Taurine supplemented cell culture medium and methods of use

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

CELL CULTURE MEDIUM SUPPLEMENTED WITH TAURINE AND METHODS OF USE

FIELD OF INVENTION

The invention relates to a medium and methods for culturing cells and for producing recombinant proteins. Specifically, the invention relates to a taurine-supplemented medium and related methods for culturing recombinant eukaryotic cells for the production of protein biotherapeutics.

BACKGROUND

The organic acid taurine, often called a β-amino acid, is found in high concentrations in most tissues and is a derivative of the amino acid cysteine (Huxtable, RJ., 1992, Physiol Rev, 72: 101-163).

Or II

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Structure of taurine

Taurine is present in many tissues of humans and other mammalian species, for example in the brain, retina, myocardium, skeletal and smooth muscle, platelets and neutrophils. Taurine is recognized to aid in osmotic regulation, membrane stabilization and anti-inflammation, and also regulates mitochondrial protein synthesis by increasing the activity of the electron transport chain.

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Mexican Institute of Industrial Property protects against superoxide generation (Jong et al., 2010, Journal of Biomedical Science 17 (sup. 1): S25; Jong et al., 2012, Amino Acids 42: 2223-2232sw). In primary neuronal cultures, taurine has been characterized as a cytoprotector because it suppresses glutamate-induced 5 toxicity. Several media for embryo culture containing taurine have been developed.

The use of cell culture techniques involving amino acid feeding has a long history in the production of recombinant proteins from cultured cells. Amino acids are biosynthetic precursors, energy sources, osmolytic agents, and the like, and their use in production cultures strongly correlates with continued cell growth and productivity.

However, the physiological events that contribute to productivity and high protein expression yield are innumerable, and competing metabolic activities and transport mechanisms make the design of feeding strategies a challenge. The type of amino acid supplementation and the timing of addition could also impact the quality of the protein produced in culture (Altamirano et al., 2006, Electron. J. Biotechnol., 9:

Byproduct accumulation is frequently problematic in production cell culture and is considered a consequence of nutrient imbalance in the cell culture, ultimately inhibiting cell growth (Fan, Y. et al., Biotechnol Bioeng., 2015 March; 112 (3): 521-35). Hypotaurine or an analogue or precursor thereof has been suggested for use in cell culture to obtain the following byproducts:

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY desired results of reducing color intensity of a composition comprising a recombinantly produced polypeptide (WO2014145098A1, published on September 18, 2014). A cell culture medium including taurine that promotes maturation of immature retinal pigmented epithelial cells into mature retinal pigmented epithelial cells has also been described (WO2013184809A1, published December 12, 2013). However, optimization of recombinant protein productivity in taurine-supplemented cultures has not been recognized in the art. Cell culture processes that increase the productivity of recombinantly expressed proteins while minimizing the production of potentially toxic byproducts of cellular metabolism, such as ammonia, are highly desirable. Any consistent gain in productivity can be equivalent to a significantly higher commercial-scale supply of a biotherapeutic product.

Thus, there is a need in the art for a medium and methods for culturing mammalian cells, where the medium allows for healthy and robust growth and maintenance of the cells and the high-titer production of biopharmaceutical substances.

BRIEF DESCRIPTION

The inventors have made the surprising discovery that the inclusion of taurine in a cell culture medium increases specific cell productivity and allows for less byproduct production.

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Mexican Institute of Industrial Property of ammonia from these cells. Several feeding strategies that include taurine increase protein production titers. Furthermore, the addition of taurine has no negative impact on culture performance or the quality of the resulting antibody.

The present invention provides a method for producing therapeutic protein in high yield, comprising culturing a recombinant cell line in a medium containing taurine, wherein the cell line comprises a stably integrated nucleic acid encoding the therapeutic protein.

The present invention relates to a cell culture medium that is serum-free and comprises from about 0.1 mM to about 10 mM taurine. The present invention relates to a cell culture medium that is serum-free and comprises from about 0.1 mM to about 1 mM taurine, from about 0.2 mM to about 1 mM taurine, from about 0.3 mM to about 1 mM taurine, from about 0.4 mM to about 1 mM taurine, from about 0.5 mM to about 1 mM taurine. The present invention relates to a cell culture medium that is serum-free and comprises from about 1 mM to about 10 mM taurine. The present invention relates to a cell culture medium that is serum-free and comprises from about 1 mM to about 5 mM taurine, from about 1 mM to about 6 mM taurine, from about 1 mM to about 7 mM taurine, from about 1 mM to about 10 mM taurine, and from about 1 mM to about 20 mM taurine.

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY approximately 8 mM of taurine, or from approximately 1 mM to approximately 9 mM of taurine.

In some embodiments, the medium also comprises additional amino acids selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

In some embodiments, the medium contains 16 g/L of hydrolysate. In some embodiments, the medium is free of any hydrolysate.

In one embodiment, the medium contains a chemically defined base medium, such as a conventional formulation or a commercially available base medium. In one embodiment, the complete medium is chemically defined and is serum-free and hydrolyzate-free.

In some embodiments, the total process including the base medium and feeds contains a total of at least 115 mM of a mixture of amino acids or amino acid salts. In one embodiment, the amino acid mixture comprises amino acids selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan, in an amount selected from Table 1.

In some embodiments, the medium contains one or more fatty acids. In a particular embodiment, the medium contains a mixture of fatty acids (or fatty acid derivatives) and alpha-tocopherol. The fatty acids or fatty acid derivatives are selected from the group

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY consisting of linoleic acid, linolenic acid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid and 5-octanoic acid.

In some embodiments, the medium contains a mixture of nucleosides. In one embodiment, the medium contains adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

In some embodiments, the medium contains a mixture of salts. 10 The salts include divalent cations such as calcium and magnesium. In one embodiment, the medium contains calcium chloride and magnesium sulfate. Other salts may include phosphate salts.

In one embodiment, the medium: (1) contains taurine at 0.1 ± 0.015 mM, 1 ± 0.015 mM, 3 ± 0.05 mM, 5 ± 0.10 mM, 7 ± 0.15 mM, or 10 ± 0.2 15 mM; (2) contains ≥16 g/L of a hydrolysate; (3) is serum-free; (4) optionally further contains a mixture of amino acids; (5) contains a mixture of fatty acids; (6) contains a mixture of nucleosides including adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine; and (7) contains calcium, magnesium, and phosphate salts.

The present invention provides a method for producing a protein of interest in high yield, comprising culturing a recombinant cell line in a cell culture medium containing at least about 0.1 mM to about 10 mM taurine, wherein the cell line comprises an integrated nucleic acid.

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Mexican Institute of Industrial Property stably encoding the protein. In other embodiments, the medium includes any of the above aspects of the invention.

In another aspect, the invention provides a method for culturing eukaryotic cells to enhance recombinant protein production, comprising the steps of: (a) propagating or maintaining cells in a defined cell culture medium during the growth phase; (b) supplementing the basal cell culture medium with about 0.1 mM to about 10 mM L-taurine, and expressing a recombinant protein of interest during the production phase; and (c) increasing the titer of the protein of interest by adding taurine. In some embodiments, the taurine supplement is provided at least once during the production phase, or twice, three times, four times, or five times during the production phase, or every day during the production phase. In other embodiments, the method also comprises supplementing the culture medium with about 0.1 mM to about 10 mM L-taurine during the growth phase. In some embodiments, the method provides for enhanced production of recombinant protein compared to eukaryotic cells lacking taurine supplementation, or with taurine supplementation of less than 0.1 mM and under otherwise identical conditions.

In another aspect, the invention provides a method for culturing cells in a cell culture medium such as any embodiment of the medium described in the previous aspect. In one embodiment, the method utilizes the steps of: propagating or maintaining one or more cells in a

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY medium that: (1) contains taurine at a concentration of at least 0.1 mM ± 0.015 mM; (2) contains 516 g/L of hydrolysate, or does not contain hydrolysate; (3) is serum-free; and (4) optionally amino acids selected from the group consisting of a mixture of amino acids selected from Table 1.

In one embodiment, the optional mixture of amino acid supplements is selected from the group consisting of the amino acids in Table 1:

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

TABLE 1

<td>Amino acid</td><td>SCALE mM (mmol/L)</td><td>SCALE (g/L)</td>

<td>Alanine</td><td> 0-11.2</td><td> 0-1</td>

<td>Arginine</td><td> 2.4-11.9</td><td> 0.5-2.5</td>

<td>Asparagine</td><td> 1.3-33.3</td><td> 0.2-5</td>

<td>Aspartic acid</td><td> 1.5-93.9</td><td> 0.2-12.5</td>

<td>Cysteine</td><td> 1.1-19.9</td><td> 0.2-3.5</td>

<td>Glutamic acid</td><td> 1.4-47.6</td><td> 0.2-7</td>

<td>Glutamine</td><td> 0-23.9</td><td> 0-3.5</td>

<td>Glycine</td><td> 0-16.7</td><td> 0-1.25</td>

<td>Histidine</td><td> 1-9.5</td><td> 0.2-2</td>

<td>Isoleucine</td><td> 1.5-22.9</td><td> 0.2-3</td>

<td>Leucine</td><td> 1.5-38.1</td><td> 0.2-5</td>

<td>Lysine</td><td> 2.7-24.6</td><td> 0.5-4.5</td>

<td>Methionine</td><td> 1.3-13.4</td><td> 0.2-2</td>

<td>Phenylalanine</td><td> 1.2-18.2</td><td> 0.2-3</td>

<td>Proline</td><td> 1.7-26.1</td><td> 0.2-3</td>

<td>Serina</td><td> 1.9-57.1</td><td> 0.2-6</td>

<td>Threonine</td><td> 1.7-33.6</td><td> 0.2-4</td>

<td>Tryptophan</td><td> 0.5-14.7</td><td> 0.1-3</td>

<td>Tyrosine</td><td> 0.9-22.2</td><td> 0.2-5</td>

<td>Valine</td><td> 1.7-34.1</td><td> 0.2-4</td>

In some embodiments, the cell(s) are mammalian cells, avian cells, insect cells, yeast cells, or bacterial cells. In one embodiment, the cells are mammalian cells useful in the production of recombinant proteins, such as CHO cells or the CHO-K1 derivative. In some embodiments, the cells express a

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Mexican Institute of Industrial Property protein of interest, such as a biotherapeutic protein. The biotherapeutic protein may be an antigen-binding protein that may contain an Fc domain. In some embodiments, the protein of interest is an Fc fusion protein, such as an ScFv molecule or a decoy molecule. Decoy molecules include, but are not limited to, VEGF-trap and IL-1-trap proteins. In some embodiments, the protein of interest is an antibody, such as a human monoclonal antibody, humanized monoclonal antibody, a bispecific antibody, or an antibody fragment.

Given the positive effects on protein production with the inclusion of taurine in various forms of serum-free medium, cells cultured according to this method result in an average increase in protein titer. In one embodiment, compared to the protein titer in a medium that has not been supplemented with taurine, cells grown in the taurine-supplemented culture according to this method produce proteins having a protein titer that is at least 8% greater than the titer of the comparison control culture (i.e., a culture that has not been supplemented with taurine). In one embodiment, cells grown in the taurine-supplemented culture, compared to the protein titer in a medium that has not been supplemented with taurine, produce a protein titer that is at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, or at least 22% greater than the protein titer in a medium that has not been supplemented with taurine.

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, or at least 29% greater than the titer of the comparison control culture.

Furthermore, the inclusion of taurine alone in a serum-free medium allows the cultured cells to produce less ammonia byproduct than without the inclusion of taurine. In one form of medium supplemented with serum-free and hydrolysate-free taurine, the cell culture is able to achieve a byproduct concentration of 10 mM ammonia (NH₃) reduced which is at least 4% lower and up to 32% lower than in a similar cell culture in a similar cell culture medium containing no complementation (i.e., less than 0.1 mM taurine or no taurine supplement).

In another embodiment, the method includes the step of adding one or more point-of-use addition ingredients to the cell culture medium. In some embodiments, the point-of-use addition ingredient is one or more of any of NaHCO₃, glutamine, insulin, glucose, CuSO₄, ZnSO₄, FeCI₃, NiSO₄, Na₄EDTA and trisodium citrate. In one embodiment, the method uses the step of adding each of the following 20 chemical ingredients at the point of use to the cell culture medium: NaHCO₃, glutamine, insulin, glucose, CuSO₄, ZnSO₄, FeCI₃, N¡SO₄, Na₄EDTA and trisodium citrate. In some embodiments, point-of-use ingredients may be included in the medium from the beginning.

²⁵ In a specific embodiment, the aspect provides a method for

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY cultivate cells in a serum-free medium that consists essentially of: (1) taurine at a concentration of at least 0.1 mM¡ (2) contains 516 g/L of a hydrolysate; (3) is serum-free; and (4) optionally further contains at least about 20 mM, or at least about 25 mM, or at least about 30 mM, or at least about 40 mM, or at least about 50 mM, or at least about 60 mM, or at least about 70 mM total of a mixture of amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

In another aspect, the invention provides a method for producing a protein of interest using the steps of: (1) introducing into a cell a nucleic acid sequence encoding a protein of interest;

(2) selecting one or more cells that express the protein of interest;

(3) culturing the selected cells in an embodiment of the serum-free cell culture medium described in any preceding aspect, or according to any embodiment of the method described herein; and (4) expressing the protein of interest in the cells, wherein the protein of interest is secreted into the medium. In some embodiments, the cell used in the production of the protein is a mammalian cell capable of producing a biotherapeutic agent, such as CHO, 293, and BHK cells, or any derivative thereof. In one embodiment, the cell is a CHO cell, such as a CHO-K1 cell.

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

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

In one embodiment, the protein of interest can be produced at a 14, 15, 16, or 17 day average titer that is at least 8% greater than the 14, 15, 16, or 17 day average titer produced by a similar cell in a serum-free culture medium containing less than 0.1 mM taurine supplementation or no taurine supplementation. In one embodiment, the protein of interest may be produced at an average titer over 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 days that is at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, or at least 25-29% greater than the average title of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY or 17 days produced by a similar cell in a serum-free culture medium containing taurine complementation less than 0.1 mM or does not contain taurine complementation.

In another embodiment, the protein of interest is produced by: (1) introducing into a CHO cell a nucleic acid sequence encoding a protein of interest, such as an antibody or other antigen-binding protein; (2) selecting cells that stably express the protein of interest; (3) culturing the selected cells in a serum-free culture medium comprising about 0.1 mM to about 10 mM taurine.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the protein titer (yield) of samples recovered daily from the production culture in an Ab3-producing cell culture, where taurine supplementation is provided (black squares connected by solid lines) compared to no taurine supplementation (x connected by dashed lines). The benefits of taurine-supplemented crops for protein yield can be observed as early as day 6 of production.

DETAILED DESCRIPTION

It is understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also understood that the

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY The terminology used herein is only intended to describe particular modalities and is not intended to be limiting, since the scope of the present invention is defined by the claims.

As used in this specification and the appended claims, the singular forms “a,” “an,” “the,” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, a reference to “a method” includes one or more methods or steps of the type described herein or that will be apparent to those skilled in the art upon reading this description.

Unless otherwise defined, all technical and scientific terms used herein have the same meanings commonly understood by those skilled in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein may be used for the practice of the present invention, particular methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

Applicants have made the surprising discovery that the addition of taurine to a cell culture medium improves protein production by a recombinant cell in a cell culture medium, relative to a cell culture medium containing very little or no taurine.

Before describing the cell cultures and methods present,

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The Mexican Institute of Industrial Property understands that this invention is not limited to the specific experimental methods and conditions described, as such methods and conditions may vary. It is also understood that the terminology used herein is intended solely to describe specific embodiments and is not intended to be limiting.

The section headings used herein are for organizational purposes only and are not intended to limit the subject matter described. The methods and techniques described herein are generally performed in accordance with conventional methods known in the art and as described in various general and more specific references that are cited and set forth throughout this specification, unless otherwise indicated; see, e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 3^toed., Coid Spring Harbor Laboratory Press, Coid Spring Harbor, NY

(2001); and Ausubel et al., “Current Protocols in Molecular Biology”,

Greene Publishing Associates (1992); Harlow and Lane “Antibodies: A Laboratory Manual”, Coid Spring Harbor Laboratory Press, Coid Spring Harbor, NY (1990); and Julio E. Celis, “Cell Biology: A Laboratory Handbook”, 2^to ed., Academic Press, New York, NY (1998); and

Dieffenbach and Dveksler, “PCR Primer: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1995). All publications mentioned in this disclosure are incorporated herein by reference in their entirety.

DEFINITIONS “Taurine” is also known as 2-aminoethanesulfonic acid

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Mexican Institute of Industrial Property (IUPAC nomenclature; CAS registry number 107-35-7). “Taurine” and “L-taurine” are used interchangeably to refer to the same organic compound. Taurine is an organic acid containing an amino group; however, it is not considered an “amino acid” as traditionally known by those skilled in the art, since amino acids contain both an amino group and a carboxyl group. Taurine biosynthesis occurs when hypotaurine, a cysteine derivative, is converted to taurine by oxidation.

The terms “complementation,” “supplement,” “supplemented with,” and the like, refer to adding an ingredient, component, molecule, etc., that can be used in a cell culture medium to maintain or promote the growth or differentiation of the cells, to extend or enhance an attribute of the culture or the cells as a whole, or to compensate for a deficiency. For this purpose, taurine supplementation involves the addition of taurine to the culture medium at a particular concentration in a solution.

The terms “peptide,” “polypeptide,” and “protein” are used interchangeably throughout the description and refer to a molecule comprising two or more amino acid residues joined to each other by means of a peptide bond. Peptides, polypeptides, and proteins may also include modifications such as glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, alkylation, hydroxylation, and ADP-ribosylation. Peptides, polypeptides and proteins may be of scientific or commercial interest, and include

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Mexican Institute of Industrial Property protein-based drugs. Peptides, polypeptides, and proteins include, among other things, antibodies and chimeric or fusion proteins. Peptides, polypeptides, and proteins are produced by recombinant animal cell lines using cell culture methods.

The term “heterologous polynucleotide sequence,” as used herein, refers to nucleic acid polymers encoding proteins of interest, such as chimeric proteins (such as trap molecules), antibodies, or antibody portions (e.g., VH, VL, CDR3) that are produced as a biopharmaceutical. The heterologous polynucleotide sequence can be manufactured by genetic engineering techniques (e.g., by coding for nucleic acids). e.g., such as a sequence encoding a chimeric protein or a codon-optimized sequence or intronless sequence, etc.), and introduced into the cell where it may reside as an episome or be integrated into the genome of the cell. The heterologous polynucleotide sequence may be a naturally occurring sequence that is introduced at an ectopic site within the genome of the production cell. The heterologous polynucleotide sequence may be a naturally occurring sequence from another organism, such as a sequence encoding a human ortholog.

“Antibody” refers to an immunoglobulin molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain has a heavy chain variable region (HCVR or 25 VH) and a heavy chain constant region. The heavy chain constant region

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The industrial heavy chain contains three domains, CH1, CH2, and CH3. Each light chain has a light chain variable region and a light chain constant region. The light chain constant region consists of a (CL) domain. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,

CDR3, FR4. The term “antibody” includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass. The term “antibody” includes antibody molecules prepared, expressed, created, or isolated by recombinant means, such as antibodies isolated from a host cell transfected to express the antibody. The term "antibody" also includes a bispecific antibody that includes a heterotetrameric immunoglobulin that can bind to more than one different epitope. Bispecific antibodies are generally described in U.S. Patent Application Publication No. 2010/0331527, which is incorporated by reference into this application.

The term “antigen-binding portion” of an antibody (or “antibody fragment”) refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of the binding fragments encompassed within the term “antigen-binding portion” of an antibody include: (i) a

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains¡ (i) an F(ab') fragment₂, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (i¡¡) an Fd fragment, consisting of the VH and

CH1¡ (iv) an Fv fragment, consisting of the VL and VH domains of a single antibody arm; (v) a dAb fragment (Ward et al. (1989), Nature 241:544-546), consisting of a VH domain; (vi) an isolated CDR; and (vii) a scFv, consisting of the two domains of the Fv fragment, VL and VH, joined by a synthetic linker to form a single protein chain in which the VL and VH regions pair to form monovalent molecules. Other forms of single-chain antibodies, such as bivalent fragments, are also encompassed under the term “antibody” (see, e.g., Holliger, et al. (1993), PNAS USA 90:6444-6448; Poljak, et al. (1994) Structure 2:1121-1123).

In addition, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule, formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov et al. (1995), Human Antibodies and Hybridomas, 6:93-101), and the use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent, biotinylated scFv molecules (Kipriyanov et al. (1994),

Mol. Immunol. 31:1047-1058). Antibody portions such as

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY Fab and F(ab') fragments₂, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies. In addition, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques commonly known (see Sambrook et al., 1989).

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

The term “recombinant human antibody” used herein includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a combinatorial collection of recombinant human antibodies, antibodies isolated from an animal (e.g., a mouse) that is transgenic

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INDUSTRIAL for human immunoglobulin genes (see, e.g., Taylor et al. (1992), Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created, or isolated by any other means involving splicing of human immunoglobulin gene sequences with other DNA sequences. These recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In some embodiments, however, these recombinant human antibodies are subjected to in vitro mutagenesis (or, when a transgenic animal is used for human Ig sequences, in vivo somatic mutagenesis), and therefore the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not exist naturally within the human germline antibody repertoire in vivo.

“Fc fusion proteins” comprise part or all of two or more proteins, one of which is an Fc portion of an immunoglobulin molecule, which are not otherwise found together in nature. The preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, for example, in Ashkenazi et al., Proc. Nati. Acad. Sci. USA 88: 10535, 1991; Byrn et al., Nature 344:677, 1990; and Hollenbaugh et al., Construction of Immunoglobulin Fusion Proteins, in 25 Current Protocols in Immunology, sup. 4, p. 10.19.1 - 10.19.11, 1992.

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OF THE PROPERTY

INDUSTRIAL

“Receptor-Fc fusion proteins” comprise one or more extracellular domains of a receptor coupled to an Fc portion, which in some embodiments comprises a hinge region, followed by a CH2 and CH3 domain of an immunoglobulin. In some embodiments, the Fc fusion protein contains two or more distinct receptor chains that bind one or more ligands. For example, an Fc fusion protein is a trap, such as an IL-1 trap (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-58, IL-59, IL-60, IL-61, IL-62, IL-63, IL-64, IL-65, IL-66, IL-67, IL-68, IL-69 ... e.g., rilonacept, which contains the IL-1RacP ligand binding region fused to the extracellular region of IL-1R1 fused to the Fc-linked receptor of hlgG1; see U.S. Pat. No. 6,927,004), or a VEGF trap (e.g., aflibercept, which contains the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to the Fc-linked receptor of hlgG1; see U.S. Pat. Nos. 7,087,411 and 7,279,159).

CELL CULTURE

The terms “cell culture medium” and “growth medium” refer to a nutrient solution used for the growth of mammalian cells, which typically provides the nutrients necessary to enhance cell growth, such as a carbohydrate energy source, essential (e.g., phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine) and nonessential (e.g., e.g., alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine and tyrosine), trace elements, energy sources, lipids, vitamins, etc. The cell culture medium may contain extracts, e.g., serum or peptones

<img file="MX390940B_D0024.tif" />

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Mexican Institute of Industrial Property (hydrolyzed), which provide raw materials that support cell growth. The medium may contain extracts derived from yeast or soy instead of animal extracts. A chemically defined medium refers to a cell culture medium in which all chemical components are known (i.e., have a known chemical structure). The chemically defined medium is completely free of animal-derived components, such as serum- or animal-derived peptones. In one embodiment, the medium is a chemically defined medium.

The solution may also contain components that enhance growth or survival above the minimum rate, including hormones and growth factors. The solution is preferably formulated at an optimal pH and salt concentration for cell survival and proliferation.

A “cell line” refers to one or more cells derived from a particular lineage through serial passage or subculture. The term “cells” is used interchangeably with “cell population.”

The term “cell” includes any cell suitable for the expression of a recombinant nucleic acid sequence. Cells include eukaryotes, such as non-human animal cells, mammalian cells, human cells, avian cells, insect cells, yeast cells, and cell fusions, such as hybridomas or quadromas. In certain embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In other embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell.

<img file="MX390940B_D0025.tif" />

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY modalities, the cell is selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney cells (e.g., e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, lymphocytes, e.g., Jurkat (T cell) or Daudi (B cell), A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2/0, NS-0, MMT cells, stem cells, tumor cells, and a cell line derived from an aforementioned cell. In some embodiments, the cell comprises one or more viral genes, e.g., a retinal cell expressing a viral gene (e.g., a retinal cell expressing a viral gene). e.g., a PER.C6® cell). In some embodiments the cell is a CHO cell. In other embodiments the cell is a CHO K1 cell.

One aspect of the invention relates to a seed culture in which a cell population is expanded prior to protein production and harvested from the production culture. Taurine may be added to the base medium in a seed culture formulation, according to the invention described herein .

Another aspect of the invention relates to a production culture in which a protein is produced and harvested. Prior to the production phase, there is typically a growth phase (also known as a seed train or seed culture), in which all cell culture components are supplied to the culture vessel at the beginning of the culture process, and the cell population is then expanded until it is ready for production scale. As such, the culture vessel is inoculated with the cells at a

<img file="MX390940B_D0026.tif" />

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Mexican Institute of Industrial Property appropriate seeding density for the initial cell growth phase, which depends on the initial cell line. In some aspects, taurine can be added to the basal culture medium in a seed culture formulation, according to the invention described herein, to further improve or increase cell productivity in the subsequent production phase.

One aspect of the invention relates to a production culture wherein the cell culture conditions are modified to increase the growth of recombinant eukaryotic cells, while improving the production of one or more recombinant proteins of interest from said cells and maintaining cell viability, in particular by adding taurine to the production culture medium or the seed train culture. In the production culture vessel or bioreactor, a basal culture medium and cells are supplied to a culture vessel after a seed or growth phase. In certain embodiments, the cell supernatant or cell lysate is harvested after the production culture. In other embodiments, the polypeptide or protein of interest is recovered from the culture medium or cell lysate, or whatever the case may be, 20 depending on the location of the protein of interest, using well-known techniques.

Culture vessels include, but are not limited to, well plates, T-flasks, shake flasks, stirred vessels, shaker flasks, hollow fiber, air-lift bioreactors, and the like. 25 A suitable cell culture vessel is a bioreactor.

<img file="MX390940B_D0027.tif" />

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Mexican Institute of Industrial Property (CD) Q Bioreactor refers to any culture vessel that is manufactured or engineered to manipulate or control environmental conditions. Such culture vessels are well known in the art.

Bioreactor processes and systems have been developed to optimize gas exchange, to supply sufficient oxygen to sustain cell growth and productivity, and to remove CO₂Maintaining gas exchange efficiency is an important criterion for ensuring successful scale-up of cell culture and protein production. Such systems are well known to those skilled in the art.

In the polypeptide production phase, a “fed-batch cell culture” or “fed-batch culture” refers to a batch culture wherein animal cells and culture medium are initially supplied to the culture vessel and during the culture additional culture nutrients are fed, slowly, continuously or in discrete increments, with or without periodic harvesting of cells or product before termination of the culture. Fed-batch culture includes “semi-continuous fed-batch culture” where the entire culture (which may include cells and medium) is periodically removed and replaced with fresh medium. Fed-batch culture is distinguished from simple “batch culture” in that in batch culture all components for cell culture (including animal cells and all culture nutrients) are supplied to the culture vessel at the beginning of the process.

<img file="MX390940B_D0028.tif" />

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Mexican Institute of Industrial Property culture process. Fed-batch culture can be further distinguished from perfusion culture in that the supernatant is not removed from the culture vessel during the process, whereas in perfusion culture, the cells are restricted in the culture, for example, by filtration, and the culture medium is continuously or intermittently introduced and removed from the culture vessel. However, withdrawal of samples for analytical purposes is contemplated during fed-batch cell culture. The fed-batch process continues until it is determined that the maximum working volume or protein production has been reached.

The phrase “continuous cell culture,” when used here, refers to a technique used to continuously develop cells, usually in a particular growth phase. For example, if a constant supply of cells is required or the production of a particular polypeptide or protein of interest is desired, it may be necessary to maintain the cell culture in a particular growth phase. Thus, conditions must be continuously monitored and adjusted accordingly to maintain the cells in that particular phase.

MEDIA

The present invention provides a cell culture medium, which is serum-free, comprising from about 0.1 mM to 10 mM taurine. “Serum-free” applies to a cell culture medium that does not contain animal serum, such as fetal bovine serum. The serum-free medium

<img file="MX390940B_D0029.tif" />

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Mexican Institute of Industrial Property serum may contain 16 g/L of hydrolysates, such as soy hydrolysate. The present invention also provides chemically defined media that are not only serum-free but also hydrolysate-free. “Hydrolysate-free” applies to a cell culture medium that does not contain exogenous protein hydrolysates, such as animal or vegetable protein hydrolysates, such as, for example, peptones, tryptones, and the like. “Base medium” is the initial medium (present in the seed train or on day 0 of cell culture production) in which cells are propagated and contains all the necessary nutrients, including a base mix of amino acids. Various recipes (i.e., formulations) for base media can be manufactured or purchased in commercially available batches. Similarly, "basic feeding media" contains mixtures of complementary nutrients commonly consumed during a production crop and are used as a feeding strategy (for so-called "fed-batch" growing). Varieties of these basic feeding media are commercially available. A “feed” includes scheduled additions to the medium at regular intervals, for example according to a protocol involving a continuous-fed culture system, such as in a chemostat (see C. Altamirano et al., Biotechnol Prog., Nov-Dec, 2001; 17(6); 1032-41), or according to a fed-batch process (Y.M. Huang et al., Biotechnol Prog., Sep-Oct, 2010; 26(5): 1400-10). For example, a crop can be fed once a day, every two days, every three days, or it can be fed when the concentration of a

<img file="MX390940B_D0030.tif" />

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY specific component of the environment, which is monitored, falls outside a desired scale.

Serum removal and the reduction or elimination of hydrolysates from cell culture medium, while reducing batch-to-batch variability and improving downstream processing steps, unfortunately decrease cell growth, viability, and protein expression. Thus, chemically defined serum-free medium with little or no hydrolysates requires additional ingredients to improve cell growth and protein production.

Thus, the cell culture medium of the invention comprises a base medium containing all the nutrients necessary for viable cell culture. Taurine can be added to the base medium in a seed culture formulation according to the invention described herein. Furthermore, taurine can be added to the base medium in a production culture formulation, which can then be fed periodically (as in so-called “fed-batch” cultures) with or without additional ingredients such as polyamines or higher concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements and the like, depending on the requirements of the cells to be cultured or the desired parameters of the cell culture.

The invention contemplates that the amino acids in the cell culture medium supplemented with taurine can be depleted during the

<img file="MX390940B_D0031.tif" />

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY during the course of protein production culture when no additional amino acid supplementation is provided, or that the amino acids in the taurine-supplemented cell culture medium may not be depleted when supplementation of the depleted amino acids is provided (“non-depleted medium”) (as described below). The inventors have observed that cultures supplemented with taurine during the production phase improve recombinant protein production under various culture conditions as described above.

The invention provides a taurine-supplemented medium containing taurine at a concentration (expressed in millimoles per liter) of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM.

In one embodiment, the medium additionally contains 100 pM ± 15 pM ornithine, or 300 pM ± 45 pM ornithine, or 600 pM ± 90 pM ornithine, or even 900 pM ± 135 pM ornithine. In another embodiment, the medium contains at least about 5 mg/L ± 1 mg/L ornithine HCI, or at least about 10 mg/L ± 2 mg/L ornithine HCI, 15 mg/L ± 2.25 mg/L ornithine HCI, or at least about 50 mg/L ± 7.5 mg/L ornithine HCI, or at least about 100 mg/L ± 15 mg/L ornithine HCI, or at least about 150 mg/L ± 22.5 mg/L ornithine HCI.

Putrescine may optionally be added to the ornithine-supplemented medium. Putrescine has been included at very low concentrations as a component in some cell culture media formulations; see, for example, WO

<img file="MX390940B_D0032.tif" />

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

2005/028626, which describes putrescine at 0.02-0.08 mg/L; U.S. Patent No. 5,426,699 (0.08 mg/L); U.S. Patent No. RE30,985 (0.16 mg/L); U.S. Patent No. 5,811,299 (0.27 mg/L); U.S. Patent No. 5,122,469 (0.5635 mg/L); U.S. Patent No. 5,063,157 (1 mg/L); WO 2008/154014 (-100 μM - -1000 pM); U.S. Patent Application No. 2007/0212770 (0.5 - 30 mg/L polyamine; 2 mg/L putrescine; 2 mg/L putrescine + 2 mg/L ornithine; 2 mg/L putrescine + 10 mg/L ornithine).

In some embodiments, the medium is further supplemented with a combination of ornithine and putrescine, wherein the putrescine may be at a concentration of at least about 150 to 720 pM. In some embodiments, the medium is further supplemented with putrescine at a concentration of about 170 to 230 pM. In one embodiment, the medium contains putrescine at 200 pM ± 30 pM, in addition to ornithine at 90 pM ± 15 pM. In one embodiment, the medium contains 30 mg/L ± 4.5 mg/L of putrescine 2HCI, in addition to 15 mg/L ± 2.25 mg/L of ornithine. In another embodiment, the medium contains 30 mg/L ± 4.5 mg/L of putrescine 2HCI, in addition to 15 mg/L ± 2.25 mg/L of ornithine HCI (see International Publication No. WO2014/144198A1, published September 18, 2014, which is incorporated herein by reference in its entirety).

In other embodiments, ornithine is present in the medium at a concentration ranging from 0.09 ± 0.014 mM to 0.9 ± 0.14 mM, such as 0.09 ± 0.014 mM, 0.3 ± 0.05 mM, 0.6 ± 0.09 mM, or 0.9 ± 0.14 mM ornithine. In some embodiments, the medium also contains putrescine.

<img file="MX390940B_D0033.tif" />

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Mexican Institute of Industrial Property at least 0.20 ± 0.03 mM. In some embodiments, the additional putrescine is at a concentration ranging from 0.20 ± 0.03 mM to 0.714 ± 0.1 1 mM, such as 0.20 ± 0.03 mM, 0.35 ± 0.06, or 0.714 ± 0.11 mM putrescine.

Other supplements may be added to the culture medium and it is within the domain of the person skilled in the art to further determine the appropriate conditions. In some embodiments, the medium is supplemented with a mixture of amino acids selected from the group consisting of aspartic acid, cysteine, glutamic acid, glycine, lysine, phenylalanine, proline, serine, threonine, valine, arginine, histidine, asparagine, glutamine, alanine, isoleucine, leucine, methionine, tyrosine, and tryptophan, so as not to be depleted or as supplemental nutrients as required.

In one embodiment, the medium is further supplemented with about 170 µM to 175 µM of nucleosides. In one embodiment, the medium contains purine derivatives in a cumulative concentration of at least 40 µM, at least 45 µM, at least 50 µM, at least 55 µM, at least 60 µM, at least 65 µM, at least 70 µM, at least 75 µM, at least 80 µM, at least 85 µM, at least 90 µM, at least 95 µM, at least 100 µM, or at least 105 µM. In one embodiment, the medium contains about 100 μM to 110 μM of purine derivatives. Purine derivatives include hypoxanthine and the nucleosides adenosine and guanosine. In one embodiment, the medium contains pyrimidine derivatives in a cumulative concentration of at least 30 μM, at least 35 μM,

<img file="MX390940B_D0034.tif" />

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY at least 40 μM, at least 45 μM, at least 50 μM, at least 55 μM, at least 60 μM, or at least 65 μM. In one embodiment, the medium contains about 65 μM to 75 μM of pyrimidine derivatives. Pyrimidine derivatives include the nucleosides thymidine, uridine, and cytidine. In a particular embodiment, the medium contains adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

In addition to the inclusion of any of the above additives, in one embodiment the medium is additionally supplemented with micromolar amounts of fatty acids (or fatty acid derivatives) and tocopherol. In one embodiment, the fatty acids include one or more of any of linoleic acid, linolenic acid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and octanoic acid. In one embodiment, the medium contains tocopherol, linoleic acid, and thioctic acid.

In one embodiment, the medium may also be supplemented with a vitamin mixture that includes other nutrients and essential nutrients at a cumulative concentration of at least about 700 µM or at least about 2 mM. In one embodiment, the vitamin mixture contains one or more of D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, pyridoxine HCl, D-pantothenic acid (hemiCa), riboflavin, thiamin HCl, vitamin B12, and the like. In one embodiment, the vitamin mixture includes everything

<img file="MX390940B_D0035.tif" />

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY following: D-biotin, choline chloride, folic acid, myoinositol, niacinamide, pyridoxine HCI, D-pantothenic acid (hemiCa), riboflavin, thiamine HCI and vitamin B12.

Various embodiments of the media of the invention include any combination of the above-described embodiments, including hydrolyzate-free and serum-free chemically defined media comprising taurine in the indicated amounts, plus, among other things, (a) amino acids; (b) optionally nucleosides; (c) salts of divalent cations; (d) fatty acids and tocopherol; and (e) vitamins. In some embodiments, small amounts of hydrolysates can be added to the taurine-supplemented medium.

Applicants contemplate that one or more of any of a variety of base media, or combinations thereof, to which taurine is added, may be used in the practice of this invention. Base media are generally known in the art and include, but are not limited to, Eagle's MEME (minimal essential medium) (Eagle, Science, 1955, 112(3168):501-504), Ham's F12 medium (Ham, Proc. Nat'l. Acad. Sci. USA, 1965, 53:288-293), F-12 K medium, Dulbecco's medium, Dulbecco's modified Eagle's medium (Proc. Nati. Acad. Sci. USA., 1952 Aug; 38(8): 747-752), DMEM/Ham's F12 1:1, Trowell's T8 medium, A2 medium (Holmes and Wolf, Biophys. Biochem. Cytol., 1961, 10:389-401), Waymouth's medium (Davidson and Waymouth, Biochem. J., 1945, 39(2):188-199), Williams's E medium (William's et al., Exp. Cell Res., 1971, 69:105 ff.), RPMI 1640 (Moore et al., J. Amer. Med. Assoc.,

<img file="MX390940B_D0036.tif" />

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

1967, 199:519-524), MCDB 104/110 medium (Bettger et al., Proc. Nat'l. Acad. Sel. USA, 1981, 78(9):5588-5592), Ventrex HL-1 medium, albumin-globulin medium (Orr et al., Appl. Microbiol., 1973, 25(1):49-54), RPMI-1640 medium, RPMI-1641 medium, Iscove's modified Dulbecco's medium, McCoy's 5A medium, Leibovitz's L-15 medium, and serum-free medium such as the EX-CELL™ 300 series (JRH Biosciences, Lenexa, Kansas), protamine-zinc-insulin medium (Weiss et al., 1974, US 4,072,565), biotin-folate medium (Cartaya, 1978, US Re30,985), transferrin-fatty acid medium (Baker, 1982, US 4,560,655), transferrin-EGF medium (Hasegawa, 1982, US 4,615,977; Chessebeuf, 1984, US 4,786,599), and other media permutations (see Inlow, US 6,048,728; Drapeau, US 7,294,484; Mather, US 5,122,469; Furukawa, US 5,976,833; Chen, US 6,180,401; Chen, US 5,856,179; Etcheverry, US 5,705,364; Etcheverry, US 7,666,416; Ryll, US 6,528,286; Singh, US 6,924,124; Luán, US 7,429,491; and the like).

In a particular embodiment, the media are chemically defined and contain, in addition to taurine: mixtures of amino acids as defined herein; CaCl₂ 2H₂EITHER; HEPES buffer, KCI; MgSO₄; NaCl; Na₂HPO₄ or other phosphate salts; pyruvate; D-biotin; choline chloride; folic acid; myoinositol; niacinamide; pyridoxine HCI; D-pantothenic acid; riboflavin; tlamin HCI; vitamin B12; p-aminobenzoic acid; ethanolamine HCI; poloxamer 188; DL-atocopherol phosphate; linoleic acid; Na₂SeOs; thioctic acid; and glucose; and optionally adenosine; guanosine; cytidine; uridine; thymldine; and hypoxanthine 2Na.

<img file="MX390940B_D0037.tif" />

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

In one embodiment, the initial osmolarity of the medium of the invention is 200-500, 250-400, 275-350, or about 300 mOsm. During growth of the cells in the media of the invention, and in particular after any feeding according to a fed-batch protocol, the osmolarity of the culture may increase to about 350, 400, 450, 500, or about 550 mOsm.

In some embodiments where the osmolarity of the defined medium is less than about 300, the osmolarity is brought to about 300 by the addition of one or more salts in excess of the specified amount. In one embodiment, the osmolarity is increased to a desired value by adding one or more osmolytes selected from sodium chloride, potassium chloride, a magnesium salt, a calcium salt, an amino acid salt, a salt of a fatty acid, sodium bicarbonate, sodium carbonate, potassium carbonate, a chelator which is a salt, a sugar (e.g., galactose, glucose, sucrose, fructose, fucose, etc.), and a combination thereof. In one embodiment, the osmolyte is added at and above its concentration to a component already present in the defined medium (e.g., a sugar is added at and above the concentration specified for a sugar component).

Any and all forms of the media described above and any other serum-free medium containing at least about 0.1 mM taurine are referred to as taurine-supplemented media. Conversely, media that do not contain taurine are referred to as taurine-supplemented media.

<img file="MX390940B_D0038.tif" />

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Mexican Institute of Industrial Property containing taurine or media containing less than 0.1 mM taurine, are referred to hereinafter as non-taurine supplemented or non-taurine supplemented media.

FED BATCH CULTURE

Cell culture feeding strategies are aimed at ensuring optimal cell growth and propagation outside of a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art; see, for example, “Animal Cell Culture: A Practical Approach,” D.

Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells can be grown in suspension or attached to a solid substrate. Fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors are available for mammalian cell culture, with or without microcarriers, and operated in batch, fed-batch, continuous, semi-batch, or perfusion modes. The cell culture medium or concentrated feed medium can be added to the culture continuously or at intervals throughout the culture. For example, a culture can be fed once a day, every two days, every three days, or it can be fed when the concentration of a specific medium component being monitored falls outside a desired range.

In addition to the inclusion of taurine, in one embodiment, the medium may be additionally supplemented with amino acids to a cumulative (total) concentration of at least 20 mM. In one embodiment, the initial concentration of amino acids included in the medium is

<img file="MX390940B_D0039.tif" />

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MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL initial cell culture medium is not included in the cumulative (total) concentration of supplemented amino acids. In one embodiment of the cell culture medium, or in the method for culturing cells or the method for producing a protein of interest, the medium may be supplemented with an amount greater than about 20 mM, greater than about 25 mM, greater than about 30 mM, greater than about 40 mM, greater than about 50 mM, or greater than about 60 mM, greater than about 70 mM, greater than about 100 mM, greater than about 200 mM, greater than about 300 mM, greater than about

400 mM, or greater than about 500 mM. See also the table herein. In one embodiment, the amount of amino acids added to the medium is about 30 mM ± 10 mM or more.

Supplemental feeding regimens can be optimized by experts in the field to support cell growth, minimize cellular stress, or provide a “non-depleted environment” during the production phase.

“Non-exhausted medium” includes cell culture medium that has been determined to have the nutrients, particularly amino acids, 20 necessary for the production of a recombinant protein of interest.

Amino acid feeds typically supplement the amino acids needed as building blocks to produce a recombinant protein in a cell culture. However, some amino acids may be depleted faster than others depending on the requirements of that particular protein produced by the cells in culture.

<img file="MX390940B_D0040.tif" />

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY cultivation. In a non-depleted medium, the feeding regime has been determined so that the necessary amino acids are replenished as they are consumed. Thus, depletion and subsequently optimal consumption rates (pg/cell-day) can be determined by the following steps: culturing eukaryotic cells expressing the protein of interest in cell culture medium; measuring each amino acid concentration in the culture medium at time points to establish a depletion level; Identify the depletion time point at which the amino acid concentration falls below the depletion level; calculate the consumption rates for each amino acid; and determine the optimal consumption rate as the consumption rate at the time point immediately preceding the depletion time point. The cell culture is then supplemented with the appropriate concentration of a particular amino acid to maintain these determined optimal consumption rates so that the culture medium is not depleted.

It is understood that the present invention provides a cell culture medium supplemented with taurine that improves protein titer in depleted cultures as well as in non-depleted cultures.

The present invention provides a cell culture comprising a cell line expressing a protein of interest in a taurine-supplemented medium as described above. Examples of cell lines that are routinely used to produce protein biotherapeutics include, but are not limited to, cells

<img file="MX390940B_D0041.tif" />

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY primary, BSC cells, HeLa cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, CHO cells, CHO-K1 cells, NS-1 cells, MRC-5 cells, WI-38 cells, 3T3 cells, 293 cells, Per.C6 cells and chicken embryo cells. In one embodiment, the cell line is a CHO cell line or one or more of several specific variants of CHO cells optimized for large-scale protein production, e.g., CHO-K1.

In one embodiment, the taurine-supplemented cell culture contains insulin, which can be added as a point-of-use ingredient to the medium, or can be included in the medium formulation. In one embodiment, the cell line comprises cells capable of producing a biotherapeutic protein.

In one embodiment, the medium is supplemented at intervals during cell culture according to a fed-batch process. Fed-batch culture is generally known in the art and is used to optimize protein production (see Y.M. Huang et al., Biotechnol Prog., Sep-Oct. 2010;

(5): 1400-10).

The cell growth or seed culture phase (i.e., a first cell culture) where no medium exchange is provided, is typically followed by a second, distinct culture, known as the polypeptide production phase. Batch processes with

<img file="MX390940B_D0042.tif" />

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY food products are typically used during the production phase.

The invention provides a cell culture medium comprising about 0.1 mM to about 10 mM taurine at the beginning of the production cell culture (day 0). Alternatively, the cell culture medium comprising about 0.1 mM to about 10 mM taurine can be supplemented on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, or day 10 of the production cell culture. The cell culture medium added to the production culture over several days comprises a total amount of taurine of between about 0.1 mM and about 10 mM. The cell culture medium comprising a total amount of taurine of between about 0.1 mM and about 10 mM can be added in any sequence.

Taurine can also be added to the basal medium during the expansion phase of the seed train.

Supplementary feeding can be performed to include additional nutrients such as vitamins, amino acids, and other nutrients as described above, at intervals as frequent as every day or every 2–3 days throughout the production crop. Supplementary feeding (the addition of supplemented medium containing nutrients) can be performed at least twice or at least eight times throughout the production crop for a crop lasting 2 weeks or longer. Alternatively, supplemental feeding can be done every day during the growing season. Alternative crop feeding programs are also considered.

<img file="MX390940B_D0043.tif" />

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MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL

Additional amino acid supplementation can also be performed to provide a non-depleted medium, with depleted amino acids determined according to methods known in the art and described herein. When using this regimen, additional amino acids are added at intervals, preferably daily or every 2-3 days during the production culture, depending on the determination of amino acid depletion. In one embodiment, the mixture of additional amino acids to maintain a non-depleted cell culture medium is added to the culture at or near day 1, at or near day 2, at or near day 3, at or near day 4, at or near day 5, at or near day 6, at or near day 7, at or near day 8, at or near day 9, at or near day 10, at or near day 11, at or near day 12, at or near day 13, and at or near day 14, for a 2 week or longer culture. Alternative crop feeding programs are also contemplated.

Animal cells, such as CHO cells, can be grown in small scale cultures, such as in 125 ml vessels that have approximately 25 ml of medium, 250 ml vessels that have approximately 50 ml to 100 ml of medium, 500 ml vessels that have approximately 100 ml to 200 ml of medium. Alternatively, cultures can be large scale, such as in 1000 ml vessels having approximately 300 ml to 1000 ml of medium, 3000 ml vessels having approximately 500 ml to 3000 ml of medium, 8000 ml vessels

<img file="MX390940B_D0044.tif" />

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Mexican Institute of Industrial Property that hold approximately 2,000 ml to 8,000 ml of medium, and 15,000 ml vessels that hold approximately 4,000 ml to 15,000 ml of medium. Manufacturing cultures can hold 10,000 L of medium or more. Large-scale cultures, such as those for the manufacture of protein therapeutics, are typically maintained for days or even weeks while the cells produce the desired protein. During this time, the culture can be supplemented with a concentrated food medium containing components such as nutrients and amino acids that are consumed during the culture. The concentrated food medium can be based on any cell culture medium formulation. Such concentrated food medium may contain most of the components of the cell culture medium, for example, at about 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100X, 200X, 400X, 600X, 800X, or even about 1000X their normal useful amount. Concentrated food media are frequently used in fed-batch culture processes.

In some embodiments, the taurine-containing cell culture is further supplemented with “point-of-use addition ingredients,” also known as additions, point-of-use ingredients, or point-of-use chemicals, during the course of cell growth or protein production. Point-of-use addition ingredients include one or more of any of a growth factor or other protein, a buffer, an energy source, a salt, an amino acid, a metal, and a chelator. Other proteins include

<img file="MX390940B_D0045.tif" />

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Mexican Institute of Industrial Property transferrin and albumin. Growth factors, including cytokines and chemokines, are generally known in the art and are known to stimulate cell growth or, in some cases, cell differentiation. A growth factor is usually a protein (e.g., insulin), a small peptide, or a steroid hormone, such as estrogen, DHEA, testosterone, and the like. In some cases a growth factor may be a non-natural chemical that promotes cell proliferation or protein production, such as tetrahydrofolate (THF), methotrexate, and the like. Non-limiting examples of protein and peptide growth factors include angiopoietins, bone morphogenic proteins (BMPs), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), glial cell line-derived neurotrophic factor 15 (GDNF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), Growth differentiation factor 9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin, insulin-like growth factor (IGF), migration-stimulating factor 20, myostatin (GDF-8), nerve growth factor (NGF) and other neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-β), Tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), 25-agonists of the WNT signaling pathway, growth factor

<img file="MX390940B_D0046.tif" />

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY placental (PIGF), fetal bovine somatotropin (FBS), interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7 and the like. In one embodiment, the cell culture medium is supplemented by the addition of insulin-like growth factor at the point of use. In one embodiment, the concentration of insulin in the medium, that is, the amount of insulin in the cell culture medium after addition, is about 0.1 μM to 10 μM.

Buffers are generally known in the art. The invention is not restricted to any particular buffer or buffers, and any person of ordinary skill in the art can select an appropriate buffer or buffer system for use with a particular cell line producing a particular protein. In one embodiment, a point-of-use addition buffer is NaHCO₃In another embodiment, the buffer is HEPES. In other embodiments, the point-of-use addition buffer comprises both NaHCO₃ such as HEPES.

Energy sources for use as point-of-use addition ingredients in cell culture are also well known. Without limitation, in one embodiment, the point-of-use addition energy source is glucose. Given the particular and specific requirements of a particular cell line and the protein to be produced, in one embodiment, glucose can be added at a concentration of approximately 1 to 20 mM to the medium. In some cases, glucose can be added in high concentrations of up to 10 g/L.

Similarly, chelators are well known in the art of

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY cell culture and protein production. Tetrasodium EDTA dehydrate and citrate are two chelators known in the art, although other chelators can be used in the practice of this invention. In one embodiment, a point-of-use chelator is tetrasodium EDTA dihydrate. In one embodiment, a point-of-use chelator is citrate, such as Na₃CeH_sEITHER₇.

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

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

In some embodiments, the protein titer produced by the cell culture in the taurine-supplemented medium is at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22% higher, at least 23% higher, at least 24% older, at least 25% older, at least 26% older, at least 27% older, at least

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28% greater or at least 29% greater than the protein titer (yield) of cells cultured without taurine supplementation. In some embodiments, the protein titer produced by the cells in the taurine-supplemented medium is at least 2%, at least 3%, at least 4%, or at least 5% greater than the protein titer (yield) of similar or identical cells cultured in medium not supplemented with taurine.

In some embodiments, ammonia accumulation in the cell culture is reduced by more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 15%, or more than 20% in the taurine-supplemented medium, compared to a cell culture in a medium not supplemented with taurine.

PROTEIN PRODUCTION

In addition to taurine-supplemented medium and methods of culturing cells in taurine-supplemented medium, the present invention provides improved methods for producing a protein, such as a therapeutically effective antibody or other biopharmaceutical drug substance in a cell cultured in taurine-supplemented medium. The present invention provides a method for producing a therapeutic protein in high yield, comprising culturing a recombinant cell line in a medium containing taurine, wherein the cell line comprises a stably integrated nucleic acid encoding the therapeutic protein.

In some embodiments, the protein titer (yield) of the 25 mammalian cells cultured in the taurine-containing medium (medium

<img file="MX390940B_D0049.tif" />

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY supplemented with taurine) is at least 100 mg/L, at least 0.5 g/L, at least 1 g/L, at least 1.2 g/L, at least 1.4 g/L, at least 1.6 g/L, at least 1.8 g/L, at least 2 g/L, at least 2.5 g/L greater than the protein titer of an identical mammalian cell grown in a medium not supplemented with taurine.

In some embodiments, the protein production yield or titer, which may be expressed in grams of protein product per liter of culture medium, of the cells cultured in the taurine-supplemented medium is at least 100 mg/L, at least 1 g/L, at least 1.2 g/L, at least 1.4 g/L, at least 1.6 g/L, at least 1.8 g/L, at least 2 g/L, at least 2.5 g/L, at least 3 g/L, at least 3.5 g/L, at least 4 g/L, at least 4.5 g/L, at least 5 g/L, at least 5.5 g/L, at least 6 g/L, at least 6.5 g/L, at least 7 g/L, at least 7.5 g/L, at least 8 g/L, at least 8.5 g/L, at least 9 g/L, at least 9.5 g/L, at least 10 g/L, at least 15 g/L, or at least 20 g/L.

In some embodiments, the protein titer produced by the cells in the taurine-supplemented medium is at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23% older, at least 24% older, at least 25% older, at least 26% older, at least 27% older,

<img file="MX390940B_D0050.tif" />

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY at least 28% greater or at least 29% greater than the protein titer (yield) of similar or identical cells cultured in a medium not supplemented with taurine.

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

In some embodiments, the antibody is selected from the group consisting of an anti-programmed cell death 1 antibody (e.g., an anti-PD1 antibody as described in U.S. Patent Application Publication No. US2015/0203579A1), an anti-programmed cell death 1 ligand (e.g., an anti-PD-L1 antibody as described in U.S. Patent Application Publication No. US2015/0203580A1), an anti-DII4 antibody, an anti-390940 antibody, or an anti-PD-L1 antibody.

<img file="MX390940B_D0051.tif" />

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY angiopoietin 2 (e.g., an anti-ANG2 antibody as described in U.S. Patent No. 9,402,898), an anti-angiopoietin-like antibody 3 (e.g., an anti-AngPtl3 antibody as described in U.S. Patent No. 9,018,356), an anti-platelet-derived growth factor receptor antibody (e.g., an antiPDGFR antibody as described in U.S. Patent No. 9,265,827), an anti-Erb3 antibody, an anti-prolactin receptor antibody (e.g., the anti-PRLR antibody described in U.S. Patent No. 9,302,015), an anti-complement 5 antibody (e.g., an anti-C5 antibody described in U.S. Patent Application Publication No. US2015/0313194A1), an anti-TNF antibody, an anti-epidermal growth factor receptor antibody (e.g., an anti-EGFR antibody described in U.S. Patent No. 9,132,192, or an anti-EGFRvIll antibody described in U.S. Patent Application Publication No. US2015/0259423A1), an anti-proprotein convertase subtilisin Kexin 9 antibody (e.g., an anti-PCSK9 antibody described in U.S. Patent No. 8,062,640 or U.S. Patent Application Publication No. US2014/0044730A1), an anti-growth and differentiation factor 8 antibody (e.g., An antibody comprising at least one of the following groups, e.g., an anti-GDF8 antibody, also known as an anti-myostatin antibody, described in U.S. Patent Nos. 8,871,209 or 9,260,515), an anti-glucagon receptor antibody (e.g., the anti-GCGR antibody described in U.S. Patent Application Publication Nos. US2015/0337045A1 or US2016/0075778A1), an anti-VEGF antibody, an anti-IL1R antibody, a glucagon receptor antibody, an anti-glucose ...

<img file="MX390940B_D0052.tif" />

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY interleukin 4 (e.g., an anti-IL4R antibody described in U.S. Patent Application Publication No. US2014/0271681A1 or U.S. Patent Nos. 8,735,095 or 8,945,559), an anti-interleukin 6 receptor antibody (e.g., an anti-IL6R antibody described in U.S. Patent Nos. 7,582,298, 8,043,617 or 9,173,880), an anti-IL1 antibody, an anti-IL2 antibody, an anti-IL3 antibody, an anti-IL4 antibody, an anti-IL5 antibody, an anti-IL6 antibody, an anti-IL7 antibody, an anti-interleukin 33 (e.g., the anti-IL33 antibody described in U.S. Patent Application Publication Nos. US2014/0271658A1 or US2014/0271642A1), an anti-respiratory syncytial virus antibody (e.g., (e.g., the anti-RSV antibody described in U.S. Patent Application Publication No. US2014/0271653A1), an anti-cluster of differentiation 3 (e.g., an anti-CD3 antibody, described in U.S. Patent Application Publication Nos. US2014/0088295A1 and US20150266966A1, and U.S. Application No. 62/222,605), an anti-cluster of differentiation 20 (e.g., an anti-CD20 antibody described in U.S. Patent Application Publication Nos. US2014/0088295A1 and US20150266966A1, and in U.S. Patent No. 7,879,984), an anti-CD19 antibody, an anti-CD28 antibody, an anti-cluster of differentiation 48 (e.g., the anti-CD48 antibody described in U.S. Patent No. 9,228,014), an anti-Feldl antibody (e.g., the one described in U.S. Patent No. 9,079,948), an anti-Middle East respiratory syndrome virus (e.g., an anti-MERS antibody described in the publication of

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY U.S. Patent Application No. US2015/0337029A1), an anti-Ebola virus antibody (e.g., the one described in U.S. Patent Application Publication No. US2016/0215040), an anti-Zika virus antibody, an anti-lymphocyte activation gene 3 antibody (e.g.,

e.g., an anti-LAG3 antibody or an anti-CD223 antibody), an anti-nerve growth factor antibody (e.g., an anti-NGF antibody described in U.S. Patent Application Publication No. US2016/0017029 and U.S. Patent Nos. 8,309,088 and 9,353,176), and an anti-activin A antibody. In some embodiments, the bispecific antibody is selected from the group consisting of an anti-CD3 x anti-CD20 bispecific antibody (described in U.S. Patent Application Publication Nos. US2014/0088295A1 and US20150266966A1), an anti-CD3 x anti-mucin 16 bispecific antibody (e.g., an anti-CD3 x anti-Muc16 bispecific antibody), and an anti-CD3 x anti-prostate-specific membrane antigen bispecific antibody (e.g., an anti-CD3 x anti-CD20 bispecific antibody). e.g., an anti-CD3 x anti-PSMA bispecific antibody). In some embodiments, the protein of interest is selected from the group consisting of alirocumab, sarilumab, fasinumab, nesvacumab, dupilumab, trevogrumab, evinacumab, and rinucumab. All publications mentioned throughout this disclosure are incorporated herein by reference in their entirety.

In some embodiments, the protein of interest is a recombinant protein containing an Fc portion and another domain (e.g., an Fc fusion protein). In some embodiments, an Fc fusion protein is a receptor-Fc fusion protein containing one or more of the following:

<img file="MX390940B_D0054.tif" />

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Mexican Institute of Industrial Property plus extracellular domains of a receptor coupled to an Fc portion. In some embodiments, the Fc portion comprises a hinge region followed by a CH2 and CH3 domain of an IgG. In some embodiments, the receptor-Fc fusion protein contains two or more distinct receptor chains that bind a single ligand or multiple ligands. For example, an Fc fusion protein is a TRAP protein, such as an IL-1 trap (e.g., rilonacept, which contains the ligand binding region of IL-1RAcP fused to the Fc-fused II-1R1 extracellular region of hlgG1; see U.S. Patent No. 6,927,004 , which is incorporated herein by reference in its entirety), or a VEGF trap (e.g., (e.g., aflibercept, which contains the Ig domain 2 of the VEGF receptor Flt1, fused to the Fc domain 3 of the VEGF receptor Flk1 fused to hlgG1; see U.S. Patent Nos. 7,087,411 and 7,279,159). In other embodiments, an Fc fusion protein is a scFv-Fc fusion protein containing one or more antigen-binding domains, such as a variable heavy chain fragment and a variable light chain fragment of an antibody, coupled to an Fc portion.

The present invention is not limited to any particular cell type for protein production. Examples of suitable cell types for protein production include mammalian cells, insect cells, avian cells, bacterial cells, and yeast cells. The cells may be stem cells or recombinant cells transformed with a vector for recombinant gene expression, or

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Mexican Institute of Industrial Property cells transfected with a virus to produce viral products. The cells may contain a recombinant heterologous polynucleotide construct encoding a protein of interest. This construct may be an episome or an element that physically integrates into the cell's genome. The cells may also produce a protein of interest without having that protein encoded on a heterologous polypeptide construct. In other words, the cell can naturally encode the protein of interest, such as a B cell that produces an antibody. The cells can also be primary cells, such as chicken embryo cells, or primary cell lines. Examples of useful cells include BSC cells, LLC-MK cells, CV-1 cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK-21 cells, chicken embryo cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 293 cells, RK cells, Per.C6 cells, and CHO cells. In various embodiments, the cell line is a derivative of CHO cells, such as CHO-K1, CHO DUX B-11, CHO DG-44, VeggieCHO, GS-CHO, S-CHO, or CHO lee mutant lines.

In one embodiment, the cell, which is a CHO cell, ectopically expresses a protein. In one embodiment, the protein comprises an immunoglobulin heavy chain region, such as a CH1, CH2, or CH3 region. In one embodiment, the protein comprises a human or rodent immunoglobulin CH2 and CH3 region. In one embodiment, the protein comprises an immunoglobulin CH1, CH2, and CH3 region.

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Mexican Institute of Industrial Property of human or rodent. In one embodiment, the protein comprises a hinge region and a CH1, CH2, and CH3 region. In a specific embodiment, the protein comprises an immunoglobulin heavy chain variable domain. In one embodiment, the protein comprises an immunoglobulin light chain variable domain. In one embodiment, the protein comprises an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain. In one embodiment, the protein is an antibody, such as a human antibody, a rodent antibody, or a chimeric human/rodent antibody (e.g., human/mouse, human/rat, or human/hamster).

A production phase can be performed at any cultivation scale, from shake flasks or Wave Bags to one-liter bioreactors, and even large-scale industrial bioreactors. Similarly, a seed train expansion phase can be performed at any cultivation scale, from shake flasks or Wave Bags to one-liter or larger bioreactors. A large-scale process can be carried out in a volume of approximately 100 liters to 20,000 liters or more. One or more of several means can be used to control protein production, such as temperature shifting or chemical induction. A growth phase can occur at a higher temperature than a production phase. For example, the growth phase may occur at a first temperature of approximately 35°C to 38°C and the production phase may occur at a second temperature of

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY approximately 29° C to 37° C, optionally from about 30° C to 36° C or from about 30° C to 34° C. In addition, chemical inducers of protein production such as caffeine, butyrate, tamoxifen, estrogen, tetracycline, doxycycline and hexamethylenebisacetamide (HMBA) may be added concurrently, before or after a temperature change. If inducers are added after a temperature change, they can be added from one hour to five days after the temperature change, for example, one to two days after the temperature change. Production cell cultures can be run as a fed-continuous culture system, as in a chemostat (see C. Altamirano et al., 2001, supra), or according to a fed-batch process (Huang, 2010, supra).

The invention is useful for improving protein production through cell culture processes. The cell lines used in the invention can be genetically manipulated to express a polypeptide of commercial or scientific interest. Genetic manipulation of the cell line involves transfecting, transforming, or transducing the cells with a recombinant polynucleotide molecule, or otherwise altering (e.g., e.g., by homologous recombination and gene activation or fusion of a recombinant cell with a non-recombinant cell), in order to cause the host cell to express a desired recombinant polypeptide. Methods and vectors for genetically engineering cells or cell lines to express a polypeptide of interest are well known to those skilled in the art.

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Mexican Institute of Industrial Property subject matter; for example, several techniques are illustrated in “Current Protocols in Molecular Biology,” Ausubel et al., eds. (Wiley &amp; Sons, New York, 1988, and quarterly updates); Sambrook et al., “Molecular Cloning: A Laboratory Manual” (Cold Spring Laboratory Press, 1989);

Kaufman, RJ, “Large Scale Mammalian Cell Culture”, 1990, pp. 15–69. A wide variety of cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and commercial suppliers. Examples of cell lines commonly used in industry include

VERO, BHK, HeLa, CVI (including Cos), MDCK, 293, 3T3, myeloma cell lines (e.g., NSO, NSI), PC12, WI38, and Chinese hamster ovary (CHO) cells. CHO cells are widely used for the production of recombinant complex proteins, such as cytokines, coagulation factors, and antibodies (Brasel et al. (1996), Blood

88:2004-2012; Kaufman et al. (1988), J. Biol. Chem 263:6352-6362;

McKinnon et al. (1991), J Mol Endocrino! 6:231-239; Wood et al. (1990), J Immunol. 145:3011-3016). The dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sel USA 77:4216-4220), DXBI-1 and DG-44, are convenient host CHO cell lines because the efficient selectable and amplifiable DHFR gene expression system allows high-level recombinant protein expression in these cells (Kaufman RJ. (1990), Meth Enzymol 185:537-566). In addition, these cells are easy to manipulate as adherent or suspension cultures and exhibit relatively good genetic stability. CHO cells and

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Mexican Institute CD of Industrial Property Q proteins recombinantly expressed by them have been extensively characterized and have been approved by regulatory agencies for use in clinical and commercial manufacturing. In some embodiments, the CHO cell lines are the cell lines described in U.S. patent application publication Nos. 2010/0304436 A1, 2009/0162901 A1, and 2009/0137416 A1, and in U.S. Patent Nos. 7,455,988 B2, 7,435,553 B2 and 7,105,348 B2.

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

EXAMPLES

EXAMPLE 1: Enhanced antibody titers due to taurine supplementation

Example 1A - High-Throughput Shake Flask Culture: 250 mL shake flasks were inoculated with a seed culture of a monoclonal antibody (Ab1)-producing cell line derived from CHO-K1. The inoculated cells were grown at 35.5°C for seventeen days and fed with glucose and other supplemental nutrients as needed. The cells grew

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY in a chemically defined base medium (hydrolysate-free and serum-free).

Each culture flask was supplemented with 1 mM taurine on day 0 (flask 1b) or was not supplemented (flask 1a).

TABLE 2: Average 17-day antibody titers (q/L) and approximate titer increase (%) from baseline

<td>Flask</td><td>Medium complement</td><td colspan="2">Ab1 Title</td>

<td>1st</td><td>Not supplemented*</td><td>7.3 g/L</td><td></td>

<td>1b</td><td>Taurine</td><td>7.9 g/L^TO</td><td> 8%</td>

*Baseline control for % titer increase; flask 1b compared to the titer in unsupplemented medium (flask 1a).

^L The difference in the final titer between supplemented and non-supplemented culture is statistically significant (p &lt; 0.05).

Titer values were calculated from protein harvested on days 15–17 and are statistically significant (p &lt; 0.05) compared to baseline. Taurine-supplemented cultures exhibited an overall 8% increase in final protein titer compared to unsupplemented cultures.

Example 1B - Laboratory Scale Bioreactors: In a similar example 20, but on a larger production scale, 2 L bioreactors were inoculated with a seed culture of a monoclonal antibody (Ab2, Ab3, or Ab4) producing cell line derived from CHO-K1. The inoculated cultures were grown at a temperature of 35.5°C, OD set point of 40.4%, and 22 mL air sparge for 14 days.

The Ab2 and Ab3 processes had pH set points of 7.0 ±

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0.15, while the Ab4 process had a pH set point of 7.13 ± 0.27. Glucose, antifoam, and basal feed were supplied to the bioreactors as needed. Cultures were grown in unsupplemented medium (bioreactors 2a, 3a, 4a) or grown in medium supplemented with approximately 1 mM taurine (Ab2 and Ab3) or medium supplemented with approximately 3 mM taurine (Ab4), added on day 0 of production (bioreactors 2b, 3b, and 4b, respectively).

The antibody yield (titer) was 6.4 g/L for Ab2-producing cells, but cells grown with taurine produced 8 g/L of protein. The 24% increase in titer compared with cells grown without taurine supplementation is statistically significant (p &lt; 0.05). The resulting final titers for Ab3-producing and Ab4-producing cultures were also significantly higher (p &lt; 0.05) after 14 days (11% and 20%, respectively), compared to cultures not supplemented with taurine. See Table 3.

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TABLE 3: 14-day average antibody titers (g/L) v approximate titer increase (%) from baseline

<td>Medium complement</td><td>Bioreactor #</td><td>Qualification Ab2</td><td>Bloreactor #</td><td>Qualification Ab3</td><td>Bioreactor #</td><td>Qualification Ab4</td>

<td>Not supplemented*</td><td>2nd</td><td>6.4 g/L</td><td>3a</td><td>6.6 g/L</td><td>4a</td><td>4.4 g/L</td>

<td>Taurine</td><td>2b</td><td>8 24% g/L^TO</td><td>3b</td><td>7.3 11% g/L^TO</td><td>4b</td><td>5.3 20% g/L^TO</td>

* Unsupplemented medium is the baseline control for % titer increase, where the % titer increase in bioreactors 2b, 3b, or 4b is compared to the titer in unsupplemented medium (bioreactors 2a, 3a, or 4a, respectively).

^L The differences in the final titer between the supplemented and non-supplemented cultures are statistically significant (p &lt; 0.05).

The time course of protein titer was plotted for the Ab3-producing cell culture and a significant improvement in protein titer was observed due to taurine supplementation every day of culture starting on day 6.

TABLE 4: Antibody titer enhancement (g/L) due to taurine supplementation at representative time points

<td>Bioreactor</td><td>Medium complement</td><td>Ab3 title on the 6th</td><td>Ab3 title on the 9th</td><td>Ab3 title on the 14th</td>

<td>3a</td><td>Not supplemented*</td><td>1.7 _J/L</td><td>4.1 g/L</td><td>6.6 g/L</td>

<td>3b</td><td>Taurine</td><td>g/L^12%</td><td>«íú^29%</td><td>g⁷/L^{At 11%}</td>

*Approximate increase (%) compared to unsupplemented medium collected on the same day^L The increase in titer with taurine supplementation is statistically significant (p &lt; 0.05) compared to the non-supplemented culture.

A significant titer increase was observed in the production culture as early as day 6 (12% increase compared to the same culture without taurine supplementation). See also Figure 1. The greatest difference in this time course was observed on day 9 (significant (p &lt; 0.05), 29% increase in bioreactor 3b compared to 3a), and a significant (p &lt; 0.05) 11% titer increase was observed on the final day of cultivation (day 14).

The protein production benefits of taurine supplementation are observed across different scales (examples 1A and 1B) and different cell lines (example 1B).

EXAMPLE 2: Consistent productivity with varying taurine concentrations in a high-throughput shake flask culture

Protein titer consistency was analyzed by varying the amount of taurine added to the culture on day 0 of production. 250 mL shake flasks were inoculated with a seed culture of a monoclonal antibody (Ab1)-producing cell line derived from CHO-K1. The inoculated cells were grown at 35.5°C for fourteen days and fed with glucose and other supplemental nutrients as needed. Cells were grown in chemically defined basal medium (hydrolysate-free and serum-free).

Each culture contained taurine at concentrations of 0.1 mM, 0.3 mM, 0.5 mM, 0.7 mM, 1 mM, 3 mM, 5 mM, 7.5 mM, or 10 mM, or did not contain taurine (unsupplemented).

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TABLE 5: Average 14-day antibody titers (g/L) of cultures supplemented with 0.1 to 10 mM taurine

<td>Shake flask</td><td>Medium complement</td><td colspan="2">Ab1 Title</td>

<td>5a</td><td>Not supplemented*</td><td>6.5 g/L</td><td></td>

<td>5b</td><td>Taurine 0.1 mM</td><td>6.7 g/L</td><td> 3%</td>

<td>5c</td><td>Taurine 0.3 mM^TO</td><td>6.8 g/L</td><td> 5%</td>

<td>5d</td><td>Taurine 0.5 mM^TO</td><td>6.9 g/L</td><td> 6%</td>

<td>5e</td><td>Taurine 0.7 mM^TO</td><td>7.0 g/L</td><td> 8%</td>

<td>5f</td><td>Taurine 1 mM^TO</td><td>7.0 g/L</td><td> 8%</td>

<td>5g</td><td>Taurine 5 mM^TO</td><td>7.1 g/L</td><td> 9%</td>

<td>5h</td><td>Taurine 7.5 mM^TO</td><td>7.1 g/L</td><td> 9%</td>

<td>5i</td><td>Taurine 10 mM^TO</td><td>7.1 g/L</td><td> 9%</td>

* Unsupplemented medium is the baseline control for % titer increase.

* The difference in final titer is statistically significant compared to the unsupplemented control (p &lt; 0.1).

^TO The difference in final titer is statistically significant compared to the unsupplemented control (p &lt; 0.05).

It is shown that varying the amount of taurine supplementation produces consistently high titers when taurine is added in the range of at least 0.1 mM to 10 mM. Final titers under taurine supplementation conditions were statistically different from those under unsupplemented conditions. For 0.1 mM taurine, p &lt; 0.1, while p &lt; 0.05 for 0.3 mM to 10 mM taurine.

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EXAMPLE 3: Analysis of variable taurine feeding programs in a high-throughput shake flask culture

EXAMPLE 3A: Addition of taurine during the seed train expansion phase:

The benefits of adding taurine to the culture during the seed train phase of expansion were evaluated in a high-throughput shake flask model. In flask 6a (Table 6), Ab1-producing CHO cells were thawed in chemically defined basal medium (hydrolysate-free and serum-free) supplemented with 1 mM taurine. The taurine concentration of the basal medium was maintained at 1 mM throughout the expansion phase. During production, the basal culture medium was supplemented with 1 mM taurine on day 0. Glucose and basal nutrient feeding were supplied as needed during the 17 days of production.

Cells in flask 6b were grown in chemically defined, taurine-free (unsupplemented) basal medium (hydrolysate-free and serum-free) throughout the seed train expansion phase. On day 0 of production, the culture basal medium was supplemented with 1 mM taurine. On day 17 of production, glucose and basal nutrient feeds were supplied as needed.

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TABLE 6: Average antibody titers of 17 days of cultures supplemented with 1 mM taurine during different phases of the process

<td>Shake flask</td><td>Addition of 1 mM taurine</td><td>Ab1 Title</td>

<td>6a</td><td>Seed and production train phases</td><td>7.7 g/L</td>

<td>6b</td><td>Production phase only</td><td>7.8 g/L</td>

The differences in the final titer (day 17) are not statistically significant (p > 0.1)

Final titer values (day 17) for the two conditions (taurine supplementation only during production or combined in the seed train and production) are similar. The resulting titers are not statistically significant (p > 0.1). The benefit of taurine addition during the seed train expansion phase is analogous to that of taurine supplementation during the production phase.

EXAMPLE 3B: Variable taurine feeding schedules during the production phase: To determine if standard variable taurine feeding schedules have any effect on protein titer on taurine supplemented cultures, additional analogous experiments were done in shake flask cultures growing Ab1-producing CHO cells. The cells were subjected to variable culture conditions similar to example 2, where the feeding schedule was the same as before, with basal glucose/nutrient feeding being added as needed.

Ab1-producing cultures were supplemented with a total of 5 mM taurine. Similar productivity was observed (7.1 g/L, 6.8 g/L and

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7.0 g/L) by varying the taurine feeding programs. The titer values compared in this experiment were not statistically different (p > 0.1) (see Table 7).

TABLE 7: Average 14-day antibody titers (g/L) of cultures supplemented with 5 mM taurine with varying programs

<td>Bullfighting program</td><td>Ab1 title on the 14th</td>

<td>5 mM, day 0</td><td>7.1 g/L</td>

<td>1 mM, days O, 3, 5, 7, 10 (5 mM total)</td><td>6.8 g/L</td>

<td>1 mM, day 0; 2 mM, days 7, 10 (5 mM total)</td><td>7.0 g/L</td>

The differences between the titer values on day 14 are not statistically significant (p >0.1).

Taurine feeding programs have no negative effects or altered outcomes where taurine supplementation is beneficial for product performance. Thus, taurine supplementation can be added once on day 0, or on subsequent days of the production phase, or at various intervals throughout the production phase.

EXAMPLE 4: Measurement of ammonia byproduct in a high-throughput shake flask culture

The byproduct ammonia was measured after 14 days of cultivation in a manner analogous to Example 2 in the taurine-supplemented cultures of Ab1-producing CHO cells. The 25 cells were subjected to variable culture conditions similar to those

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TABLE 8: Average ammonia (mM) on day 17 vs. decrease (%)

<td>Medium complement</td><td colspan="2">Ammonia in Ab1</td>

<td>Not supplemented*</td><td>2.56 mM</td><td></td>

<td>Taurine</td><td>1.73 mM^TO</td><td> -32%</td>

*Baseline control for % decrease in ammonia; taurine supplementation condition compared to ammonia in unsupplemented medium.

^L The decrease in ammonia concentration is statistically significant (p &lt; 0.1).

In Ab1-producing cells, taurine supplementation in the medium supports a sustainable, healthy culture, with a 32% reduction in the ammonia byproduct. The decrease in ammonia concentration due to taurine supplementation is statistically significant (p &lt; 0.1).

The present invention may be incorporated in other specific embodiments.