WO2014022102A1 - Methods of using anti-apoptotic compounds to modulate one or more properties of a cell culture - Google Patents

Abstract

Methods of modulating the properties of a cell culture expressing a protein of interest are provided. In various embodiments the methods relate to the addition of anti-apoptotic compounds to growing cell cultures.

Description

METHODS OF USING ANTI-APOPTOTIC COMPOUNDS TO MODULATE ONE OR MORE PROPERTIES OF A CELL CULTURE

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119 of U.S. Provisional

Application Serial No. 61/679,464, filed August 3, 2012 and U.S. Provisional Application Serial No. 61/678,527, filed August 1, 2012, which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to compounds and processes for modulating one or more properties of a cell culture, including mammalian cell cultures such as CHO cell cultures.

BACKGROUND OF THE INVENTION

Apoptosis, or programmed cell death, is a cellular suicide process in which damaged or harmful cells are eliminated from multicellular organisms. Apoptosis is clearly distinguished from cell death (necrosis) resulting from environmental deterioration. Apoptosis is morphologically characterized by chromosome aggregation in cell nuclei, fragmentation of cell nuclei, disappearance of microvillus on the cell surface layers, aggregation of cytoplasm, and the like. When cells initiate apoptosis, they become atrophied. Intracellular contents are immediately incorporated by macrophages and surrounding cells without being released outside the cells.

Cells undergoing apoptosis have distinct morphological changes including cell shrinkage, membrane blebbing, chromatin condensation, apoptotic body formation and fragmentation. This cell suicide program is evolutionarily conserved across animal and plant species. Apoptosis also acts as a host defense mechanism. For example, virally infected cells are eliminated by apoptosis to limit the propagation of viruses.

Apoptosis can be of significant concern to those involved in the biotechnology industry. One common goal of this industry is to manufacture high levels of a particular protein of interest, which typically involves the use of high density cell cultures. In the context of high density cell culture technology, such as a cell growth process performed in a typical fed-batch bioreactor, apoptosis can account for a significant portion of cell death in the culture. In nature, apoptosis can be a results of a combination of one or many physiological factors; in a bioreactor apoptosis may be induced in response to stressors such as nutrient and growth factor deprivation, oxygen depletion, toxin accumulation, and shear stress (see, e.g., Goswami et al., (1999) Biotechnol. Bioeng. 62:632-640). Uncontrolled apoptosis can limit the maximum viable cell density, accelerate the onset of the death phase and potentially decrease the titer of a protein of interest, whether naturally occurring or recombinant {see, e.g., Chiang and Sisk, (2005) Biotechnol. Bioeng. 91 :779-792; Figueroa et al., (2001) Biotechnol. Bioeng. 73:21 1-222; Figueroa et al., (2003) Metab. Eng. 5:230-245; Figueroa et al., (2004) Biotechnol Bioeng. 85:589-600; and Mercille and Massie, (1994) Biotechnol. Bioeng. 44: 1 140-1 154). This decline can lead to a loss of productivity and an increase in the cellular contaminants that can complicate a production operation, both at commercial scale (typically -500L cultures and larger), at bench scale (typically up to ~20L) or at pilot scale (typically -20—200L).

Various methods of apoptosis prevention and inhibition have been used to maintain cell viability during extended production runs in mammalian cell culture {see, e.g., Arden and Betenbaugh, (2004) Trends Biotechnol. 22: 174-180; and Vives et al., (2003) Metab. Eng. 5: 124-132). For example, by altering the extracellular environment through media supplementation of growth factors, hydrolysates, and limiting nutrients it has been reported that these ameliorative measures provided increased protein production and decreased apoptosis (see, e.g., Burteau et al., (2003) In Vitro Cell Dev. Biol. 39A:291-296; and Zhang & Robinson, (2005) Cytotechnology 48:59-74).

Others in the industry have adopted chemical and genetic strategies to inhibit the apoptotic signaling cascade from within the cell {see, e.g., Sauerwald et al., (2002) Biotechnol. Bioeng. 77:704-716; and Sauerwald et al., (2003) Biotechnol. Bioeng. 81 :329- 340).

Despite these varied approaches, apoptosis-limited cell culture performance is still a problem, particularly in the area of the manufacture of proteins in large quantities. The disclosed methods and compositions address this shortfall. SUMMARY OF THE INVENTION

In one aspect, the instant disclosure provides a method of modulating the viability the cells of a cell culture, the titer of a protein of interest that is expressed by the cells of a cell culture, or both the viability of the cells of a cell culture and the titer of a protein of interest expressed by cells of a cell culture is provided. In one embodiment the method comprising: (a) inoculating cell culture medium with cells; and (b) maintaining the cell culture under conditions suitable for cell growth in the presence of one or more anti-apoptotic compound for a desired period of time. In various embodiments, the one or more anti-apoptotic compound comprises one or more of MDL 28170, cypermethrin, cyclosporine A, BBMP, Bongkrekic acid, S-15176 difumerate, cyclic pifithrin-a, pifithrin mu, BI-6C9, NSCI, NS3694 and Necrostatin- 1. In one embodiment, the one or more anti-apoptotic compound is present in the cell culture at a concentration of greater than or equal to 1 μg/mL. In still another embodiment the cell culture is a suspension culture. In a specific embodiment the suspension culture is a fed-batch culture. In yet another embodiment the cell culture is a perfusion culture. In a particular example of a perfusion culture the perfusion culture is an Alternating Tangential Flow (ATF) perfusion culture. In some embodiments, the cells of the cell culture are animal cells, and in particular embodiments the cells of the cell culture are mammalian cells. In specific embodiments the mammalian cells are one or more of VERO cells, MDCK cells, murine 3T3 cells, Chinese hamster ovary (CHO) cells, NSO₀, human HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS- 1), human hepatocellular carcinoma cells (e.g., Hep G2), human embryonic kidney cells (e.g., HEK 293 cells), and mouse myeloma NSO cells human HKB cells. When the cells of the cell culture are CHO cells, in various embodiments the CHO cells are one or more of BHK21, BHK TK , CHO, CHO-K-1, CHO-DUKX, PA- DUKX; CHO-S; CHO pro3-; CHO pro5; CHO P12; CHO dhfr-(CHO DX B l l, CHO DG44), CHO-DUK-B 1 1, CHO-Lecl O, CHO-Lecl, CHO-Lecl3. In some embodiments the protein of interest comprises an antigen binding protein. In other embodiments the protein of interest comprises an antibody. In particular embodiments the antibody comprises a non-human antibody, a humanized antibody or a fully human antibody. In a further embodiment the anti-apoptotic compound is added to the cell culture coincident with the inoculation. In still a further embodiment the anti-apoptotic compound is added to the cell culture at a time subsequent to the addition of cells. In still a further embodiment the anti-apoptotic compound is present in the cell culture medium prior to inoculation.

In another aspect, the instant disclosure provides a cell culture media comprising one or more anti-apoptotic compound. In one embodiment the one or more anti-apoptotic compound comprises one or more of MDL 28170, cypermethrin, cyclosporine A, BBMP, Bongkrekic acid, S-15176 difumerate, cyclic pifithrin-a, pifithrin mu, BI-6C9, NSCI, NS3694 and Necrostatin- 1. In a further embodiment the one or more anti-apoptotic compound is present in the media at a concentration of greater than or equal to 1 μg/mL.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a plot showing the viable cell density of a cell culture expressing antibody A that were achieved in the presence and absence of the anti-apoptotic compounds cyclic pifithrin, pifithrin, RBI, BBMP, BI-6C9 and Bongkrekic acid at several concentrations.

Figure 2 is a plot showing the cell viability of a cell culture expressing antibody A that were achieved in the presence and absence of the anti-apoptotic compounds cyclic pifithrin, pifithrin, RBI, BBMP, BI-6C9 and Bongkrekic acid at several concentrations.

Figure 3 is a plot showing the viable cell density of a cell culture expressing antibody B that were achieved in the presence and absence of the anti-apoptotic compounds cyclic pifithrin, pifithrin, RBI, BBMP, BI-6C9 and Bongkrekic acid at several concentrations. Figure 4 is a plot showing the cell viability of a cell culture expressing antibody B that were achieved in the presence and absence of the anti-apoptotic compounds cyclic pifithrin, pifithrin, RBI, BBMP, BI-6C9 and Bongkrekic acid at several concentrations.

Figure 5 shows the structures of three of the anti-apoptotic compounds studied; structures are shown for NSCI, NS3694 and Necrostatin- 1.

Figure 6 is a plot showing the viable cell density of a cell culture expressing antibody A that were achieved in the presence and absence of the anti-apoptotic compounds NSCI, NS3694 and Necrostatin- 1 at several concentrations.

Figure 7 is a plot showing the cell viability of a cell culture expressing antibody A that were achieved in the presence and absence of the anti-apoptotic compounds NSCI, NS3694 and Necrostatin- 1 at several concentrations.

Figure 8 is a plot showing the viable cell density of a cell culture expressing antibody B that were achieved in the presence and absence of the anti-apoptotic compounds NSCI, NS3694 and Necrostatin- 1 at several concentrations.

Figure 9 is a plot showing the cell viability of a cell culture expressing antibody B that were achieved in the presence and absence of the anti-apoptotic compounds NSCI, NS3694 and Necrostatin- 1 at several concentrations.

Figure 10 is a plot showing the viability of cells expressing Antibody A that were achieved in the presence and absence of the anti-apoptotic compounds NSCI, NS3694 and NSCI at a concentration of 10 μΜ after 17 days.

Figure 1 1 is a plot showing the viability of cells expressing Antibody A that were achieved in the presence and absence of the anti-apoptotic compounds NSCI, NS3694 and NSCI at a concentration of 10 μΜ after 17 days.

Figure 12 a plot showing the titer of cells expressing Antibody A that were achieved in the presence and absence of the anti-apoptotic compounds NSCI, NS3694 and NCBI at a concentration of 10 μΜ after 17 days.

Figure 13 is a plot showing the viability of cells expressing Antibody A that were achieved in the presence and absence of the anti-apoptotic compound combinations NS3694 and Necrostatin- 1, NSCI and Necrostatin- 1 and NSCI and NS3694 at a concentration of 5 μΜ after 17 days.

Figure 14 is a plot showing the viability of cells expressing Antibody A that were achieved in the presence and absence of the anti-apoptotic compound combinations NS3694 and Necrostatin- 1, NSCI and Necrostatin- 1 and NSCI and NS3694 at a concentration of 5 μΜ after 17 days.

Figure 15 a plot showing the titer of cells expressing Antibody A that were achieved in the presence and absence of the anti-apoptotic compound combinations NS3694 and Necrostatin-1 , NSCI and Necrostatin- 1 and NSCI and NS3694 at a concentration of 5 μΜ after 17 days.

Figure 16 is a plot showing the viability of cells expressing Antibody A that were achieved in the presence and absence of the anti-apoptotic compound combinations NS3694 and Necrostatin, NSCI and Necrostatin, NSCI and NS3694, and NSCI, NS3694 and Necrostatin at a concentration of 10 μΜ after 17 days.

Figure 17 is a plot showing the viability of cells expressing Antibody A that were achieved in the presence and absence of the anti-apoptotic compound combinations NS3694 and Necrostatin- 1, NSCI and Necrostatin-1, NSCI and NS3694, and NSCI, NS3694 and Necrostatin-1 at a concentration of 10 μΜ after 17 days.

Figure 18 a plot showing the titer of cells expressing Antibody A that were achieved in the presence and absence of the anti-apoptotic compound combinations NS3694 and Necrostatin-1 , NSCI and Necrostatin-1, NSCI and NS3694, and NSCI, NS3694 and Necrostatin-1 at a concentration of 10 μΜ after 17 days.

Figure 19 is a bar graph showing the effect on the viable and total cell density of five different cell lines each expressing a different antibody were obtained (Molecule A, Molecule B, Molecule C, Molecule D, Molecule E) of treatment with a combination of anti-apoptotic compounds (NSCI (10 μΜ) and NS3694 (10 μΜ) and Necrostatin- 1 (10 μΜ)) after 13 days.

Figure 20 is a bar graph showing the effect on the titer of five different cell lines each expressing a different antibody were obtained (Molecule A, Molecule B, Molecule C, Molecule D, Molecule E) of treatment with a combination of anti-apoptotic compounds (NSCI (10 μΜ) and NS3694 (10 μΜ) and Necrostatin- 1 (10 μΜ)) after 13 days.

Figure 21 comprises three plots and shows the effect of 10 μΜ or 30 μΜ of each of NSCI, NS3694 and Necrostatin-1 on the viable cell density of cultures comprising Hybridomas 1, 2, and 3.

Figure 22 comprises three plots and shows the integral viable cell densities of cultures comprising Hybridomas 1, 2, and 3.

Figure 23 comprises three plots and shows the effect of each of 10 μΜ or 30 μΜ of NSCI, NS3694 and Necrostatin-1 on the viable cell density of cultures comprising Hybridomas 1, 2, and 3.

Figure 24 comprises three plots which demonstrate the effect of the combination of NSCI, NS3694 and Necrostatin-1 at concentrations of 10 μΜ, 20 μΜ or 30 μΜ of on the cultures.

Figure 25 shows the results of a JMP model study that was performed to evaluate the effect of NSCI and NS3694 on viable cell density and culture viability. DETAILED DESCRIPTION OF THE INVENTION

Various compounds targeting different apoptotic mechanisms were identified. These compounds were found to improve cell viability and productivity. The compounds include the caspase 3 inhibitor NSCI, apoptosome formation inhibitor NS3694 and non-apoptotic death inhibitor Necrostatin- 1. The compounds were observed to be effective in reducing the loss of cell viability with improved productivity in fed-batch cultures expressing an antigen binding protein of interest.

Accordingly, the instant disclosure provides methods of modulating the properties of cell cultures expressing a "protein of interest;" as used herein a "protein of interest" specifically includes antigen binding proteins, such as antibodies and antibody fragments, and molecules that specifically bind a given antigen. In various embodiments the cell culture comprises a mammalian cell culture, such as CHO cells. In some embodiments the method comprises adding one or more anti-apoptotic compounds to a growing cell culture.

It is noted that the disclosed methods can be applied to cultures expressing any type of protein of interest, including naturally occurring proteins, recombinant proteins, engineered proteins (e.g., proteins that do not occur in nature and which have been designed and/or created by humans). A protein of interest can, but need not be, a protein that is known or suspected to be therapeutically relevant. Particular examples of a protein of interest include antigen binding proteins (as described and defined herein), peptibodies (i.e., a molecule comprising peptide(s) fused either directly or indirectly to other molecules such as an Fc domain of an antibody, where the peptide moiety specifically binds to a desired target; the peptide(s) may be fused to either an Fc region or inserted into an Fc-Loop, a modified Fc molecule. Fc-Loops are described in U.S. Patent Application Publication No. US2006/0140934 incorporated herein by reference in its entirety), fusion proteins (e.g., Fc fusion proteins, wherein a Fc fragment is fused to a protein or peptide) and hormones and other naturally occurring secreted proteins, and mutant forms of naturally occurring proteins.

The disclosed method is applicable to suspension cultures grown in stirred tank reactors (including traditional batch and fed-batch cell cultures, which may but need not comprise a spin filter), perfusion systems (including alternating tangential flow ("ATF") cultures, acoustic perfusion systems, depth filter perfusion systems, and other systems), hollow fiber bioreactors (HFB, which in some cases may be employed in perfusion processes) as well as various other cell culture methods (see, e.g., Tao et al., (2003) Biotechnol. Bioeng. 82:751-65; Kuystermans & Al-Rubeai, (201 1) "Bioreactor Systems for Producing Antibody from Mammalian Cells" in Antibody Expression and Production. Cell Engineering 7:25-52, Al-Rubeai (ed) Springer; Catapano et al., (2009) "Bioreactor Design and Scale-Up" in Cell and Tissue Reaction Engineering: Principles and Practice, Eibl et al. (eds) Springer-Verlag, incorporated herein by reference in their entireties). L Definitions

As used herein, the terms "a" and "an" mean one or more unless specifically indicated otherwise.

As used herein, the term "antigen binding protein" is used in its broadest sense and means a protein comprising a portion that binds to an antigen or target and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes binding of the antigen binding protein to the antigen. Examples of antigen binding proteins include a human antibody, a humanized antibody; a chimeric antibody; a recombinant antibody; a single chain antibody; a diabody; a triabody; a tetrabody; a Fab fragment; a F(ab')2 fragment; an IgD antibody; an IgE antibody; an IgM antibody; an IgGl antibody; an IgG2 antibody; an IgG3 antibody; or an IgG4 antibody, and fragments thereof. The antigen binding protein can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. See, e.g. , Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). In addition, peptide antibody mimetics ("PAMs") can be used, as well as scaffolds based on antibody mimetics utilizing fibronectin components as a scaffold.

An antigen binding protein can have, for example, the structure of a naturally occurring immunoglobulin. An "immunoglobulin" is a tetrameric molecule. In a naturally occurring immunoglobulin, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.

Naturally occurring immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain can be done in accordance with the definitions of Kabat et al. in Sequences of Proteins of Immunological Interest, 5^th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, (1991). As desired, the CDRs can also be redefined according an alternative nomenclature scheme, such as that of Chothia (see Chothia & Lesk, (1987) J. Mol. Biol. 196:901-917; Chothia et al, (1989) Nature 342:878-883 or Honegger & Pluckthun, (2001) J. Mol. Biol. 309:657-670).

In the context of the instant disclosure an antigen binding protein is said to

"specifically bind" or "selectively bind" its target antigen when the dissociation constant (K_D) is <10^"8 M. The antibody specifically binds antigen with "high affinity" when the K_D is <5x 10^"9 M, and with "very high affinity" when the K_D is <5x 10^"10 M.

As used herein, the term "antibody" refers to an intact immunoglobulin or to an antigen binding portion thereof that competes with the intact antibody for specific binding, unless otherwise specified. Antigen binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies and can form an element of a protein of interest. Antigen binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), fragments including complementarity determining regions (CDRs), single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.

A Fab fragment is a monovalent fragment having the V_L, V_H, C_L and C_H1 domains; a F(ab')2 fragment is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment has the V_H and C_H1 domains; an Fv fragment has the V_L and V_H domains of a single arm of an antibody; and a dAb fragment has a V_H domain, a V_L domain, or an antigen-binding fragment of a V_H or V_L domain (U.S. Pat. Nos. 6,846,634, 6,696,245, U.S. App. Pub. Nos. 05/0202512, 04/0202995, 04/0038291, 04/0009507, 03/0039958, Ward et al., (1989) Nature 341 :544-546).

A single-chain antibody (scFv) is an antibody in which a V_L and a V_H region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain wherein the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site (see, e.g., Bird et al., Science 242:423-26 (1988) and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-83). Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises V_H and V_L domains joined by a linker that is too short to allow for pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, e.g., Holliger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-48; and Poljak et al., (1994) Structure 2: 1 121-23). If the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical antigen binding sites. Polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites. Similarly, tribodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen binding sites, respectively, which can be the same or different.

Complementarity determining regions (CDRs) and framework regions (FR) of a given antibody can be identified using the system described by Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, (1991). As desired, the CDRs can also be redefined according an alternative nomenclature scheme, such as that of Chothia (see Chothia & Lesk, (1987) J. Mol. Biol. 196:901-917; Chothia et al, 1989, Nature 342:878-883 or Honegger & Pluckthun, (2001) J. Mol Biol. 309:657-670. One or more CDRs can be incorporated into a molecule either covalently or noncovalently to make it an antigen binding protein. An antigen binding protein can incorporate the CDR(s) as part of a larger polypeptide chain, can covalently link the CDR(s) to another polypeptide chain, or can incorporate the CDR(s) noncovalently. The CDRs permit the antigen binding protein to specifically bind to a particular antigen of interest.

An antigen binding protein can have one or more binding sites. If there is more than one binding site, the binding sites can be identical to one another or can be different. For example, a naturally occurring human immunoglobulin typically has two identical binding sites, while a "bispecific" or "bifunctional" antibody has two different binding sites.

For purposes of clarity, and as described herein, it is noted that an antigen binding protein can, but need not, be of human origin (e.g., a human antibody), and in some cases will comprise a non-human protein, for example a rat or murine protein, and in other cases an antigen binding protein can comprise a hybrid of human an non- human proteins (e.g., a humanized antibody).

A protein of interest can comprise a human antibody. The term "human antibody" includes all antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (a fully human antibody). Such antibodies can be prepared in a variety of ways, including through the immunization with an antigen of interest of a mouse that is genetically modified to express antibodies derived from human heavy and/or light chain-encoding genes, such as a mouse derived from a Xenomouse®, UltiMab™, or Velocimmune® system. Phage-based approaches can also be employed.

Alternatively, a protein of interest can comprise a humanized antibody. A "humanized antibody" is a specific form of an antigen binding protein and forms another aspect of the instant disclosure. A humanized antibody has a sequence that differs from the sequence of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and/or additions, such that the humanized antibody is less likely to induce an immune response, and/or induces a less severe immune response, as compared to the non- human species antibody, when it is administered to a human subject. In one embodiment, certain amino acids in the framework and constant domains of the heavy and/or light chains of the non-human species antibody are mutated to produce the humanized antibody. In another embodiment, the constant domain(s) from a human antibody are fused to the variable domain(s) of a non-human species. Examples of how to make humanized antibodies can be found in U.S. Pat. Nos. 6,054,297, 5,886,152 and 5,877,293.

An "Fc" region, as the term is used herein, comprises two heavy chain fragments comprising the C_H2 and C_H3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the C_H3 domains. Proteins of interest comprising an Fc region, incuding antigen binding proteins and Fc fusion proteins, form another aspect of the instant disclosure.

A "hemibody" is an immunologically functional immunoglobulin construct comprising a complete heavy chain, a complete light chain and a second heavy chain Fc region paired with the Fc region of the complete heavy chain. A linker can, but need not, be employed to join the heavy chain Fc region and the second heavy chain Fc region. In particular embodiments a hemibody is a monovalent form of an antigen binding protein disclosed herein. In other embodiments, pairs of charged residues can be employed to associate one Fc region with the second Fc region. A hemibody can be a protein of interest in the context of the instant disclosure.

The term "host cell" means a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present. A cell culture can comprise one or more host cells.

As used herein, the terms "culture" and "cell culture" are used interchangeably and refer to a cell population that is maintained in a medium under conditions suitable to survival and/or growth of the cell population. As will be clear to those of ordinary skill in the art, these terms as used herein also refer to the combination comprising the cell population and the medium in which the population is suspended.

The terms "polypeptide" and "protein" (e.g., as used in the context of a protein of interest or a polypeptide of interest) are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residues is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms can also encompass amino acid polymers that have been modified, e.g., by the addition of carbohydrate residues to form glycoproteins, or phosphorylated. Polypeptides and proteins can be produced by a naturally-occurring and non-recombinant cell, or polypeptides and proteins can be produced by a genetically-engineered or recombinant cell. Polypeptides and proteins can comprise molecules having the amino acid sequence of a native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. The terms "polypeptide" and "protein" encompass molecules comprising only that 20 naturally occurring amino acids, as well as molecules that comprise non-naturally occurring amino acids. Examples of non-naturally amino acids (which can be substituted for any naturally-occurring amino acid found in any sequence disclosed herein, as desired) include: 4- hydroxyproline, γ-carboxyglutamate, ε-Ν,Ν,Ν-trimethyllysine, ε-Ν-acetyllysine, O- phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5 -hydroxy lysine, σ- N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention. A non-limiting list of examples of non-naturally occurring amino acids that can be inserted into an antigen binding protein sequence or substituted for a wild- type residue in an antigen binding sequence include β-amino acids, homoamino acids, cyclic amino acids and amino acids with derivatized side chains. Examples include (in the L-form or D-form; abbreviated as in parentheses): citrulline (Cit), homocitrulline (hCit), Na- methylcitrulline (NMeCit), Na-methylhomocitrulline (Na-MeHoCit), ornithine (Orn), Na- Methylornithine (Na-MeOrn or NMeOrn), sarcosine (Sar), homolysine (hLys or hK), homoarginine (hArg or hR), homoglutamine (hQ), Na-methylarginine (NMeR), Na- methylleucine (Na-MeL or NMeL), N-methylhomolysine (NMeHoK), Na-methylglutamine (NMeQ), norleucine (Nle), norvaline (Nva), 1, 2,3, 4-tetrahydroisoquino line (Tic), Octahydroindole-2-carboxylic acid (Oic), 3-(l-naphthyl)alanine (1-Nal), 3-(2- naphthyl)alanine (2-Nal), 1,2,3, 4-tetrahydroisoquino line (Tic), 2-indanylglycine (Igl), para- iodophenylalanine (pl-Phe), para-aminophenylalanine (4AmP or 4-Amino-Phe), 4-guanidino phenylalanine (Guf), glycyllysine (abbreviated "K(Ns-glycyl)" or "K(glycyl)" or "K(gly)"), nitrophenylalanine (nitrophe), aminophenylalanine (aminophe or Amino-Phe), benzylphenylalanine (benzylphe), γ-carboxyglutamic acid (γ-carboxyglu), hydroxyproline (hydroxypro), p-carboxyl-phenylalanine (Cpa), a-aminoadipic acid (Aad), Na-methyl valine (NMeVal), N-a-methyl leucine (NMeLeu), Na-methylnorleucine (NMeNle), cyclopentylglycine (Cpg), cyclohexylglycine (Chg), acetylarginine (acetylarg), α, β- diaminopropionoic acid (Dpr), a, γ-diaminobutyric acid (Dab), diaminopropionic acid (Dap), cyclohexylalanine (Cha), 4-methyl-phenylalanine (MePhe), β, β-diphenyl-alanine (BiPhA), aminobutyric acid (Abu), 4-phenyl-phenylalanine (or biphenylalanine; 4Bip), a-amino- isobutyric acid (Aib), beta-alanine, beta-aminopropionic acid, piperidinic acid, aminocaprioic acid, aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic acid, N- ethylglycine, N-ethylaspargine, hydroxylysine, allo-hydroxylysine, isodesmosine, allo- isoleucine, N-methylglycine, N-methylisoleucine, N-methylvaline, 4-hydroxyproline (Hyp), γ-carboxyglutamate, ε-Ν,Ν,Ν-trimethyllysine, ε-Ν-acetyllysine, O-phosphoserine, N- acetylserine, N-formylmethionine, 3-methylhistidine, 5 -hydroxylysine, ω-methylarginine, 4- Amino-O-Phthalic Acid (4APA), and other similar amino acids, and derivatized forms of any of those specifically listed.

As used herein, the term "anti-apoptotic compound" refers to any compound known or suspected to decrease or eliminate the rate of apoptosis-mediated cell death in a cell culture relative to a the rate of apoptosis-mediated cell death in a cell culture which has not been exposed to the compound. Examples of anti-apoptotic compounds include but are not limited to MDL 28170, cypermethrin, cyclosporine A, BBMP, Bongkrekic acid, S-15176 difumerate, cyclic pifithrin-a, pifithrin mu, BI-6C9, NSCI, NS3694, Necrostatin- 1 and combinations thereof. Additional examples of anti-apoptotic compounds are described herein and are known in the art.

As used herein, the term "bioreactor" means any vessel useful for the growth of a cell culture. The cell cultures of the instant disclosure can be grown in a bioreactor, which can be selected based on the application of a protein of interest that is produced by cells growing in the bioreactor. A bioreactor can be of any size so long as it is useful for the culturing of cells; preferably a bioreactor is sized appropriate to the volume of cell culture being grown inside of it. Typically, a bioreactor will be at least 1 liter and may be 10, 50, 100, 200, 250, 500, 1,000, 1500, 2000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or more, or any volume in between. The internal conditions of the bioreactor, including, but not limited to pH and temperature, can be controlled during the culturing period. Those of ordinary skill in the art will be aware of, and will be able to select, suitable bioreactors for use in practicing the present invention based on the relevant considerations.

As used herein, the term "cell density" means the number of cells present in a given volume of medium.

As used herein, the term "cell viability" means the ability of cells in culture to survive under a given set of culture conditions or experimental variations. The term as used herein also refers to that portion of cells which are alive at a particular time in relation to the total number of cells, living and dead, in the culture at that time.

As used herein, the term "titer" means the total amount of a polypeptide or protein of interest (which may be a naturally occurring or recombinant protein of interest) produced by a cell culture in a given amount of medium volume. Titer can be expressed in units of milligrams or micrograms of polypeptide or protein per milliliter of medium.

As used herein, the term "fed-batch culture" refers to a form of suspension culture and means a method of culturing cells in which additional components are provided to the culture at a time or times subsequent to the beginning of the culture process. The provided components typically comprise nutritional supplements for the cells which have been depleted during the culturing process. Additionally or alternatively, the additional components may include supplementary components (e.g., an anti-apoptotic compound). A fed-batch culture is typically stopped at some point and the cells and/or components in the medium are harvested and optionally purified.

As used herein, the term "hybridoma" means a cell or progeny of a cell resulting from fusion of an immortalized cell and an antibody-producing cell. The resulting hybridoma is an immortalized cell that produces antibodies. The individual cells used to create the hybridoma can be from any mammalian source, including, but not limited to, hamster, rat, pig, rabbit, sheep, goat, and human. The term also encompasses trioma cell lines, which result when progeny of heterohybrid myeloma fusions, which are the product of a fusion between human cells and a murine myeloma cell line, are subsequently fused with a plasma cell. The term is meant to include any immortalized hybrid cell line that produces antibodies such as, for example, quadromas (see, e.g., Milstein et al., (1983) Nature, 537:3053).

As used herein, the terms "integrated viable cell density" or "IVCD" are used interchangeably and mean the average density of viable cells over the course of the culture multiplied by the amount of time the culture has run.

As used herein, the terms "medium," "cell culture medium" and "culture medium" are used interchangeably and mean a solution containing nutrients that nourish growing cells. In certain embodiments, a culture medium is useful for growing mammalian cells. Typically, a culture medium provides essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for minimal growth and/or survival. A culture medium may also contain supplementary components that enhance growth and/or survival above the minimal rate, including, but not limited to, hormones and/or other growth factors, particular ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds usually present at very low final concentrations), amino acids, lipids, and/or glucose or other energy source; as described herein, anti-apoptotic compounds can be added to a culture medium. In certain embodiments, a medium is advantageously formulated to a pH and salt concentration optimal for cell survival and proliferation. In certain embodiments, the medium is a feed medium that is added after the beginning of the cell culture. In certain embodiments, the cell culture medium is a mixture of a starting nutrient solution and any feed medium that is added after the beginning of the cell culture. IL General Considerations

When expressing a protein of interest in the cells of a cell culture, it is desirable to establish and maintain culture conditions which provide high levels of cell viability and high levels of protein expression. This goal can be hindered by the observation that various cell lines can behave differently under the same conditions, and it can be extremely difficult to identify conditions under which all of these properties reach desired levels. What is needed, therefore, is a method by which the properties of cell viability, protein expression level or both the cell viability and protein expression level of a cell culture can be easily maintained at desired levels. The method should be easily implemented, applicable at all scales, e.g., commercial scale (typically -500, -1,000, -1500, -2000, -2,500, -5,000, -8,000, -10,000, 12,000L and greater), bench scale (typically -1, -2, -5, -10, -15 and up to -20L) or pilot scale (typically -20, -50, -75, -100, -125, -150, -175 and up to -200L), and is applicable to any type of cell culture growth apparatus or method, including cultures grown in a fed-batch mode and cultures grown in an alternating tangential flow (ATF) mode. The disclosed method addresses these and other problems associated with cell culture performance.

As described herein, the disclosed methods are applicable to any type of protein of interest. As described herein, a protein of interest may be, for example, a recombinant fusion polypeptide or an antigen binding protein, such as a human or humanized antibody. A protein of interest can comprise any form of antigen binding protein, including antibody fragments, such as Fab fragments, heavy and light chains, etc. A protein of interest can, but need not, comprise a recombinant therapeutic protein. A protein of interest can comprise two or more subunits or chains (e.g., an antigen binding protein comprising a heavy and a light chain) and can be expressed using one, two or more plasmids with each plasmid encoding one subunit or chain.

III. Method of Modulating the Properties of a Cell Culture Thus, in one aspect, the instant disclosure provides a method of modulating the viability the cells of a cell culture, the titer of a protein of interest that is expressed by the cells of a cell culture, or both the viability of the cells of a cell culture and the titer of a protein of interest expressed by cells of a cell culture, the method comprising: (a) inoculating cell culture medium with cells; and (b) maintaining the cell culture under conditions suitable for cell growth in the presence of one or more anti-apoptotic compounds for a desired period of time. The method can be employed at any scale, e.g., commercial scale (typically -500L cultures and larger), at bench scale (typically up to -20L) or at pilot scale (typically -20- -200L). The method is also adapted to be performed as a component of a suspension culture (e.g., fed-batch) process or as a component of a perfusion process (e.g., an ATF process). In one embodiment, the method comprises inoculating cell culture medium with cells. Methods of inoculating cell culture medium with cells is well known to those of skill in the art and can be performed using any of known technique, with the proviso that at the completion of the inoculation one or more cells are present in the cell culture medium.

Continuing, the method comprises maintaining the cell culture under conditions suitable for cell growth in the presence of one or more anti-apoptotic compounds for a desired period of time. The specific conditions for optimal or desired cell growth (conditions need not be optimized for maximum cell growth in the context of the disclosed methods) may vary from cell line to cell line and can be empirically determined using readily available techniques and approaches known to those of skill in the art of biotechnology. Factors that can be considered when determining conditions for cell growth include cell culture medium composition, pH and buffer system, temperature, presence of agitation, dissolved oxygen concentration and cell culture vessel configuration and mode of operation.

Used as defined herein, the term "cell culture medium" encompasses all compounds that comprise a substrate upon which a cell culture can grow. Examples of compounds that can be present in or absent from a cell culture medium include a carbon source such as glucose, trace elements such as copper and manganese. A cell culture medium can comprise serum (e.g., Fetal Bovine Serum, "FBS") or it can be serum- free. A cell culture medium can be chemically defined, or can comprise complex molecules treated as an individual media component, such as bacterial or yeast tryptone.

The method comprises maintaining the cell culture in the presence of one or more anti-apoptotic compounds for a desired period of time. While it is not the intent of the inventor to be bound by any particular theory of operation, it is believed that the anti- apoptotic compound added has the effect of maintaining the cells in the culture in a productive and viable state longer than a culture incubated in the absence of such a compound. Examples of anti-apoptotic compounds include, but are not limited to, MDL 28170, cypermethrin, cyclosporine A, BBMP, Bongkrekic acid, S-15176 difumerate, cyclic pifithrin-a, pifithrin mu, BI-6C9, NSCI, NS3694, Necrostatin- 1 and combinations thereof. For example combinations can comprise Necrostatin- 1 and NSCI; Necrostatin- 1 and NS3694; Necrostatin- 1, NSCI and NS3694; and NSCI and NS3694, to list a few of the possible combinations of anti-apoptotic compounds.

The precise period time during which the cells are in contact with the one or more anti-apoptotic compounds can vary. Selection of this time period can be empirically determined based on cell culture performance (e.g., cell viability), titer of a protein of interest or by factors such as convenience or need.

Anti-apoptotic compounds that can be employed in the disclosed methods, including those described herein, encompass several broad classes of compounds, including caspase inhibitors, apoptosome formation inhibitors, necroptosis inhibitors, p53 inhibitors, tBID inhibitors and mitochondrial permeability inhibitors. It is intended that, while particular examples of these classes of compounds are provided herein, any particular compound from these classes of compounds can be employed in the disclosed methods.

When performing the disclosed methods, the amount of the anti-apoptotic compound present in a cell culture can vary. In some cases (e.g. , for certain cell lines) one concentration will be useful to provide the desired cell culture modulation; in other cases (e.g., other cell lines) a higher or lower concentration that used in conjunction with a different cell line will be useful. Thus, in some embodiments an anti-apoptotic compound (or combination thereof) is present in the cell culture at a concentration of greater than or equal to 1 μg/mL, for example about 5 μg/mL, about 10 μg/mL, about 15 μg/mL, about 20 μg/mL, about 25 μg/mL, about 30 μg/mL, about 35 μg/mL, about 40 μg/mL, about 45 μg/mL, about 50 μg/mL, about 100 μg/mL or greater than 100 μg/mL.

When a combination of anti-apoptotic compounds are employed in the disclosed methods, the concentrations of each compound in the combination can be the same or they can vary relative to one another. Thus, in one example, a first compound can be present in the cell culture medium at a concentration of about 10 μg/mL and a second compound can also be present in the cell culture medium at a concentration of 10 μg/mL. In another example, first compound can be present in the cell culture medium at a concentration of about 10 μg/mL and a second compound can also be present in the cell culture medium at a concentration of 30 μg/mL. Having provided representative concentrations, those of skill in the art can readily determine appropriate concentrations of single and combinations of anti-apoptotic compounds that can be employed in the disclosed methods and media.

As stated herein, the disclosed method can be performed in the context of any type of cell growth operation or process. Indeed, this forms one aspect of the instant disclosure, namely the effectiveness of the method when applied to a range of applications. Thus, in one embodiment the cell culture to which the anti-apoptotic compound is exposed is a culture grown using a suspension (e.g. , fed-batch) approach. In another embodiment, the cell culture to which the anti-apoptotic compound is exposed is a culture grown using a perfusion approach, such as an ATF cell culture.

As is known in the art, protein production can be performed using any of a variety of cells, including non-mammalian cells, and any type of cell can be employed in the disclosed methods. Examples of non-mammalian cells include but are not limited to bacterial, yeast, insect, and plants and are further described herein.

For many applications, it will be desirable to use eukaryotic cells to express a protein.

Accordingly, in one embodiment the cells in a cell culture to which an anti-apoptotic compound is exposed comprise animal cells, and in a further embodiment the animal cells comprise mammalian cells.

The disclosed methods will find particular applicability when applied to cultures comprising mammalian cells. Mammalian cells are readily available as hosts for expression and are well known in the art; the terms "mammalian cell lines" and "mammalian cells" encompasses many immortalized cell lines (e.g., hybridomas), including but not limited to VERO cells, MDCK cells, murine 3T3 cells, Chinese hamster ovary (CHO) cells, NSO₀, human HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS-1), human hepatocellular carcinoma cells (e.g., Hep G2), human embryonic kidney cells (e.g., HEK 293 cells), mouse myeloma NSO cells human HKB cells, or any number of other cell lines described herein. These and other cell lines that can be of use in practicing the disclosed methods can be readily obtained commercially or from American Type Culture Collection (ATCC).

For many recombinant applications, such as the production of antigen binding proteins (including antibodies), CHO cells are generally preferred. Accordingly, the disclosed method can be performed using any type of CHO cells, with the choice of the cell line being dictated by the desired properties of the recombinant protein being produced. Specific examples of CHO cell lines that can be employed in the method include BHK21, BHK TK , CHO, CHO-K-1, CHO-DUKX, PA- DUKX; CHO-S; CHO pro3-; CHO pro5; CHO PI 2; CHO dhfr-(CHO DX Bl l, CHO DG44), CHO-DUK-B 1 1, CHO-LeclO, CHO- Lecl, CHO-Lecl3, and cells or the derivatives/progenies of any of such cell line.

As noted, the disclosed method can be of particular utility when applied to a cell culture expressing any protein of interest. In some applications the cells of the cell culture have been transformed with a recombinant vector encoding a protein of interest. In these applications, the cell may not naturally express the protein of interest, or may only express the protein of interest naturally at very low levels (which can drive the selection of a recombinant approach). In other applications the cells of a cell culture may normally express the protein of interest and it may be desired to simply express higher titers of the protein. The disclosed methods are applicable to either situation.

When the cell culture comprises CHO cells expressing a recombinant protein the protein can comprise an antigen binding protein, as that term is used and defined herein. In a specific embodiment, an antigen binding protein can comprise an antibody. The antibody can comprise any form of antibody, including a fully human antibody (such as those produced by the Xenomouse® system), a humanized antibody, or a non-human antibody such as a murine antibody.

When performing the method, the anti-apoptotic compound (or combination of anti- apoptotic compounds) can be added to a cell culture at any point during a cell culture operation, for example before or after inoculation of cell culture media with cells, or coincident with inoculation of the cell culture media with cells. Accordingly, in one embodiment the anti-apoptotic compound (or combination of anti-apoptotic compounds) is added to the cell culture media coincident with the addition of cells. In another embodiment the anti-apoptotic compound (or combination of anti-apoptotic compounds) is added to the cell culture media following inoculation of the cell culture media with cell(s). In yet another embodiment the anti-apoptotic compound is added to the cell culture media prior to inoculation of the cell culture media with cells. This embodiment is described further herein. IV. Cell Culture Media Comprising an Anti-apoptotic Compound

In another aspect, the instant disclosure provides a cell culture media comprising an anti-apoptotic compound. The cell culture media can comprise any components, with the proviso that it also comprises an anti-apoptotic compound. Examples of compounds that can be present in a cell culture media in addition to an anti-apoptotic compound include trace nutrients, a carbon source, vitamins and amino acids. Examples of media that may be of use in the context of the disclosed methods include RPMI and DMEM media. Other suitable media formulations are known in the art and many are commercially available.

The media to which an anti-apoptotic compound (or combination of anti-apoptotic compounds) is added can be either liquid or solid (e.g., powdered) in form. For example, in one embodiment cell culture media can be prepared in a liquid base and the anti-apoptotic compound (or combination of anti-apoptotic compounds) can be added directly to the liquid media. In another embodiment, the anti-apoptotic compound (or combination of anti- apoptotic compounds) can be added in dry form to a pre-formulated cell culture media powder, which is subsequently hydrated. A cell culture media can optionally comprise phenol red or other components that can indicate the pH and other properties of the media.

The anti-apoptotic compound (or combination of anti-apoptotic compounds) can be present in the media at any concentration. The precise concentration can be determined empirically for a given cell culture, and can vary from one culture to another. For example, a first cell culture may deliver a desired profile (e.g., viability, protein titer, etc) using one concentration of anti-apoptotic compound, while a second cell culture may deliver a desired profile (e.g., viability, protein titer, etc) at a different concentration of anti-apoptotic compound. When performing the disclosed methods, the amount of the anti-apoptotic compound present in a cell culture can vary. In some cases (e.g. , for certain cell lines) one concentration will be useful to provide the desired cell culture modulation; in other cases (e.g., other cell lines) a higher or lower concentration that used in conjunction with a different cell line will be useful. Thus, in some embodiments an anti-apoptotic compound (or combination thereof) is present in the cell culture at a concentration of greater than or equal to 1 for example about 5 μg/mL, about 10 μg/mL, about 15 μg/mL, about 20 μg/mL, about 25 μg/mL, about 30 μg/mL, about 35 μg/mL, about 40 μg/mL, about 45 μg/mL, about 50 μg/mL, about 100 μg/mL or greater than 100 μg/mL.

When a combination of anti-apoptotic compounds are employed in the disclosed methods, the concentrations of each compound in the combination can be the same or they can vary relative to one another. Thus, in one example, a first compound can be present in the cell culture medium at a concentration of about 10 μg/mL and a second compound can also be present in the cell culture medium at a concentration of 10 μg/mL. In another example, first compound can be present in the cell culture medium at a concentration of about 10 μg/mL and a second compound can also be present in the cell culture medium at a concentration of 30 μg/mL. Having provided representative concentrations, those of skill in the art can readily determine appropriate concentrations of single and combinations of anti-apoptotic compounds that can be employed in the disclosed methods.

Examples of anti-apoptotic compounds that can form a component of the disclosed cell culture media include, but are not limited to, MDL 28170, cypermethrin, cyclosporine A, BBMP, Bongkrekic acid, S-15176 difumerate, cyclic pifithrin-a, pifithrin mu, BI-6C9, NSCl, NS3694, Necrostatin- 1 and combinations thereof. For example combinations can comprise Necrostatin- 1 and NSCl; Necrostatin- 1 and NS3694; Necrostatin- 1 , NSCl and NS3694; and NSCl and NS3694, to list a few of the possible combinations of anti-apoptotic compounds that can comprise elements of the disclosed cell culture media.

Anti-apoptotic compounds that can be employed in the disclosed media, including those described herein, encompass several broad classes of compounds, including caspase inhibitors, apoptosome formation inhibitors, necroptosis inhibitors, p53 inhibitors, tBID inhibitors and mitochondrial permeability inhibitors. It is intended that, while particular examples of these classes of compounds are provided herein, any particular compound from these classes of compounds can be employed in the disclosed media.

Various references have been provided in the instant disclosure. All references cited herein, including the Examples, are incorporated in their entireties for any purpose.

EXAMPLES

The Examples that follow are illustrative of various embodiments of the disclosed invention(s) and various uses thereof. They are set forth for explanatory purposes only, and should not be construed as limiting the scope of the disclosed invention(s) in any way. Example 1

Anti-apoptotic compounds were identified using the following general approach. Initially, the apoptotic pathway in mammalian cells, notably CHO cells, was examined. This analysis provided insight into various pathway steps involved in the apoptotic process, as well as various approaches to modulate a cell's apoptotic response. Representative steps identified as points at which intervention may modulate the apoptotic response include modulation of calcium flux in and out of the cell; variations in the permeability of the mitochondrial membrane; modulation of the formation of the apoptosome; variation in caspase activity; variation in p53 binding and inhibition of tBid binding, and pathways that lead to cell death but not through a traditional apoptosis pathway (e.g., cell necroptosis).

Example 2

A list of candidate compounds was compiled which was correlated with the points of intervention identified in Example 1 and is shown in Table 1

Table 1

A range of compounds were tested for their ability to enhance viable cell density and cell viability. In each case a cell culture expressing one of two fully human antibodies (Antibody A and Antibody B) was incubated with one candidate anti-apoptotic compound, which was added at varying concentrations to cell culture media.

Example 2A

In one set of experiments, the anti-apoptotic compounds tested included cyclic pifithrin (10 μΜ and 50 μΜ), pifithrin mu (10 μΜ and 50 μΜ), RBI (10 μΜ and 50 μΜ), BBMP (10 μΜ and 50 μΜ), BI-6C9 (10 μΜ and 50 μΜ) and Bongkrekic acid (50 μΜ). The anti-apoptotic compound was present in the culture at time t = 0 days in this set of experiments. See Figures 1-4. Figure 1 shows the observed effect of the anti-apoptotic compounds tested on the viable cell density of cells expressing Antibody A. As shown in Figure 1, the tested compounds did not provide the desired levels of enhancement in viable cell density of cells expressing Antibody A after 12 days.

Figure 2 shows the observed effect of the anti-apoptotic compounds tested on the cell viability of cells expressing Antibody A. As shown in Figure 2, the tested compounds did not provide the desired levels of enhancement in the viability of cells expressing Antibody A after 12 days.

Figure 3 shows the observed effect of the anti-apoptotic compounds tested on the viable cell density of cells expressing Antibody B. As shown in Figure 3, the tested compounds did not provide the desired levels of enhancement in viable cell density of cells expressing Antibody B after 10 days.

Figure 4 shows the observed effect of the anti-apoptotic compounds tested on the cell viability of cells expressing Antibody B. As shown in Figure 4, the tested compounds did not provide the desired levels of enhancement in the viability of cells expressing Antibody B after 10 days.

Example 2B

In a second set of experiments, the anti-apoptotic compounds tested included NSCI (10 μΜ and 50 μΜ), NS3694 (1 μΜ, 10 μΜ and 50 μΜ) and Necrostatin- 1 (2 μΜ, 10 μΜ and 50 μΜ). The structure of these compounds are shown in Figure 5. The anti-apoptotic compound was present in the culture at time t = 0 days in this set of experiments. See Figures 6-9.

Figure 6 shows the observed effect of the anti-apoptotic compounds tested on the viable cell density of cells expressing Antibody A. As shown in Figure 6, the tested compounds provided a modest enhancement in viable cell density of cells expressing Antibody A after 12 days.

Figure 7 shows the observed effect of the anti-apoptotic compounds tested on the cell viability of cells expressing Antibody A. As shown in Figure 7, the tested compounds provided a modest enhancement in cell viability of cells expressing Antibody A after 12 days.

Figure 8 shows the observed effect of the anti-apoptotic compounds tested on the viable cell density of cells expressing Antibody B. As shown in Figure 8, the tested compounds provided a modest enhancement in viable cell density of cells expressing Antibody B after 12 days. Figure 9 shows the observed effect of the anti-apoptotic compounds tested on the cell viability of cells expressing Antibody B. As shown in Figure 9, the tested compounds provided a modest enhancement in the viability of cells expressing Antibody B after 12 days. Example 3

It was decided to target the mitochondrial membrane depolarization to apoptosome formation pathway and the activation of caspase 3 and caspase 7. From this list NSCI, NS 3694 and Necrostatin- 1 were selected for further study based on a set of criteria including an assessment of experimental efficacy and evaluated concentration, time of addition and combinations.

Accordingly, in another set of experiments, several anti-apoptotic compounds were tested again as a component to a cell culture expressing Antibody A; only one anti-apoptotic compound was added to each culture. The anti-apoptotic compounds studied included NSCI (10 μΜ), NS3694 (10 μΜ) and Necrostatin- 1 (10 μΜ). The structures of these compounds are shown in Figure 5. The anti-apoptotic compound was present in the culture at time t = 0 days in this set of experiments. See Figures 10-12.

Figure 10 shows the observed effect of the anti-apoptotic compounds tested on the viability of cells expressing Antibody A. As shown in Figure 10, the tested compounds provided an enhancement in viable cell density of cells expressing Antibody A after 17 days.

Figure 1 1 shows the observed effect of the anti-apoptotic compounds tested on the viable cell of cells expressing Antibody A. As shown in Figure 1 1, the tested compounds provided a modest enhancement in cell viability of cells expressing Antibody A after 17 days.

Figure 12 shows the observed effect of the anti-apoptotic compounds tested on the titer of cells expressing Antibody A. As shown in Figure 12, the tested compounds provided a modest enhancement in cell viability of cells expressing Antibody A after 17 days.

Example 4

In another set of experiments, several anti-apoptotic compounds were tested again as a component to a cell culture expressing Antibody A. In contrast to previous experiments, in these experiments combinations of two anti-apoptotic compound were added to each culture. The anti-apoptotic compound combinations studied included NS3694 (5 μΜ) and Necrostatin- 1 (5 μΜ); NSCI (5 μΜ) and Necrostatin- 1 (5 μΜ); and NSCI (5 μΜ) and NS3694 (5 μΜ). The structures of these compounds are shown in Figure 5. The anti- apoptotic compound was present in the culture at time t = 0 days in this set of experiments. See Figures 13- 15. Figure 13 shows the observed effect of the anti-apoptotic compounds tested on the viability of cells expressing Antibody A. As shown in Figure 13, the tested compounds provided an enhancement in viable cell density of cells expressing Antibody A after 17 days.

Figure 14 shows the observed effect of the anti-apoptotic compounds tested on the viable cell of cells expressing Antibody A. As shown in Figure 14, the tested compounds provided a modest enhancement in cell viability of cells expressing Antibody A after 17 days.

Figure 15 shows the observed effect of the anti-apoptotic compounds tested on the titer of cells expressing Antibody A. As shown in Figure 15, the tested compounds provided a modest enhancement in cell viability of cells expressing Antibody A after 17 days.

Example 5

In another set of experiments, several anti-apoptotic compounds were tested again as a component to a cell culture expressing Antibody A. In contrast to previous experiments, in these experiments combinations of two or three anti-apoptotic compound were added to each culture. The anti-apoptotic compound combinations studied included NS3694 (10 μΜ) and Necrostatin-1 (10 μΜ); NSCI (10 μΜ) and Necrostatin- 1 (10 μΜ); NSCI (10 μΜ) and NS3694 (10 μΜ) and NSCI (10 μΜ) and NS3694 (10 μΜ) and Necrostatin-1 (10 μΜ). The structures of these compounds are shown in Figure 5. The anti-apoptotic compound was present in the culture at time t = 0 days in this set of experiments. See Figures 16-18.

Figure 16 shows the observed effect of the anti-apoptotic compounds tested on the viability of cells expressing Antibody A. As shown in Figure 16, the tested compounds provided an enhancement in viable cell density of cells expressing Antibody A after 17 days.

Figure 17 shows the observed effect of the anti-apoptotic compounds tested on the viable cell of cells expressing Antibody A. As shown in Figure 17, the tested compounds provided a modest enhancement in cell viability of cells expressing Antibody A after 17 days.

Figure 18 shows the observed effect of the anti-apoptotic compounds tested on the titer of cells expressing Antibody A. As shown in Figure 18, the tested compounds provided a modest enhancement in cell viability of cells expressing Antibody A after 17 days. Example 6

Based on the results achieved using the anti-apoptotic compounds alone or in combination it was of interest to examine the effect of various cell lines on the observed effects. Accordingly, five different cell lines each expressing a different antibody were obtained (Molecule A, Molecule B, Molecule C, Molecule D, Molecule E). Each culture was either treated with a combination of anti-apoptotic compounds (NSCI (10 μΜ) and NS3694 (10 μΜ) and Necrostatin-1 (10 μΜ)) or untreated and used as a control. At Day 13 of the experiment the total cell density, viable cell density and viability were measured. The results are presented in Figures 19 and 20. Collectively, these figures demonstrate that cell cultures producing Molecules A, B and C showed similar cell growth and viability at Day 13. The cell culture producing Molecule D exhibited reduced growth with treatment of the combination of anti-apoptotic compounds at Day 6; this culture also exhibited reduced productivity relative to controls. The cell culture producing Molecule A exhibited a higher productivity compared with controls. The cell culture producing Molecule E showed significantly higher viability and greater productivity compared with controls.

Table 2 summarizes the data of Figures 19 and 20 in a tabular form and demonstrates the effect of the anti-apoptotic compounds on cell density, viable cell density, viability and titer. In Table 2, "control" refers to a culture to which no anti-apoptotic compound was added and "treated" refers to a culture to which an anti-apoptotic compound was added.

Table 2

Example 7

A study of the effect of several anti-apoptotic compounds on fed-batch shake flask cultures was performed. The study involved three hybridomas, Hybridoma 1, Hybridoma 2 and Hybridoma 3. In this set of experiments the anti-apoptotic compounds NSCI, NS3694 and Necrostatin- 1 were added alone, or in combinations of two or more, to each culture at 10 and 30 um concentrations on Day 3 of culture. The cultures were studied with respect to viable cell density (VCD) and cell viability.

Briefly, 125 ml shake flasks were inoculated with 2e5 c/ml of hybridomas 1, 2 or 3 in

25 ml of Hybridoma SFM™ (Life Technologies) and incubated at 36 °C with 5% C0₂ and 160rpm. The cultures were fed on Day 3 and Day 5 with yeast extract and glutamine and glucose were added daily if necessary to maintain concentrations above 2mM and 2 g/L respectively.

Anti-apoptotic compounds were dissolved in dimethylsulfoxide (DMSO) to 10 mg/mL, sterile filtered and frozen at -20 °C. The diluted compounds were thawed and added to the cultures on Day 3 along with 50 of DMSO (0.2% volume/volume) itself as a control for any solvent effects. The results of the experiments are shown in Figures 21 25.

Figure 21 comprises three plots and shows the effect of each of NSCI, NS3694 and Necrostatin- 1 on the viable cell density of cultures comprising Hybridomas 1, 2, and 3. As Figure 21 demonstrates, cultures comprising Hybridomas 1 and 3 achieved higher peak viable cell densities. Figure 22 shows the integral viable cell densities.

Figure 23 comprises three plots and shows the effect of each of NSCI, NS3694 and Necrostatin- 1 on the viable cell density of cultures comprising Hybridomas 1, 2, and 3. As Figure 23 demonstrates, cultures comprising Hybridomas 1 and 2 achieved higher cell viability on Days 4 and 5 following treatment with NSCI on Day 3.

Following the experiments performed using individual anti-apoptotic compounds, the effect of combinations of the anti-apoptotic compounds NSCI, NS3694 and Necrostatin- 1 on cultures comprising Hybridoma 1 was studied. In these experiments the cultures were grown under appropriate conditions and the combination of NSCI, NS3694 and Necrostatin- 1 was added to the cultures at day 3. Figure 24 highlights the results of that study.

Briefly, NSCI, NS3694 and Necrostatin- 1 were evaluated using a response surface experimental design with concentrations from 10-30 μΜ with axial points at 3 μΜ and 37 μΜ with treatment on Day 3. Fed-batch shake flask cultures of Hyridoma 1 were studied.

125 ml shake flasks were inoculated with 2e5 c/ml of hybridomas 1 in 25 ml of

Hybridoma SFM™ (Life Technologies) and incubated at 36 °C with 5% C0₂ and 160rpm. The cultures were fed on Day 3 and Day 5 with yeast extract and glutamine and glucose were added daily if necessary to maintain concentrations above 2mM and 2 g/L respectively.

Anti-apoptotic compounds were dissolved in dimethylsulfoxide (DMSO) to 10 mg/mL, sterile filtered and frozen at -20 °C. The diluted compounds were thawed and added to the cultures on Day 3 along with 50 uL of DMSO (0.2% volume/volume) itself as a control for any solvent effects. The results of these combination studies are shown in Figures 24 and 25.

Figure 24 comprises three plots which demonstrate the effect of the combination of NSCI, NS3694 and Necrostatin- 1 on the cultures. Collectively, these plots demonstrate that treatment with the combination of compounds facilitated higher late culture viable cell densities and higher total numbers of viable cells. The overall culture viability of the treated cultures was similar to the control cultures.

Figure 25 shows the results of a JMP model study that was performed to evaluate the effect of NSCI and NS3694 on viable cell density and culture viability. The plots comprising Figure 25 demonstrate that, with respect to Hybridoma 1 , there was a positive impact of the anti-apoptotic compounds on viable cell density and culture viability by Day 5.

Example 8

The methods and compositions disclosed herein can be applied to suspension cultures as well as perfusion cultures, such as ATF cultures. Perfusion culture of a CHO cell line can be simulated using spin tube bioreactors with daily removal and addition of culture media. This design can be used to simulate stirred tank perfusion of a CHO cell line employing a hollow fiber-based cell retention system. This type of simulation can not only be indicative of how a given culture is likely to respond under perfusion conditions, but also represents a convenient and non-resource intensive way to optimize culture conditions, including variations in media composition, and to obtain preliminary data on the effect of a particular apoptotic compound or combination of apoptotic compounds on the cells of the culture.

This simulation design can also be used to study various culture conditions and how changes in the perfusion process, media, etc, affect the culture. For example, the recovery of a distressed culture can be studied. For example, a culture can be distressed and then conditions can be altered (e.g. , by the addition of one or more anti-apoptotic compounds such as NSCI, NS3694 and Necrostatin- 1 and combinations thereof) to try to rescue the culture. Alternatively, the sustainability of a healthy culture can also be studied (e.g., by the addition of one or more anti-apoptotic compounds such as NSCI, NS3694 and Necrostatin- 1 and combinations thereof).

In one embodiment, cells expressing a protein of interest can be inoculated into 50 ml spin tube reactors (TPP, Switzerland) at a cell density of 10e6 c/ml in 20ml of defined culture medium. The cultures can then incubated at 36 °C, 5% CO₂ and spun at 225 rpm with a 50mm radius of rotation. On each day of culture, the spin tube bioreactors can be centrifuged at lOOg for 5 minutes to pellet the cells; following pelleting, a portion of the medium can be removed and an equal volume of fresh medium can be added. This step of removal and addition of fresh media is representative of the continuous replacement of spent media with fresh media. The pelleted cells can then be resuspended and incubated.

NSCI, NS3694 and Necrostatin- 1 can be dissolved in DMSO or other appropriate solvent and can be added to the cultures at a desired concentration, e.g., 50 μΜ or as a combination of all three compounds at 30 μΜ each to the spin tube reactors. These compounds can be added at any point in the simulation, for example as a component of the media on which the cell cultures were grown, at the same time the media is inoculated with the cells or at a point subsequent to the inoculation. For example, the compounds can be added at a point at which the cell culture reaches a desired cell density.

Claims

CLAIMS What is claimed is:

1. A method of modulating the viability the cells of a cell culture, the titer of a protein of interest that is expressed by the cells of a cell culture, or both the viability of the cells of a cell culture and the titer of a protein of interest expressed by cells of a cell culture, the method comprising:

(a) inoculating cell culture medium with cells; and

(b) maintaining the cell culture under conditions suitable for cell growth in the presence of one or more anti-apoptotic compounds for a desired period of time.

2. The method of claim 1, wherein the oen or more anti-apoptotic compound comprises one or more of MDL 28170, cypermethrin, cyclosporine A, BBMP, Bongkrekic acid, S- 15176 difumerate, cyclic pifithrin-a, pifithrin mu, BI-6C9, NSCI, NS3694 and Necrostatin- 1.

3. The method of claim 1, wherein the one or more anti-apoptotic compound is present in the cell culture at a concentration of greater than or equal to 1 μg/mL.

4. The method of claim 1, wherein the cell culture is a suspension culture.

5. The method of claim 1, wherein the suspension culture is a fed-batch culture.

6. The method of claim 1, wherein the cell culture is a perfusion culture.

7. The method of claim 6, wherein the perfusion culture is an Alternating Tangential Flow (ATF) perfusion culture.

8. The method of claim 1, wherein the cells of the cell culture are animal cells.

9. The method of claim 1, wherein the cells of the cell culture are mammalian cells.

10. The method of claim 9, wherein the mammalian cells are one or more of VERO cells, MDCK cells, murine 3T3 cells, Chinese hamster ovary (CHO) cells, NSO₀, human HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS-1), human hepatocellular carcinoma cells (e.g., Hep G2), human embryonic kidney cells (e.g., HEK 293 cells), and mouse myeloma NSO cells human HKB cells.

1 1. The method of claim 10, wherein the CHO cells are one or more of BHK21 , BHK TK , CHO, CHO-K-1, CHO-DUKX, PA- DUKX; CHO-S; CHO pro3-; CHO pro5; CHO PI 2; CHO dhfr-(CHO DX Bl l, CHO DG44), CHO-DUK-B l l, CHO-LeclO, CHO- Lecl, CHO-Lecl3.

12. The method of claim 1, wherein the protein of interest comprises an antigen binding protein.

13. The method of claim 12, wherein the protein of interest comprises an antibody.

14. The method of claim 13, wherein the antibody comprises a non-human antibody, a humanized antibody or a fully human antibody.

15. The method of claim 1, wherein the anti-apoptotic compound is added to the cell culture coincident with the inoculation.

16. The method of claim 1, wherein the anti-apoptotic compound is added to the cell culture at a time subsequent to the addition of cells.

17. The method of claim 1, wherein the anti-apoptotic compound is present in the cell culture medium prior to inoculation.

18. A cell culture media comprising one or more anti-apoptotic compound.

19. The cell culture media of claim 18, wherein the one or more anti-apoptotic compound comprises one or more of MDL 28170, cypermethrin, cyclosporine A, BBMP, Bongkrekic acid, S-15176 difumerate, cyclic pifithrin-a, pifithrin mu, BI-6C9, NSCI, NS3694 and Necrostatin- 1.

20. The cell culture media of claim 19, wherein the one or more anti-apoptotic compound is present in the media at a concentration of greater than or equal to 1 μg/mL.