WO2002004598A2

WO2002004598A2 - Improvement of cell culture performance by inhibitors of pyruvate dehydrogenase kinase

Info

Publication number: WO2002004598A2
Application number: PCT/US2001/021864
Authority: WO
Inventors: Brian D. Follstad
Original assignee: Immunex Corp
Current assignee: Immunex Corp
Priority date: 2000-07-12
Filing date: 2001-07-10
Publication date: 2002-01-17
Anticipated expiration: 2003-01-12
Also published as: AU2001275897A1; WO2002004598A3

Abstract

The invention provides methods and compositions for improved in vitro culture of animal cells that make use of inhibitors of pyruvate dehydrogenase kinase. Particularly preferred inhibitors are salts of dichloroacetic acid such as sodium dichloroacetate.

Description

IMPROVEMENTOFCELLCULTUREPERFORMANCE

This application claims the benefit of U.S. Provisional Application No. 60/217,899, filed 12 July 2000, the disclosure of which is incorporated by reference herein.

FIELD OF THE INVENTION

This invention is in the general field of animal cell culture. More particularly, the invention concerns improved medium and methods for the cultivation of animal cell lines and the production of natural and recombinant products derived therefrom.

BACKGROUND OF THE INVENTION

A variety of medical conditions in humans are associated with high lactic acid levels in the blood and tissues. These conditions include strenuous exercise, stroke, cardiac arrest and suffocation. In addition, congenital lactic acidosis can be caused by inherited or spontaneously acquired mutations in genes encoding enzymes of mitochondrial glucose metabolism or oxidative phosphorylation. In aerobic respiration, glucose is broken down into pyruvate, which in turn is metabolized in the mitochondria to carbon dioxide and water via the tri-carboxylic acid (TCA) cycle. In lactic acidosis, pyruvate is instead shunted into anaerobic glycolysis and converted into lactic acid or lactate.

Dichloroacetate has been used for many years as a drug in humans to treat conditions associated with lactic acidosis as well as diabetes and hyperlipoproteinemia (reviewed in Stacpoole et al, 1998, Drag. Metabol. Rev. 30(3):499-539; see also US Patents Nos. 4,122,188 to Stacpoole, 4,631,294 to Barsan, and 5,587,397 to Fox). Dichloroacetate inhibits Pyruvate Dehydrogenase (PDH) kinase activity, which in turn leads to the activation of the PDH complex and the redirection of pyruvate away from lactate formation and into the TCA cycle (Crabb et al, 1981, Metabolism 30(10): 1024-1039). However, dichloroacetate is toxic to humans and other animals when administered at high or chronic doses.

Many commercially important proteins are produced in animal cells that are adapted for long term growth in culture. One of the limits to growing mammalian cell lines at high concentrations in culture is the production of lactate. Lactate accumulates in mammalian cell cultures, leading to acidification of the medium and inhibition of cell growth. In controlled pH cell culture, base is added in order to circumvent (at least in part) this problem.

Several authors have speculated that the accumulation of lactate in continuous mammalian cell cultures is due to a lack of enzyme activities connecting glycolysis with the TCA cycle (Fitzpatrick et. al., 1997, Appl. Biochem. Biotechnol. 43:93-116; Neermann and Wagner, 1996, J. Cell Physiol. 166:152-169; Petch et al, 1994, J. Cell Physiol. 161:71-76). Accordingly, Irani et al, 1999, Biotechnol. Bioeng. 66(4):238-46, circumvented the endogenous pathways and genetically engineered mammalian cells to express an exogenous yeast cytosolic pyruvate carboxylase. hi this metabolic scheme, the authors proposed that the cells converted pyruvate to malate, which then entered the TCA cycle.

Nevertheless, there remains a need in the art for simpler methods of improving the primary metabolism of animal cell cultures so as to reduce lactate production, thereby avoiding the consequent negative impacts on proliferation and expression characteristics of lactate accumulation. The invention fullfils this need by providing a simple, easy and inexpensive way of reducing the production of lactate by cultured animal cells.

SUMMARY OF THE INVENTION

In the invention provided herein, chemical inhibitors of pyruvate dehydrogenase kinase are added to medium used for culturing animal cells in vitro. Animal cell cultures grown in such medium demonstrated significantly reduced production of lactate. Consequently, the pH shifts that typically accompany cultured cell growth were reduced in non-pH controlled cell cultures, and less base addition was required in pH controlled cell cultures. Thus, culture robustness and performance was improved.

Accordingly, in one aspect, the invention provides a method for culturing animal cells in vitro, the method comprising culturing the animal cells in tissue culture medium containing an effective amount of an inhibitor of pyruvate dehydrogenase kinase. Preferred cells are mammalian cells, and particularly CHO cells. The inhibitor of pyruvate dehydrogenase kinase can be, for example, dichloroacetate or related salts of dichloroacetetic acid. Alternatively, the inhibitor of pyruvate dehydrogenase kinase can be (R)-3, 3, 3-trifluoro-2-hydroxy-2- methylpropionic acid, or an anilide or amide derivative thereof. The invention finds particular use in the culturing of animal cells that are genetically engineered to express a protein of interest. In a related aspect, the invention also provides a tissue culture medium containing an effective amount of an inhibitor of pyruvate dehydrogenase kinase.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Viable cell concentration (solid lines) and percent cell viability (broken lines) measured as a function of time in culture. CHO cells were grown in shake flasks containing medium supplemented with the indicated concentration of dichloroacetate.

Figure 2. Lactate accumulation (solid lines) and culture pH (broken lines) measured as a function of time in culture. CHO cells were grown in shake flasks containing medium supplemented with the indicated concentration of dichloroacetate. Figure 3. Viable cell concentration (solid lines) and percent cell viability (broken lines) measured as a function of time in culture. CHO cells were grown in bioreactors containing medium supplemented with the indicated concentration of dichloroacetate.

Figure 4. Lactate accumulation (solid lines) and sodium carbonate base added (broken lines) measured as a function of time in culture. CHO cells were grown in bioreactors containing medium supplemented with the indicated concentration of dichloroacetate.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, in part, on the discovery that inhibitors of pyruvate dehydrogenase kinase can be used in vitro to improve the performance and growth of animal cell cultures. Specifically, inhibitors of pyruvate dehydrogenase kinase act to reduce lactate formation in production cell culture, thereby enhancing culture robustness.

In illustrative, non-limiting examples described below, dichloroacetate was used as a model compound to inhibit pyruvate dehydrogenase kinase activity in a mammalian cell culture system. Using a non-pH controlled culture system (shake flasks), the reduction in lactate formation significantly reduced the pH shifts that accompanied cell growth which in turn led to a dramatic increase in the viable cell concentration and viability, hi a pH-controlled bioreactor, the lactate levels were again lower in the culture containing dichloroacetate but without a corresponding increase in the viable cell concentration. However, the amount of base required to maintain the pH set-point was noticeably reduced. These results demonstrate that inhibitors of pyruvate dehydrogenase kinase can be used to substantially improve the performance and reliability of in vitro animal cell culture systems.

The invention can improve the culture performance of generally any type of animal cell that can be grown in in vitro culture including but not limited to mammalian cells, insect cells, avian cells and reptilian cells. By animal cell is meant a cell whose progenitors were derived from a multicellular animal. Preferably, the animal cell lines are mammalian cell lines. The invention is particularly advantageous for growing industrially important mammalian cell lines that have been adapted to grow in long term culture.

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, IL). Some of the more established cell lines typically used in the industrial or academic laboratory and which are preferred are CHO, VERO, BHK, HeLa, Cos, CVl, MDCK, 293, 3T3, PC12, hybridoma, myeloma, and WI38 cell lines, to name but a few examples. The dihydrofolate reductase (DHFR)-deficient mutant cell line (Urlaub et al, 1980, Proc Natl Acad Sci USA 77:4216-4220), DXB 11 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 protein 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. Thus, for purposes herein, in vitro cell culture excludes the in vitro culturing of tissues isolated from an organism (e.g., a perfused heart tissue). Typically, in vitro cell culture is performed under sterile, controlled temperature and atmospheric conditions in tissue culture plates (e.g., 10 cm plates, 96 well plates, etc.), roller bottles or other adherent culture (e.g.. , on microcarrier beads) or in suspension culture. 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, DE; Cell Trends, Inc., Middletown, MD) as well as university and/or government- sponsored organizations (e.g., The Cell Culture Center, Minneapolis, MN) offer cell culture services on a contract basis.

Animal cell cultures can be cultured in small to large scale processes, hi small, non-pH controlled processes (where pH is controlled by the initial buffer components in the medium, and not adjusted during the culture process except by the changing of the medium), the invention finds particular use because it reduces pH shifts due to lactate production. Hence, the cell cultures have increased levels of viable cell concentration and percent viability. In pH controlled processes (e.g., bioreactors), where the pH is continually adjusted, less base components are necessary over the life of the culture. Therefore, the invention provides a number of advantages regardless of the size or type of cell culture used. Tissue 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, tissue culture medium contains a buffer, salts, energy source, amino acids, vitamins and trace essential elements. In addition, the medium can oftentimes require additional components such as growth factors, lipids, and/or other serum components (e.g., transferrin). Any media capable of supporting growth of animal cells in culture can be used; the invention is broadly applicable to animal cells in culture, particularly mammalian cells, and the choice of media is not crucial to the invention. Tissue culture media suitable for use in the invention are commercially available from ATCC (Manassas, VA). For example, any one or combination of the following media can be used: RPMI-1640 Medium, Dulbecco's Modified Eagle's Medium, Minimum Essential Medium Eagle, F-12K Medium, Iscove's Modified

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

Added to the medium is an effective amount of an inhibitor of pyruvate dehydrogenase kinase. An effective amount is that amount sufficient to reduce lactate formation by at least 10%, and preferably 20%, over 48 hours in culture. An inhibitor of pyruvate dehydrogenase kinase is defined as an organic molecule that prevents PDH kinase from phosphorylating the specific serine residues on the El (alpha subunit) of the PDH complex involved in regulating PDH activity. Excluded from the definition of an inhibitor of pyruvate dehydrogenase kinase for purposes of the invention are non-specific inhibitory conditions such as alterations in ionic strength and/or phosphate concentrations, and non-specific kinase and/or enzymatic inactivating agents such as DEPC. Also excluded from the definition of an inhibitor of pyruvate dehydrogenase kinase for purposes of the invention is pyruvate, since addition of pyruvate also increases the undesired production of lactate.

A variety of specific inhibitors of pyruvate dehydrogenase kinase are known in the art. For example, salts of dichloroacetic acid are well known and commercially available. Generally, such salts of dichloroacetic acid will have the following formula:

wherein X is any mono or divalent metallic cation, a is an integer from 1 to 2 inclusive, and b is an integer from 1 to 2 inclusive.

Specific salts include those formed by the alkali metal and alkaline earth metal ions such as sodium, potassium, calcium, and magnesium, ammonium, and substituted ammonium where the substituent is a mono- or di-lower alkyl radical of 1-4 carbon atoms and ethylene di- ammonium. Pharmaceutically acceptable salts, with minimum cell cytotoxicity, are preferred. Specific pharmaceutical salts useful in this invention include sodium dichloroacetate, potassium dichloroacetate, and diisoproyl ammonium dichloroacetate. The sodium dichloroacetate and free base forms are highly preferred. Generally, salt and free base forms of dichloroacetate are particularly preferred for use in the invention because of their ready availability and economical price. Salts of dichloroacetic acid can be used at a concentration between about 0.5 mM to about 100 mM, preferably about 50 mM. Preferably such compounds are used at a concentration of at least about 1 mM, more preferably at least about 4 mM, and most preferably at about 10 mM. The invention also includes the use of other specific inhibitors of pyruvate dehydrogenase kinase that are known or yet to be discovered. For example, difluoroacetate, 2-chloroproprionate, 3- chloroproprionate, and 2,2' -dichloroacetate are inhibitors (Crabb et al, 1981, cited above). In addition, (R)-trifluoro-2-hydroxy-2-methylpropionic acid is a mild inhibitor. Bebemitz et al. , 2000, J. Med. Chem. 43(ll):2248-57, describes a series of anilides of (R)-trifluoro-2-hydroxy-2- methylpropionic acid that act as improved inhibitors of pyruvate dehydrogenase kinase. These include N-phenyl-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide 1, and derivatives substituted at the ortho position with a small electron-withdrawing group, such as, for example, chloro, acetyl, or bromo. A particularly preferred compound is N-(2-Chloro-4-isobutylsulfamoylphenyl)- (R)-3, 3, 3-trifluoro-2-hydroxy-2-methylpropionamide. Further, Aicher et al, 2000, J. Med. Chem. 43(2):236-249, describe a series of secondary amides that are also derived from (R)- trifluoro-2-hydroxy-2-methylpropionic acid.

The concentration of such compounds to use in the invention can be determined by those skilled in the art by, for example, comparing the inhibitory activity against pyruvate dehydrogenase kinase of the compound to that of dichloroacetate, and extrapolating appropriate concentrations therefrom. The extrapolated concentrations can then be used as a starting point to determine the range of effective amounts of compound that should be added to a culture medium, which amounts can then be determined using small scale experiments such as those described herein.

The invention finds particular utility in improving the production of proteins via cell culture processes. The cell lines used in the invention can be genetically engineered to express a protein of commercial or scientific interest. By genetically engineered is meant that the cell line has been transfected, transformed or transduced with a recombinant polynucleotide molecule, and/or otherwise altered (e.g., by homologous recombination and gene activation) so as to cause the cell to express 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 of skill 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).

Particularly preferred proteins for expression are protein-based drugs, also known as biologies. Preferably, the proteins are expressed 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, RANTES, growth hormone, insulin, insulinotropin, insulin-like growtii factors, parathyroid hormone, interferons, nerve growth factors, glucagon, interleukins 1 through 18, colony stimulating factors, lymphotoxin-β, 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 Cvtokines: Handbook for Basic and Clinical Research. Vol. II (Aggarwal and Gutterman, eds. Blackwell Sciences, Cambridge MA, 1998); Growth Factors:A Practical Approach (McKay and Leigh, eds., Oxford University Press Inc., New York, 1993) and The Cvtokine Handbook (AW Thompson, ed.; Academic Press, San Diego CA; 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), Interleukin-1 receptors (type 1 and 2), rnterleukin-4 receptor, rnterleukin-15 receptor, Interleukin-17 receptor, Interleukin-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-Inducing Receptor (AIR). A particularly preferred receptor is a soluble form of the B -l receptor type II; such proteins are described in US Patent 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 VI (Proceedings of the Vlth 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 4 IBB and OX40; the ligands are often members of the TNF family (as are 4 IBB 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 VJJJ, Factor LX, apolipoprotein E, 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 Bl; Boss et al, U.S. Pat. No. 4,816,397; Boss et al, European Patent No. 0,120,694 Bl; Neuberger, M. S. et al, WO 86/01533; Neuberger, M. S. et al, European Patent No. 0,194,276 Bl; Winter, U.S. Pat. No. 5,225,539;

Winter, European Patent No. 0,239,400 Bl; Queen et al, European Patent No. 0451 216 Bl; and Padlan, E. A. et al, EP 0 519 596 Al. For example, the invention can be used to induce the expression 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, CDlla, CD18, NGF, CD20, CD45, Ep-cam, other cancer cell surface molecules, TNF-alpha, TGF-bl, 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 IgGl. 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.

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 (HJ.C; using such resins as phenyl ether, butyl ether, or propyl ether), HPLC, or some combination of above.

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).

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

Example: Shake Flask

In this experiment, the effect of adding various concentrations of dichloroacetate was tested in mammalian cell shake flask cultures.

A Chinese Hamster Ovary (CHO) cell line that had been genetically engineered to produce TNFR:Fc was grown in serum-free medium containing lipids.

Shake flasks were inoculated at 5.0xl0⁵ cells/ml with either 0, 0.5, 5.0, or 10.0 mM of dichloroacetate (Cat.# 157638, ICN Biomedicals, Inc., Costa Mesa, CA). The dichloroacetate was added to the medium and pH adjusted with NaOH before adding the cells. Shake flasks were maintained in an incubator set at 37°C on a shake flask platform set at 150 rpm. Measurements for cell concentration and viability were taken using a hemacytometer and the trypan blue dye exclusion method. Glucose and lactate levels were determined using a 2700 Select YSI analyzer (Yellow Springs, OH). pC0₂, p0₂, and pH were determined using a blood gas analyzer (Chiron, Emeryville, CA).

Viable cell concentration and viability values for the cultures are shown in Figure 1. The higher dichloroacetate concentrations resulted in increasing viable cell concentrations and viabilities. The most dramatic results were observed using 10.0 mM dichloroacetate in the culture, in which the viable cell concentration was almost twice that of the control with a significantly higher viability. This difference in culture viable cell concentration and viability could be explained by measuring both the culture lactate levels and pH. The measured lactate concentrations and pH correlated directly with the dichloroacetate levels as shown in Figure 2. Here, 10.0 mM dichloroacetate resulted in an almost one-third drop in the culture lactate levels and an almost 0.3 pH unit difference compared to the control.

Example: Small-Scale Bioreactor

The pH changes in the shake flask experiments most likely played a significant role in the observed changes in the culture viable cell concentrations and viabilities. Therefore, a small-scale bioreactor experiment was performed to test the effect of adding dichloroacetate under pH controlled conditions.

The bioreactor experiments were performed using 2.0 L reactors with a 1.0 L working volume (Applikon, Foster City, CA). A DCU controller (B. Braun Biotech International, AUentown, PA) was used to maintain the reactor temperature at 37 °C, the pH at 7.0 using sodium carbonate base addition, and the dissolved oxygen concentration at 30% air saturation. Agitation was set at 150 rpm. Oxygen was supplied through headspace aeration and sparging. Reactor inoculum was obtained from spinner flask cultures grown under conditions identical to those described above for the shake flask cultures. Dichloroacetate was added to the medium and pH adjusted with NaOH before adding the cells. Reactors were inoculated at 5.0xl0⁵ cells/1 with a viability greater than 94%. The cell concentration, viability, glucose, lactate, pC0₂, p0₂, and pH were measured as described above.

In this experimental set-up, the culture viable cell concentrations and viabilities did not differ noticeably for the control and dichloroacetate containing cultures as shown in Figure 3. However, the lactate levels and the amount of sodium carbonate base added to adjust the culture pH did differ as shown in Figure 4. In this case, the culture with dichloroacetate had a lower amount of lactate and a corresponding reduction in the amount of base required to neutralize this acid. However, after 100 hours the ρC0₂ levels increased faster in the culture containing dichloroacetate versus the control. The additional acidity caused by rising pC0₂ levels caused the base addition requirement to increase at that point. Using more effective methods to eliminate the accumulation of dissolved C0₂ in the dichloroacetate culture would most likely result in further reduction in the amount of base required.

The results from these two experiments show how adding an inhibitor of pyruvate dehydrogenase kinase, in this case dichloroacetate, can be applied in mammalian cell culture for a number of purposes. In non-pH controlled cultures, dichloroacetate clearly reduces the amount of lactate produced and thus significantly reduces the pH changes that occur during a culture. This in turn has a positive impact on the culture viability and viable cell concentration. In pH controlled environments, dichloroacetate still has the ability to reduce lactate production and therefore culture base addition requirements. In short, adding an inhibitor of pyruvate dehydrogenase kinase, dichloroacetate, helps to create a more robust mammalian cell culture process by reducing the effects of lactate production.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method for culturing animal cells in vitro, the method comprising culturing the animal cells in tissue culture medium containing an effective amount of an inhibitor of pyruvate dehydrogenase kinase.

2. The method of claim 1, wherein the animal cells are mammalian cells.

3. The method of claim 2, wherein the mammalian cells are selected from the group consisting of CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, a myeloma cell line, a hybridoma cell line, and WI38 cells.

4. The method of any one of claims 1-3, wherein the inhibitor of pyruvate dehydrogenase kinase is a compound of the formula:

5. The method of claim 4, wherein the inhibitor of pyruvate dehydrogenase kinase is dichloroacetate.

6. The method of claim 5, wherein the dichloroacetate is at a concentration of between about 1 mM to about 100 mM.

7. The method of claim 5, wherein the dichloroacetate is at a concentration of above about 1 mM.

8. The method of claim 5, wherein the dichloroacetate is at a concentration of above about 4 mM.

9. The method of claim 5, wherein the dichloroacetate is at a concentration of about 10 mM.

10. The method of any one of claims 1-3, wherein the inhibitor of pyruvate dehydrogenase kinase is an anilide derivative of (R)-3, 3, 3-trifluoro-2-hydroxy-2-methylpropionic acid.

11. The method of claim 10, wherein the anilide derivative is N-(2-Chloro-4- isobutylsulfamoylphenyl)-(R)-3, 3, 3-trifluoro-2-hydroxy-2-methylpropionamide.

12. The method of any one of claims 1-11, wherein the animal cells are genetically engineered to express a protein of interest.

13. The method of claim 12, wherein the protein of interest is selected from the group consisting of a soluble TNF receptor, a soluble JL-4 receptor, a soluble JL-1 type II receptor, a soluble flt3 ligand, a soluble CD40 ligand, an erythropoeitin, an antibody, an Fc-fusion protein, a calcitonin, a growth hormone, an insulin, an insulinotropin, insulinlike growth factors, a parathyroid hormone, an interferons, a nerve growth factor, a glucagon, an interleukins, a colony stimulating factor, a glucocerebrosidase, a superoxide dismutase, a tissue plasminogen activator, a Factor VIII, a Factor LX, an apolipoprotein E, an apolipoprotein A-I, a globin, an JL-2 receptor, an IL-2 antagonist, alpha-1 antitrypsin, and an alpha-galactosidase A.

14. The method of any one of claims 1-13, wherein the animal cells are cultured in bioreactors.

15. The method of any one of claims 1-14, further comprising collecting the protein of interest.

16. A tissue culture medium containing an effective amount of an inhibitor of pyruvate dehydrogenase kinase.

17. The tissue culture medium of claim 16, wherein the inhibitor of pyruvate dehydrogenase kinase is a compound of the formula:

18. The tissue culture medium of claim 17, wherein the inhibitor of pyruvate dehydrogenase kinase is dichloroacetate.

19. The tissue culture medium of claim 18, wherein the dichloroacetate is at a concentration of between about 1 mM to about 100 mM.

20. The tissue culture medium of claim 18, wherein the dichloroacetate is at a concentration of above about 1 mM.

21. The tissue culture medium of claim 18, wherein the dichloroacetate is at a concentration of above about 4 mM.

22. The tissue culture medium of claim 18, wherein the dichloroacetate is at a concentration of about 10 mM.

23. The tissue culture medium of claim 16, wherein the inhibitor of pyruvate dehydrogenase kinase is an anilide derivative of (R)-3, 3, 3-trifluoro-2-hydroxy-2-methylpropionic acid.

24. The tissue culture medium of claim 23, wherein the anilide derivative is N-(2-Chloro-4- isobutylsulfamoylphenyl)-(R)-3, 3, 3-trifluoro-2-hydroxy-2-methylpropionamide.

25. The tissue culture medium of any one of claims 16-24, further comprising an animal cell line.

26. The tissue culture medium of claim 25, wherein the animal cells are mammalian cells.

27. The tissue culture medium of claim 26, wherein the mammalian cells are selected from the group consisting of CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, a myeloma cell line, a hybridoma cell line, and WI38 cells.

28. The tissue culture medium of any one of claims 25-27, wherein the animal cells are genetically engineered to express a protein of interest.