are titrated at concentrations up to four times (40X) that of typical feed media concentrations (10x). This model was applied to three cell lines expressing different recombinant proteins.

Four parameters that were measured were peak cell density, viability of cells, final titer of recombinant protein produced, and Qp₁ or specific rate of protein production. It was noted that the amino acid group had the most significant impact on the parameters. The salt group had no observed effect, while increasing the concentration of the vitamin/nucleoside group to 4OX decreased peak cell density by as much as 52%. The results indicate that optimal medium formulation can be predicted using a statistical design to balance different groups of media components. From this study, it demonstrates that this highly concentrated peptone-free feed (formulation: 34x-40x AA, 10x Salts, 34x Vitamins/Nucleosides; feed volume 7%, 10%, 10%) can achieve improved final titers, which are comparable to that.of titers found in an optimized process using peptone-containing media. In particular, one cell line showed a 50% increase in titer.

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

What is claimed is:

1. A fed batch cell culture method comprising culturing a mammalian cell genetically engineered to produce a protein and feeding the culture during a production phase, wherein the feed media is chemically defined and highly concentrated, and wherein production of the recombinant protein by the cell culture is increased when compared to a batch cell culture fed with a chemically defined media.

2. The method of claim 1 , wherein the concentrated feed media is at least

3Ox concentrated over growth media.

3. The method of claim 2, wherein the concentrated media is at least 4Ox concentrated over growth media.

4. The method of claim 1 , wherein the recombinant protein production is increased 10%.

5. The method of claim 1, wherein the recombinant protein production is increased 25%.

6. The concentrated feed media of claim 1 wherein the feed media is made from two separate powders, the first powder comprising acid soluble components and the second powder comprising basic soluble components.

7. A concentrated fed batch culture feed media comprising at least 30 fold concentrated components, and wherein the media is chemically defined.

8. The feed media of claim 7 wherein the components are amino acids, salts, vitamins and essential metals.

9. A method of making a concentrated fed batch culture media comprising dissolving basic soluble components in the solution, and subsequently dissolving acid soluble components in the solution, followed by adjusting the pH to physiological acceptable levels.

10. The method of claim 9, wherein the acidic conditions are between pH 3 and 6.

11. The method of claim 9, wherein the basic conditions are between pH 8 and 11.