EA041694B1 - CELL CULTIVATION MEDIUM WITH TAURINE SUPPLEMENT AND METHODS OF APPLICATION - Google Patents

Description

The field of technology to which the present invention relates

The present invention relates to media and methods for culturing cells and obtaining recombinant proteins. In particular, the present invention relates to a medium supplemented with taurine and methods for culturing recombinant eukaryotic cells therein to obtain protein-based biologics.

Background of the Invention

The organic acid taurine, often referred to as the β-amino acid, occurs in high concentrations in most tissues and is a derivative of the amino acid cysteine (Huxtable, RJ., 1992, PhysiolRev, 72:101-163).

ABOUT

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Structure of taurine

Taurine is present in many human and other mammalian tissues, such as the brain, retina, myocardium, skeletal and smooth muscle, platelets, and neutrophils.

It is recognized that taurine promotes osmoregulation, membrane stabilization and anti-inflammatory effects, and also regulates mitochondrial protein synthesis by increasing the activity of the electron transport chain, which protects against the formation of superoxides (Jong et al., 2010, Journal of Biomedical Science 17(Suppl 1):S25 ; Jong et al., 2012, Amino Acids 42:2223-2232sw). In primary cultures of neurons, taurine has been described as a cytoprotectant by suppressing glutamate-induced toxicity. Various embryonic culture media containing taurine have been developed.

Cell culture techniques involving amino acid feeds have long been used to obtain recombinant proteins from cultured cells. Amino acids are biosynthesis precursors, energy sources, osmolytes, etc., and their use in producer cultures is significantly associated with continuous cell growth and productivity.

At the same time, the physiological events that promote productivity and high-efficiency protein expression are innumerable, and competing activities and transport mechanisms make the design of feeding strategies a difficult problem. The type of amino acid supplement and the time of addition can also influence the quality of the protein produced in culture (Altamirano, et al., 2006, Electron. J. Biotechnol. 9:61-67). Accumulation of by-products is often problematic in cell culture and is thought to result from nutrient imbalances in cell culture, ultimately inhibiting cell growth (Fan, Y. et al. BiotechnolBioeng. 2015 Mar; 112(3):521-35). Hypotaurin, or an analogue or precursor thereof, has been proposed in cell culture to achieve the desired results of reducing the color intensity of a composition containing a recombinantly produced polypeptide (WO 2014145098 A1, published September 18, 2014). A cell culture medium has also been described that promotes the maturation of immature retinal pigment epithelial cells into mature retinal pigment epithelial cells (WO 2013184809 A1, published December 12, 2013). However, optimization of the productivity of recombinant proteins in cultures supplemented with taurine is not generally recognized in the art. Cell culture methods that increase the productivity of recombinantly expressed proteins while minimizing the release of potentially toxic by-products of cellular metabolism, such as ammonia, are highly desirable. Any sustained increase in productivity can equate to a much higher supply of a biotherapeutic product on an industrial scale.

Thus, there is a need in the art for a medium and methods for culturing mammalian cells, where the medium provides normal and stable growth and maintenance of the cells and the production of high titers of the biopharmaceutical drug substance.

Brief summary of the present invention

The inventors of the present invention have made the surprising discovery that the inclusion of taurine in the cell culture medium increases the specific productivity of the cells and contributes to the reduced formation of the ammonia by-product by these cells. Various feeding strategies, including taurine, contribute to the production of increased protein titers. In addition, the addition of taurine does not adversely affect the characteristics of the culture or the quality of the resulting antibodies.

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

The present invention provides a cell culture medium that is serum-free and contains about 0.1 mM to about 10 mM taurine. The present invention provides a cell culture medium that is serum-free and contains about 0.1 mM to about 1 mM taurine, about 0.2 to about 1 mM taurine, about 0.3 to about 1 mM taurine, about 0.4 to about 1 mM taurine, or about 0.5 to about 1 mM taurine. The present invention provides a cell culture medium that is serum-free and contains from about 1 mM to about 10 mM taurine. The present invention provides a cell culture medium that is serum free and contains about 1 mM to about 5 mM taurine, about 1 mM to about 6 mM taurine, about 1 mM to about 7 mM taurine, about 1 mM to about 8 mM taurine or about 1 mM to about 9 mM taurine.

In some embodiments, the medium further 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 accordance with some embodiments, the implementation of the environment contains <16 g/l of hydrolyzate. In accordance with some embodiments, the medium does not contain a hydrolyzate.

In accordance with one embodiment, the medium contains a basic medium that is chemically defined, such as a custom formulation, or a commercially available basic medium. In accordance with one embodiment, the complete medium is chemically detectable, does not contain serum, and does not contain hydrolysate.

In accordance with some embodiments, the entire medium, including the base medium and feeds, contains a total of at least 115 mM of a mixture of amino acids or amino acid salts. According to one embodiment, the amino acid mixture contains 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 the amount selected from the table. 1.

In accordance with some embodiments, the medium contains one or more fatty acids. According to one 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 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 octanoic acid.

In accordance with some embodiments, the medium contains a mixture of nucleotides. According to one embodiment, the medium contains adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

In accordance with some embodiments, the medium contains a mixture of salts. Salts include divalent cations such as calcium and magnesium. In accordance with one embodiment, the medium contains calcium chloride and magnesium sulfate. Other salts may include phosphates.

In accordance with one embodiment, the medium (1) contains 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 mM taurine, (2) contains <16 g/l hydrolysate, (3) is serum-free, (4) optionally additionally 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 high yield protein of interest, 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 contains a stably integrated nucleic acid encoding a protein . In accordance with other embodiments, the implementation of the environment includes any of the above aspects of the present invention.

According to another aspect, the present invention provides a method for culturing eukaryotic cells to enhance production of a recombinant protein, comprising the steps of (a) expanding or maintaining the cells in a defined cell culture medium during the growth phase, (b) adding a basic cell culture medium containing about 0.1 mM to about 10 mM L-taurine, and expression of the recombinant protein of interest during the product formation phase, and (c) raising the titer of the protein of interest by adding taurine. According to some embodiments, the addition of taurine is provided at least once during the product formation phase, or twice, three times, four times, or five times during the product formation phase, or every day during the product formation phase. According to other embodiments, the method further comprises adding about 0.1 mM to about 10 mM L-taurine culture medium during the growth phase. In some embodiments, the method provides for increased production of the recombinant protein compared to eukaryotic cells without taurine supplementation or with less than 0.1 mM taurine supplementation and under otherwise identical conditions.

According to another aspect, the present invention provides a method for culturing cells in a cell culture medium, such as any embodiment of the medium described in the above aspect. In accordance with one embodiment, the method comprises the steps of propagating or maintaining a cell or cells in a medium that (1) contains taurine at a concentration of at least 0.1+0.015 mM, (2) contains <16 g/L of hydrolyzate or not contains a hydrolyzate, (3) does not contain sera, (4) optionally contains amino acids selected from the group consisting of a mixture of amino acids selected from the table. 1.

In accordance with one embodiment, the optional mixture of amino acid supplements is selected from the group consisting of amino acids in table. 1.

Table 1

Amino acid mM range (mmol/L) Range (g/l) Alanine 0-11.2 0-1 Arginine 2.4-11.9 0.5-2.5 Asparaginin 1.3-33.3 0.2-5 Aspartic acid 1.5-93.9 0.2-12.5 Cysteine 1.1-19.9 0.2-3.5 Glutamic acid 1.4-47.6 0.2-7 Glutamine 0-23.9 0-3.5 Glycine 0-16.7 0-1.25 G istidine 1-9.5 0.2-2 Isoleucine 1.5-22.9 0.2-3 Leucine 1.5-38.1 0.2-5 Lysine 2.7-24.6 0.5-4.5 Methionine 1.3-13.4 0.2-2 Phenylalanine 1.2-18.2 0.2-3 Proline 1.7-26.1 0.2-3 Serene 1.9-57.1 0.2-6 Threonine 1.7-33.6 0.2-4 tryptophan 0.5-14.7 0.1-3 Tyrosine 0.9-22.2 0.2-5 Valine 1.7-34.1 0.2-4

In some embodiments, the cell or cells are mammalian cells, avian cells, insect cells, yeast cells, or bacterial cells. According to one embodiment, the cells are mammalian cells suitable for the production of recombinant proteins, such as CHO cells or a Cho-Κι derivative. In some embodiments, cells express a protein of interest, such as a biotherapeutic protein. The biotherapeutic protein may be an antigen binding protein which may contain an Fc domain. In some embodiments, the protein of interest is an Fc fusion protein, such as a ScFv molecule or a decoy molecule. Trap molecules include, but are not limited to, a VEGF Trap protein and an IL-1 Trap protein. In some embodiments, the protein of interest is an antibody, such as a human monoclonal antibody, a humanized monoclonal antibody, a bispecific antibody, or an antibody fragment.

Given the positive effects on protein production when taurine is included in various forms of serum-free media, cells cultured in accordance with this method form an increased protein titer on average. In accordance with one embodiment, when compared with the protein titer in medium that was not supplemented with taurine, cells grown in medium supplemented with taurine in

- 3 041694 according to this method, proteins are formed having a protein titer that is at least 8% greater than the titer of the control reference culture (ie the culture that was not supplemented with taurine). In accordance with one embodiment, cells grown in culture supplemented with taurine, when compared with the protein titer in the medium that was not supplemented with taurine, form 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%, 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% more than the reference culture.

Similarly, incorporation of taurine alone into serum-free media results in lower levels of ammonia by-product in cultured cells than without incorporation of taurine. According to one serum-free and hydrolyzate-free medium embodiment with the addition of taurine in cell culture, a reduced level of ammonia by-product (mM NH3) can be achieved that is at least 4% lower to 32% lower than in the same cell culture, which contains no additive (i.e., less than 0.1 mM taurine or no added taurine).

According to another embodiment, the method comprises the step of adding one or more targeted additives to the cell culture medium. In some embodiments, the target supplement is any one or more of NaHCO ₃ , glutamine, insulin, glucose, CuSO ₄ , ZnSO ₄ , FeCl ₃ , NiSO ₄ , Na ₄ EDTA, and Na ₃ citrate. According to one embodiment, the method comprises the step of adding each of the following target chemicals to the cell culture medium: NaHCO ₃ , glutamine, insulin, glucose, CuSO4, ZnSO4, FeCl3, NiSO4, Na4EDTA, and Na3 citrate. In accordance with some variants of implementation of the target additives can be included in the environment at the initial stage.

In accordance with a specific embodiment, the aspect provides a method of culturing cells in a serum-free medium, which consists essentially of (1) taurine at a concentration of at least 0.1 mm; (2) contains <16 g/L hydrolysate, (3) is serum-free, (4) optionally additionally contains a total of at least about 20 mM, or at least about 25 mM, or at least about 30 mM, or at least at least about 40 mM, or at least about 50 mM, or at least about 60 mM, or at least about 70 mM 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.

According to another embodiment, the present invention provides a method for producing a protein of interest, comprising the steps of (1) introducing into a cell a nucleic acid sequence that encodes a protein of interest; (2) selecting a cell or cells expressing the protein of interest; (3) culturing the selected cell according to an embodiment of the serum-free cell culture medium described in the previous aspect or according to any embodiment of the method described herein; and (4) expression of the protein of interest in the cell, where 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 a CHO, 293, and BHK cell, or any derivatives thereof. According to one embodiment, the cell is a CHO cell, such as a CHO-K1 cell.

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

In one embodiment, the protein of interest is capable of being formed at a mean titer on days 14, 15, 16, or 17 that is at least 8% greater than the mean titer on days 14, 15 day 16 or 17 formed by a similar cell in serum-free medium containing less than 0.1 mM or no addition of taurine. In accordance with one embodiment, the protein of interest is capable of being formed at an average titer of 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th , 15th, 16th or 17th day, which is at least

- 4 041694 by 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 29% more than the average titer in the 6th, 7th, 8th, 9th th, 10th, 11th, 12th, 13th, 14th, 15th, 16th or 17th day, formed by a similar cell in a serum-free cell culture medium that contains less than 0, 1 mm taurine or does not provide for the addition of such.

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

Brief description of the drawing

The figure shows the titer (yield) of the protein from the samples extracted each day from the producer culture in the cell culture forming Ab3, where the addition of taurine is provided (solid squares connected by solid lines) compared with the situation where the addition of taurine is not provided (x, connected dotted lines). The benefits of culture with the addition of taurine for protein yield can be observed already on the 6th day of cultivation with the formation of the product.

Detailed disclosure of the present invention

It is to be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is only intended to describe specific embodiments, and is not limiting as the scope of the present invention is defined by the claims.

As used in the present description and the accompanying form of the invention, the singular forms of the noun include the plural forms, unless the context clearly specifies otherwise. Thus, for example, reference to a method includes one or more of the methods and/or steps described herein and/or which will become apparent to those skilled in the art upon reading this disclosure.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention belongs. While any methods and materials similar or equivalent to those described herein may be used in the practice of the present invention, preferred methods and materials are described below. All publications referred to in this document are incorporated herein by reference in their entirety.

The present inventors have made the surprising discovery that the addition of taurine to cell culture medium increases protein production by the recombinant cell in cell culture compared to cell culture medium that contains very little or no taurine.

Before describing the cell cultures and methods of the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is only intended to describe specific embodiments and is not limiting.

The section headings used herein are for organizational purposes only and are not intended to limit the subject matter being described. The methods and techniques described herein are generally carried out in accordance with standard methods known in the art and described in various general and more specific sources, which are mentioned and described throughout this description, unless otherwise indicated. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), Julio E. Celis, Cell Biology: A Laboratory Handbook, 2nd ed., Academic Press, New York, N.Y. (1998), and Dieffenbach and Dveksler, PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1995). All publications referred to throughout this disclosure are incorporated herein by reference in their entirety.

Definitions

Taurine is also known as 2-aminoethanesulfonic acid (IUPAC nomenclature; CAS registration number 107-35-7). The terms taurine and L-taurine are used interchangeably to refer to the same organic compound. Taurine is an organic acid containing an amino group, but is not considered an amino acid in the traditional sense of those skilled in the art when the amino acids contain both an amino group and a carboxyl group. Taurine biosynthesis occurs when hypotaurine, which is a derivative of cysteine, is converted to taurine through oxidation.

The terms additive, addition, with addition, etc. refers to the addition of an element, component, molecule, etc., that can be used in a cell culture medium to maintain and/or promote cell growth and/or differentiation, to enhance or enhance a property of the culture as a whole, or to compensate for a deficiency. From this point of view, the addition of taurine involves the addition of taurine at a certain concentration in a solution to the culture medium.

The terms peptide, polypeptide and protein are used interchangeably throughout the specification and refer to a molecule containing two or more amino acid residues connected to each other by a peptide bond. Peptides, polypeptides, and proteins may also include modifications such as glycosylation, lipid addition, sulfation, gamma-carboxylation of glutamic acid residues, alkylation, hydroxylation, and ADP-ribosylation. Peptides, polypeptides, and proteins may be of scientific or commercial interest, including protein-based drugs. Peptides, polypeptides, and proteins include, but are not limited to, antibodies and chimeric or fusion proteins. Peptides, polypeptides, and proteins are generated from recombinant animal cell lines using cell culture techniques.

The term heterologous polynucleotide sequence as used herein refers to nucleic acid polymers encoding proteins of interest, such as chimeric proteins (similar to decoy molecules), antibodies, or antibody portions (e.g., VH, VL, CDR3) that are formed as biopharmaceutical drug substance. A heterologous polynucleotide sequence can be obtained using genetic engineering techniques (eg, such as a chimeric protein coding sequence or a codon-optimized sequence, an intron-deprived sequence, etc.) and introduced into a cell where it can be positioned as an episome or integrated into the cell's genome. The heterologous polynucleotide sequence may be a naturally occurring sequence that is incorporated into an ectopic region in the genome of the producing cell. The heterologous polynucleotide sequence may be a naturally occurring sequence from another organism, such as a sequence encoding a human orthologue.

The term antibody refers to an immunoglobulin molecule composed of four polypeptide chains, two heavy (H) chains, and two light (L) chains linked together by disulfide bonds. Each heavy chain has a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region 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 single domain (CL). The VH and VL regions can be further divided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, ordered from amino to carboxy 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 produced, expressed, generated, 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 distinct epitope. Bispecific antibodies are generally described in US Patent Application Publication No. 2010/0331527, which is incorporated by reference herein.

The term antigen-binding site of an antibody (or antibody fragment) refers to one or more antibody fragments that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed by the term antibody antigen-binding site include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of the VL and VH domains of one antibody arm, (v) dAb fragment (Ward et al. (1989) Nature 241:544-546) which consists of the VH domain, (vi ) an isolated CDR, (vii) scFv, which consists of two domains of the Fv fragment, VL and VH, connected by a synthetic linker to form a single protein chain, in which the VL and VH regions are paired to form monovalent molecules. Other forms of single chain antibodies, such as diabodies, are also covered by the term antibody (see, for example, Holliger et al. (1993) PNAS USA 90:6444-6448; Poljak et al. (1994) Structure 2: 1121-1123).

Moreover, an antibody or antigen binding site thereof may be part of a larger immunoadhesive molecule formed by covalent or non-covalent association of an antibody or antibody site with one or more other proteins or peptides. Examples of such immunoadhesive molecules include the use of a streptavidin core to form a tetrameric scFv molecule (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93101) and the use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to form bivalent and biotinylated scFvs. -molecules (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody regions such as Fab and F(αb')2 fragments can be prepared from whole antibodies using standard techniques such as papain or pepsin digestion of whole antibodies. Moreover, antibodies, antibody regions and immunoadhesive molecules can be produced using standard recombinant DNA techniques well known in the art (see Sambrook et al., 1989).

The term human antibody includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation), for example, in CDRs, and in particular , CDR3. However, the term human antibody as used herein does not include antibodies in which CDR sequences derived from the germline of other mammalian species, such as mice, have been grafted onto human framework sequences.

The term human recombinant antibody as used herein includes all antibodies that are produced, expressed, generated or isolated by recombinant means, such as antibodies expressed by a recombinant expression vector transfected into a host cell, antibodies isolated from a combinatorial library of recombinant human antibodies, an antibody isolated from an animal (e.g. mouse) that is transgenic for human immunoglobulin genes (see e.g. Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies obtained, expressed, formed or isolated by any other method, including the connection of human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such human recombinant antibodies are subjected to in vitro mutagenesis (or, when using an animal transgenic for human Ig sequences, somatic mutagenesis in vivo), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences , which, while derived from or assigned to human germline VH and VL sequences, may not occur naturally in the human germline antibody repertoire in vivo.

Fc fusion proteins contain some or all of two or more proteins, one of which is the Fc region of an immunoglobulin molecule, that do not otherwise occur together in nature. The production of fusion proteins containing heterologous polypeptides fused to various regions of antibody-derived polypeptides (including the Fc domain) has been described, for example, by Ashkenazi et al., Proc. Natl. Acad. ScL USA 88: 10535, 1991; Byrn et al., Nature 344:677, 1990; and Hollenbaugh et al., Construction of Immunoglobulin Fusion Proteins, in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992. Receptor Fc fusion proteins contain one or more receptor domains associated with an Fc fragment, which, in some embodiments, contains a hinge region followed by CH2- and CH3- immunoglobulin domain. In some embodiments, the Fc fusion protein contains two or more separate receptor chains that bind to one or more ligands. For example, the Fc fusion protein is a decoy protein, such as, for example, an IL-1 decoy protein (e.g., rilonacept, which contains the RAcP ligand-binding region of IL-1 fused to the extracellular R1 region of IL-1 fused to Fc hIgG1; see US Pat. No. 6,927,004), or a VEGF decoy protein (e.g., aflibercept, which contains domain 2 of Ig Flt1 of the VEGF receptor fused to domain 3 of Ig Flk1 of the VEGF receptor fused to Fc hIgG1; see US Patent Nos. No. 7087411 and 7279159).

Cell culture

The terms cell culture medium and culture medium refer to a nutrient solution used to grow mammalian cells, which usually provides the necessary nutrients to enhance cell growth, such as a carbohydrate source of energy, essential (for example, phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine and histidine) and non-essential (e.g. alanine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, proline, serine and tyrosine) amino acids, trace elements, energy sources, lipids, vitamins, etc. Environment for cell culture may contain extracts, such as whey or peptones (hydrolysates), which provide starting materials that support cell growth. The media may contain yeast extracts or soy extracts instead of animal extracts. The chemically defined medium refers to

- 7 041694 to a cell culture medium in which all of the chemical components are known (ie have a known chemical structure). The chemically determined medium does not contain any components of animal origin such as serum or animal origin peptones. In accordance with one embodiment, the environment is a chemically defined environment.

The solution may also contain components that enhance growth and/or survival beyond a minimum amount, including hormones and growth factors. The solution is preferably formulated according to the pH and salt concentration that are optimal for cell survival and proliferation.

The term cell line refers to a cell or cells that are derived from a particular line by serial passage or passage of cells. The term cell is used interchangeably with the term cell population.

The term cell includes any cell that is suitable for the expression of a recombinant nucleic acid sequence. The cells include eukaryotic cells such as non-human animal cells, mammalian cells, human cells, avian cells, insect cells, yeast cells, or cell fusions such as, for example, hybridomas or quadromas. In certain embodiments, the cell is a human, monkey, great ape, hamster, rat, or mouse cell. According to other embodiments, the cell is selected from the following cells: CHO cells (e.g. CHO-Ki, DXB-11 CHO, Veggie-CHO), COS e.g. COS-7), retinal cells, Vero, CVI, kidney cells (e.g. HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL60, lymphocyte, e.g. Jurkat (T-lymphocyte) or Daudi (B-lymphocyte), A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2/0, NS-0, MMT cells, stem cell, tumor cell and a cell line derived from the above cell. In some embodiments, the cell contains one or two viral genes, such as a retinal cell that expresses a viral gene (eg, a PER.C6® cell). In some embodiments, the cell is a CHO cell. According to other embodiments, the cell is a CHO-Ki cell.

One aspect of the present invention relates to a seed culture in which a cell population is expanded prior to protein production and harvest of the producer culture. Taurine can be added to the base medium in a seed culture formulation according to the present invention as described above.

Another aspect of the present invention relates to a producer culture in which the protein is produced and harvested. The product formation phase is usually preceded by a growth phase (also known as seed fermenter system culture or seed culture) where all cell culture components are fed into the culture vessel at the start of the culture process, then the cell population is expanded to be ready for industrial volume. Accordingly, the culture vessel is seeded with cells at a suitable seeding density for the initial phase of cell growth, depending on the original cell line. In some aspects, taurine can be added to the base culture medium in a seed culture formulation according to the present invention, as described herein, to further improve or increase cell productivity in a subsequent product formation phase.

One aspect of the present invention relates to a producer culture, where culture conditions are modified to enhance the growth of recombinant eukaryotic cells, while increasing the production of one or more recombinant proteins of interest by such cells and maintaining cell viability, in particular by adding taurine to the producer culture medium and /or culture for seed fermenter system. In the product culture vessel or bioreactor, the initial culture medium or cells are fed into the culture vessel after the seed culture phase or the growth phase. According to certain embodiments, the cell supernatant or cell lysate is collected after culture to form a product. According to other embodiments, the polypeptide or protein of interest is recovered from the culture medium or cell lysate, or otherwise depending on the location of the protein of interest, using techniques well known in the art.

Culture vessels include, but are not limited to, well plates, T-flasks, shake flasks, stirrer flasks, spinner flasks, air-lift hollow fiber bioreactors, and the like. A suitable cell culture vessel is a bioreactor. A bioreactor refers to a culture vessel that is manufactured or designed to manipulate or control environmental conditions. Such culture vessels are widely known in the art.

Bioreactor processes and systems have been designed to optimize gas exchange, deliver sufficient oxygen to support cell growth and productivity, and remove _CO2 .

- 8 041694

Maintaining the efficiency of gas exchange is an important criterion for ensuring efficient expansion of cell culture volume and protein production. Such systems are well known to the person skilled in the art.

In the polypeptide product formation phase, the term fed-batch cell culture or fed-batch cell culture refers to batch culture where animal cells and culture medium are initially fed into the culture vessel and additional culture nutrients are fed slowly, continuously or stepwise, to the culture during culture time, with or without intermittent collection of cells and/or product, until completion of culture. Fed-batch culture includes semi-continuous fed-batch culture where periodically the entire culture (which may include cells and media) is removed and replaced with fresh media. Fed-batch culture differs from simple batch culture, where all cell culture components (including animal cells and all culture nutrients) are supplied to the culture vessel at the start of the batch culture process. Fed-batch culture can further differ from perfusion culture in that the supernatant is not removed from the culture vessel during the process, while in perfusion culture the cells are retained in the culture, for example by filtration, and the culture medium is continuously or intermittently injected and removed from the culture vessel. At the same time, sample removal is provided for verification purposes during fed-batch cell culture. The batch feeding process continues until it is determined that the maximum working volume and/or protein production has been reached.

The phrase continuous cell culture as used herein refers to a technique used to continuously grow cells, usually in a particular growth phase. For example, if a continuous supply of cells is required or the formation of a particular polypeptide or protein of interest is required, the cell culture may need to maintain a particular growth phase. Thus, the conditions must be constantly monitored and adjusted accordingly in order to maintain the cells in this particular phase.

environments

The present invention provides a cell culture medium that is serum-free and contains from about 0.1 to 10 mM taurine. The term serum-free is applied to a cell culture medium that does not contain animal sera, such as fetal bovine serum. Serum-free medium may contain <16 g/l of hydrolysates such as soy hydrolysate. The present invention also provides a chemically defined medium that is not only serum-free, but also free of hydrolysate. The term hydrolyzate-free is used to refer 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. The term basic medium is the initial medium (presented in the seed fermenter system and/or day 0 of cell culture) in which the cells are propagated and contains all the necessary nutrients, which include the basic mixture of amino acids. Various formulations (eg, formulations) for basic media can be made or purchased in commercially available batches. Similarly, basic fed-batch media contains mixtures of additional nutrients that are typically consumed during product culture and used in fed-batch strategies (for so-called fed-batch culture). Many basic fed media are commercially available. The term fed includes scheduled additions or additions to the medium at regular intervals, e.g. according to a protocol, including a fed-batch culture system as in a chemostat (see C. Altamirano et al., Biotechnol Prog. 2001 Nov-Dec ; 17(6):1032-41), or according to the fed-batch method (Y.M. Huang et al., Biotechnol Prog. 2010 Sep-Oct;26(5):1400-10). For example, the culture can be fed once a day, every other day, every three days, or can be fed when the concentration of a particular medium component to be controlled falls outside the desired range.

The removal of serum or the reduction or removal of hydrolysates from the cell culture medium, while reducing batch-to-batch variability and improving post-processing steps, unfortunately reduces cell growth, cell survival, and protein expression. Thus, a chemically defined serum-free and low or no hydrolysate medium needs additional components to improve cell growth and protein formation.

In this regard, the cell culture medium of the present invention contains a basic medium containing all the necessary nutrients for culturing viable cells. Taurine can be added to the base medium in a seed culture formulation according to the present invention as described above. In addition, taurine can be added to the main medium in

- 9 041694 composition based on the producer culture, which can then be periodically fed (as in so-called batch cultures) with additional components or without the use of such, such as polyamines, or increased concentrations of components, for example, amino acids, salts, sugars, vitamins , hormones, growth factors, buffers, antibiotics, lipids, trace elements, and the like, depending on the needs of the cells to be cultured or the desired cell culture parameters.

The present invention contemplates that taurine-supplemented cell culture medium can be depleted during protein-forming culture if additional amino acid supplementation is not provided, or taurine-supplemented culture medium can be inexhaustible if amino acid supplementation is provided to replace consumed amino acids (as described below). ). The present inventors have observed that cultures that are supplemented with taurine during the product formation phase are characterized by an increase in recombinant protein production under various culture conditions as described above.

The present invention provides a taurine supplemented medium that contains 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.

According to one embodiment, the medium further contains 100±15 µM ornithine, or 300±45 µM ornithine, or 600±90 µM ornithine, or even 900±135 µM ornithine. In accordance with another embodiment, the medium contains at least about 5±1 mg/l ornithine-HCl, or at least about 10±2 mg/l ornithine-HCl, or at least 15±2.25 mg/l ornithine -HCl, or at least about 50±7.5 mg/l ornithine-HCl, or at least about 100±15 mg/l ornithine-HCl, or at least about 150±22.5 mg/l ornithine- HCl.

Putrescine can optionally be added to the complementary medium. Putrescine has been included at very low concentrations as a component in some cell culture medium formulations; see for example WO 2005/028626 which describes the inclusion of 0.02-0.08 mg/l putrescine; US patent No. 5426699 (0.08 mg/l); US Patent No. RE30985 (0.16 mg/l); US patent No. 5811299 (0.27 mg/l); US patent No. 5122469 (0.5635 mg/l); US patent No. 5063157 (1 mg/l); WO 2008/154014 (~100 μM - ~1000 μM); US Patent Application No. 2007/0212770 (0.5-30mg/l polyamine; 2mg/l putrescine; 2mg/l putrescine+2mg/l ornithine; 2mg/l putrescine+10mg/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 μM. In some embodiments, the medium is further supplemented with putrescine at a concentration of from about 170 to 230 μM. According to one embodiment, the medium contains 200±30 µM putrescine in addition to >90±15 µM ornithine. According to one embodiment, the medium contains <30±4.5 mg/l putrescine-2HCl in addition to <15±2.25 mg/l ornithine. According to another embodiment, the medium contains >30±4.5 mg/l putrescine-2HCl in addition to >15±2.25 mg/l ornithine-HCl (see International Publication No. WO 2014/144198 A1, published September 18 2014, which is incorporated herein by reference in its entirety).

In still 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 accordance with some embodiments, the medium also contains at least 0.20±0.03 mM putrescine. In some embodiments, the additional putrescine is present at a concentration ranging from 0.20±0.03 mM to 0.714±0.11 mM, such as 0.20±0.03 mM, 0.35±0.06, or 0.714 ±0.11 mM putrescine.

Various other additives may be added to the culture medium and determination of further suitable conditions is within the skill of the art. 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 that it is inexhaustible, or when additional nutrients are needed.

According to one embodiment, the medium is further supplemented with about 170 to 175 μM nucleosides. In accordance with one embodiment, the medium contains purine derivatives in a total 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 accordance with one embodiment, the environment contains from about 100 to 110 μm of purine derivatives. Purine derivatives include gi

- 10 041694 poxanthine and nucleosides adenosine and guanosine. In accordance with one embodiment, the medium contains pyrimidine derivatives in a total concentration of at least about 30 μM, at least 35 μM, 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. According to one embodiment, the medium contains from about 65 to 75 μM pyrimidine derivatives. Pyrimidine derivatives include the nucleosides thymine, uridine, and cytidine. In accordance with one specific embodiment, the medium contains adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

In addition to including any of the above additives, in accordance with one embodiment, the medium is further supplemented with micromolar amounts of fatty acids (or fatty acid derivatives) and tocopherol. According to one embodiment, the fatty acids include any one or more 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. According to one embodiment, the medium contains tocopherol, linoleic acid, and thioctic acid.

In one embodiment, the medium may also be further supplemented with a vitamin mixture that includes other nutrients and essential nutrients at a total concentration of at least about 700 μM to at least about 2 mM. In accordance with 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, thiamine HCl, vitamin B12, etc. P. In one embodiment, the vitamin blend includes all of D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, pyridoxine HCl, D-pantothenic acid (hemiCa), riboflavin, thiamine HCl, and vitamin B12.

Various embodiments of the medium of the present invention include any of the combinations of the above embodiments, including chemically defined, hydrolyzate-free serum-free medium containing taurine in the indicated amounts, together with, 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, all of the small amounts of hydrolysates may be added to the taurine-supplemented medium.

The present inventors contemplate that any one or more of a variety of basic media or combinations thereof to which taurine is added may be used in the practice of the present invention. Basic media are well known in the art and include but are not limited to MEME (Minimum Culture Medium) Needle (Eagle, Science, 1955, 112(3168):501-504), F-12 Ham (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. Natl. Acad. Sci. USA., 1952 August; 38 (8):747-752), Ham's DMEM/F12 medium 1:1, Trowell's T8 medium, Holmes and Wolf's A2 medium (Biophys. Biochem. Cytol., 1961, 10:389-401), Weymouth's medium (Davidson and Waymouth, Biochem. J., 1945, 39(2): 188-199), William's E medium (William's et al., Exp. Cell Res., 1971, 69: 105 et seq.), RPMI1640 medium (Moore et al., J. Amer. Med. Assoc, 1967, 199:519-524), MCDB medium 104/110 (Bettger et al., Proc. Nat'l. Acad. Sci. USA, 1981, 78(9): 5588-5592), HL-1 Ventrex medium, albumin and globulin medium (Orr et al., Appl. Microbiol., 1973, 25(l):49-54), RPMI-1640 medium, RPMI-1641 medium, Dulbecco modified by Iscove Wednesday 5 A McCoy , L-15 Leibovitz medium, and serum-free media such as EXCELL™ 300 Series (JRH Biosciences, Lenexa, KS), Protamine Zinc Insulin Medium (Weiss et al., 1974, US 4072565), Biotin Folate Medium ( Cartaya, 1978, US Re30985), transferrin and fatty acid medium (Baker, 1982, US 4,560,655), transferrin and EGF medium (Hasegawa, 1982, US 4,615,977; Chessebeuf, 1984, US 4786599) and other media combinations (see Inlow, US 6048728; Drapeau, US 7294484; Mather, US 5122469; Furukawa, US 5976833; Chen, US 6180401; Chen, US 5856179; Etcheverry, US 5705364; Etcheverry, US 5705364 , US 7666416; Ryll, US 6528286; Singh, US 6924124; Luan, US 7429491, etc.).

In accordance with a certain embodiment, the medium is chemically defined and contains, in addition to taurine, mixtures of amino acids as defined herein, CaCl22H2O; buffer HEPES, KCl; MgSO ₄ ; NaCl; Na ₂ HPO ₄ or other phosphate salts; pyruvate; D-biotin; choline chloride; folic acid; myo-inositol; niacinamide; pyridoxine HCl; D-pantothenic acid; riboflavin; thiamine HCl; vitamin B12; p-aminobenzoic acid; ethanolamine HCl; poloxamer 188; DL-a-tocopherol phosphate; linoleic acid; Na ₂ SeO ₃ ; thioctic acid and glucose; and optionally adenosine; guanosine; cytidine; uridine; thymidine; and hypoxanthine 2Na.

In accordance with one embodiment, the initial osmolarity of the medium of the present invention is 200-500, 250-400, 275-350, or approximately 200 mOsm. During cell growth

- 11 041694 in the medium according to the present invention, in particular, after any feeding in accordance with the protocol of periodic feeding, the osmolarity of the culture can increase to about 350, 400, 450,

500 or up to approximately 550 mOsm.

In some embodiments, where the osmolarity of the medium is less than about 300, the osmolarity is adjusted to about 300 by adding one or more salts in excess of a detectable amount. According to one embodiment, the osmolarity is increased to the desired level by adding one or more osmolytes selected from sodium chloride, potassium chloride, magnesium salt, calcium salt, amino acid salt, fatty acid salt, sodium bicarbonate, sodium carbonate, potassium carbonate, chelator , which is a salt, sugar (eg, galactose, glucose, sucrose, fructose, fucose, etc.) and combinations thereof. According to one embodiment, the osmolyte is added in addition to its concentration in the component that is already present in the medium being determined (eg, sugar is added in addition to its concentration determined for the sugar component).

Without exception, all of the media embodiments described above, as well as any other serum-free media containing at least about 0.1 mM taurine, are referred to as taurine-supplemented media. In contrast, a medium containing no taurine or a medium containing less than 0.1 mM taurine is hereinafter referred to as a medium without taurine supplementation or without taurine supplementation.

Fed-Bed Cultivation

Feeding strategies for cell culture aim to ensure optimal growth and multiplication of cells outside of a multicellular organism or tissue. Suitable conditions for culturing 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 cultured in suspension or attached to a solid substrate. Fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or mechanically agitated bioreactors, with or without microcarriers, and controlled in batch, fed-batch, continuous, semi-continuous, or perfusion mode, are available for mammalian cell culture. Cell culture medium or concentrated fed-batch medium can be added to the culture continuously or at intervals during culture. For example, the culture can be fed once a day, every other day, every three days, or can be fed when the concentration of a particular medium component to be controlled falls outside the desired range.

In addition to the inclusion of taurine, in accordance with one embodiment, the environment can be further supplemented with amino acids in a total (total) concentration of at least 20 mm. In accordance with one embodiment, the concentration of the original amino acids included in the original cell culture medium is not provided in such a total (total) concentration of supplemented amino acids. In accordance with one embodiment of the cell culture medium, or in accordance with a cell culture method or a method for producing a protein of interest, the medium may be supplemented in an amount of more than about 20 mM, more than about 25 mM, more than about 30 mM, more than about 40 mM, more than about 50 mM, more than about 60 mM, more than about 70 mM, more than about 100 mM, more than about 200 mM, more than about 300 mM, more than about 400 mM, or more than about 500 mM, see also Table 1 herein. In accordance with one embodiment, the amount of amino acids added to the medium is approximately 30±10 mM or more.

Feed addition regimens can be optimized by those skilled in the art to support cell growth, minimize cellular stress, or provide a non-depleting environment during the product formation phase.

The term inexhaustible medium includes a cell culture medium that has been defined to have nutrients, in particular amino acids, necessary for the formation of the recombinant protein of interest. Amino acid feeds typically supplement the amino acids required as building blocks for the formation of the recombinant protein in cell culture. At the same time, some amino acids may be consumed faster than others, depending on the needs of a particular protein produced by cells in culture. In a non-depleting environment, the feeding schedule has been defined in such a way that essential amino acids are replenished as they are consumed. Thus, levels of consumption and subsequent optimal consumption (pg/cell-day) can be determined using the following steps: culturing the eukaryotic (eukaryotic) cell(s) expressing(expressing) the protein of interest in the cell culture medium; measuring the concentration of each amino acid in the culture medium at time points to determine the consumption rate; identifying a flow point at which the amino acid concentration falls below the flow rate; calculation of consumption levels for each amino acid; and determining the optimal consumption level as the consumption level at the time point just before the time point of consumption. The cell culture is then supplemented with an appropriate concentration of the specific amino acid to maintain such optimal intake levels, as determined, so that the culture medium is inexhaustible.

The present invention is intended to provide a taurine-supplemented cell culture medium that increases titer in depleted as well as non-depleted cultures.

The present invention provides a cell culture containing a cell line expressing a protein of interest in a medium supplemented with taurine as described above. Examples of cell lines that are widely used to produce biotherapeutics include, but are not limited to, primary cells, 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 cells -1, MRC-5 cells, WI-38 cells, 3T3 cells, 293 cells, Per.C6 cells and chick embryo cells. In one embodiment, the cell line is a CHO cell line or one or more of several specific CHO cell variants optimized for high volume protein production, such as CHO-Ki.

According to one embodiment, the taurine-supplemented cell culture contains insulin, which can be added as a target component to the medium, or can be formulated based on the medium. In accordance with one embodiment, the cell line contains cells capable of forming a biotherapeutic protein.

According to one embodiment, the medium is supplemented at intervals during cell culture in accordance with a fed-batch process. Fed-batch culture is well known in the art and is used to optimize protein production (see Y.M. Huang et al., Biotechnol Prog. 2010 Sep-Oct; 26(5): 1400-10).

The cell growth phase or cell culture (eg, first cell culture) where medium exchange is not provided is usually followed by a different second culture, known as the polypeptide formation phase. Fed-batch processes are typically used during the product formation phase.

The present invention provides a cell culture medium containing from about 0.1 mM to about 10 mM taurine at the start of cell culture to form a product (day 0). Alternatively, cell culture medium containing 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, day 7, day 8, day 9 and/or day 10 of cell culture to form a product. The cell culture medium added to the producer culture over several days contains a total amount of taurine from about 0.1 mM to about 10 mM. Cell culture medium containing a total amount of taurine from about 0.1 mM to about 10 mM can be added in any sequential manner.

Taurine can also be added to the base medium during the growth phase of the seed fermenter system.

Supplemental feeding may be performed to include additional nutrients such as vitamins, amino acids, and other nutrients described herein above at intervals of every day or every 2-3 days during product culture. Additional feeding can be performed (additional media containing nutrients are added) at least 2 times or at least 8 times during the entire cultivation to form a product over a 2-week or longer cultivation period. According to another embodiment, additional feeding is performed every day during cultivation. Alternative crop feeding schedules are also provided.

It is also possible to perform additional addition of amino acids to obtain a non-depleting environment, where consumed amino acids are determined in accordance with methods known in the art and described herein. When using this mode, additional amino acids are supplemented or added at intervals, preferably at a frequency of every day or every 2-3 days during cultivation with the formation of the product, depending on the determination of the consumption of amino acids. In accordance with one embodiment, a mixture of additional amino acids to maintain an inexhaustible cell culture medium is added on or about day 1, on or about day 2, on or about day 3, on or about 4- day, on or about day 5, on or about day 6, on or about day 7, on or about day 8, on or about day 9, on or on or about day 10, on or about day 11, on or about day 12, on or about day 13, and on or about day 14, for 2 weeks or more long-term cultivation. Alternative crop feeding schedules are also provided.

Animal cells, such as CHO cells, can be cultured in small volume cultures such as 125 ml containers having approximately 25 ml of medium, 250 ml containers having approximately 50 to 100 ml of medium, 500 ml containers having from approximately 100 to 200 ml of medium. Alternatively, the cultures may be of high volume, such as, for example, 1000 ml containers having from about 300 to 1000 ml of medium, 3000 ml containers having from about 500 to 3000 ml of medium, 8000 ml containers having from about 2000 to 8000 ml of medium, and 15000 ml containers having from about 4000 to 15000 ml of medium. Cultures for production may contain from 10,000 liters of media and more. High volume cell cultures, for example for the clinical production of protein-based therapeutics, are typically maintained for days or even weeks while the cells produce the desired protein(s). At this time, the culture can be supplemented with a concentrated fed-batch medium containing components such as nutrients and amino acids that are consumed during culture. The fed-batch medium may be based on a formulation based on any cell culture medium. Such a concentrated fed-batch medium may contain most of the components of the cell culture medium, for example, approximately 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 fed-batch media are commonly used in fed-batch culture methods.

In some embodiments, the culture medium containing taurine is further supplemented with targeted supplements, also known as additives, target components, or target chemicals, during cell growth or protein formation. Target additives include one or more of growth factors or other proteins, a buffer, an energy source, a salt, an amino acid, a metal, and a chelator. Other proteins include transferrin and albumin. Growth factors, which include cytokines and chemokines, are well known in the art and are known to stimulate cell growth or, in some cases, cell differentiation. The growth factor is typically a protein (eg, insulin), a small peptide, or a steroid hormone such as estrogen, DHEA, testosterone, and the like. In some cases, the growth factor may be a non-naturally occurring chemical compound that promotes cell proliferation or protein formation, such as tetrahydrofolate (THF), methotrexate, and the like. Non-limiting examples of protein and peptide growth factors include angiopoietins, bone morphogenetic proteins (BMP), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), glial cell neurotrophic factor. lines (GDNF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), growth and differentiation factor 9 (GDF9), hepatocyte growth factor (HGF), hepatoma growth factor (HDGF), insulin, insulin-like growth factor (IGF), migration-stimulating factor, myostatin (GDF-8), nerve growth factor (NGF) and other neurotrophins, platelet growth factor (PDGF), thrombopoietin (TRO), transforming growth factor alpha (TGF-α) , transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), Wnt signaling pathway agonists, placental growth factor (PIGF), fetal bovine catfish atotropin (FBS), interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, and the like. In accordance with one embodiment, the cell culture medium can be supplemented with targeted additional growth factor insulin. In accordance with one embodiment, the concentration of insulin in the medium, for example, the amount of insulin in the cell culture medium after addition, may be from about 0.1 to 10 μM.

Buffers are well known in the art. The present invention is not limited to any particular buffer or buffers, and one skilled in the art can select an appropriate buffer or buffer system for use with a particular cell line producing a particular protein. In accordance with one embodiment, the target additional buffer is NaHCO ₃ . According to another embodiment, the buffer is HEPES. According to other embodiments, the target additional buffer contains both NaHCO3 and HEPES.

Energy sources for use as a targeted supplement in cell culture are also well known in the art. Without limitation, according to one embodiment, the target additional energy source is glucose. Given the specific and specific requirements of a particular cell line and resulting protein, in accordance with one embodiment, glucose can be added at a concentration of from about 1 to 20 mm in the environment. In some cases, glucose can be added at high levels of 20 g/l or more.

Similarly, chelators are well known in the field of cell culture and protein production. Tetrasodium dihydrate and citrate EDTA are two common chelators used in the art, while other chelators can be used in the practice of the present invention. According to one embodiment, the target additional chelator is tetrasodium dihydrate EDTA. According to one embodiment, the target additional chelator is citrate, such as Na ₃ C ₆ H ₅ O7.

According to one embodiment, the cell culture medium can be further supplemented with one or more targeted additional amino acids as an energy source, such as glutamine. In accordance with one embodiment, the cell culture medium is supplemented with the desired additional glutamine at a final concentration of from about 1 to 13 mM.

Other targeted additives include one or more of various metal salts such as iron, nickel, zinc and copper salts. According to one embodiment, the cell culture medium is supplemented with any one or more of copper sulfate, zinc sulfate, ferric chloride, and nickel sulfate.

In some embodiments, the protein titer resulting from culturing cells in a medium supplemented with taurine 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 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 29% more than the titer (yield) of the protein from cells cultured in a medium without the addition of taurine. In some embodiments, the protein titer generated from cells in taurine-supplemented medium is at least 2%, at least 3%, at least 4%, or at least 5% greater than the titer (yield) of protein from similar or the same cells cultured in a medium without the addition of taurine.

According to some embodiments, ammonia accumulation in 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 by 10%, more than 15% or more than 20% in the medium with the addition of taurine compared with cell culture in the medium without the addition of taurine.

Protein formation

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

In accordance with some embodiments, the titer (yield) of the protein formed by mammalian cells cultured in a medium containing taurine (medium with the addition of 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 more than the protein titer formed by the same mammalian cell cultured in a medium without the addition of taurine.

In accordance with some embodiments, the yield or titer of protein production, which can be expressed in grams of protein product per liter of culture medium, from cells cultured in medium supplemented with taurine is at least 100 mg/l, at least 1 g/l 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 l or at least 20 g/l.

In some embodiments, the protein titer generated from cells in 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%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28% or at least 29% more than the titer (yield) of protein from similar or the same cells cultured in a medium without the addition of taurine.

- 15 041694

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 fragment of an antibody, a single chain antibody, a diabody, triabody, or tetrabody, a Fab fragment, or F(ab')2 fragment, IgD antibody, IgE antibody, IgM antibody, IgG antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody, or IgG4 antibody. In accordance with one embodiment, the antibody is an IgG1 antibody. According to one embodiment, the antibody is an IgG2 antibody. In accordance with one embodiment, the antibody is an IgG4 antibody. In accordance with one embodiment, the antibody is an IgG2/IgG4 chimeric antibody. In accordance with one embodiment, the antibody is a chimeric IgG2/IgG1 antibody. In accordance with one embodiment, the antibody is an IgG2/IgG1/IgG4 chimeric antibody.

In some embodiments, the antibody is selected from the group consisting of an antibody to programmed cell death protein 1 (e.g., an antibody to PD1 as described in U.S. Patent Application Publication No. US 2015/0203579A1), an antibody to a programmed cell death protein ligand 1 (e.g. anti-PD-L1 antibodies as described in U.S. Patent Application Publication No. US 2015/0203580A1), anti-Dll4 antibodies, anti-angiopoietin 2 antibodies (e.g. anti-ANG2 antibodies as described in U.S. Patent No. 9402898), antibodies to angiopoietin-like protein 3 (for example, antibodies to AngPtl3, as described in US patent No. 9018356), antibodies to platelet-derived growth factor receptor (for example, antibodies to PDGFR, as described in US patent No. 9265827), antibodies to Erb3, antibodies to the receptor prolactin (e.g., anti-PRLR antibodies as described in US Pat. No. 9,302,015), antibodies to complement 5 (e.g., anti-C5 antibodies as described in US Pat. No. US 2015/0313194 A1), antibodies to TNF, antibodies to epidermal growth factor (e.g. anti-EGFR antibodies as described in US Pat. No. 9,132,192 or anti-EGFRvIII antibodies as described in US Pat. No. US 2015/0259423 A1), antibodies to subtilisin/kexin proprotein convertase (e.g. as described in US Pat. ), anti-glucagon receptor antibodies (e.g., anti-GCGR antibodies as described in US Pat. No. US 2015/0337045 Al or US 2016/0075778 A1), anti-VEGF antibodies, to IL4R as described in US Pat. No. US 2014/0271681 Al or US Pat. to IL1, antibodies to IL2, antibodies to IL3, antibodies to IL4, antibodies to IL5, antibodies to IL6, antibodies to IL7, antibodies to interleukin 33 (e.g., antibodies to IL33 as described in US Pat. anti-RSV antibodies as described in US Pat. No. 62/222605), anti-cluster of differentiation 20 antibodies (e.g. anti-CD20 as described in US Pat. Nos. US 2014/0088295 A1 and US 20150266966 A1 and US Pat. , antibodies to cluster of differentiation 48 (e.g., anti-CD48 antibodies as described in US Pat. No. 9,228,014), antibodies to Fel d1 (e.g., as described in US Pat. No. 9,079,948), antibodies to Middle East respiratory syndrome virus (e.g., antibodies to MERS , as described in US Patent No. US2015/0337029A1), antibodies to Ebola virus (for example, as described in U.S. Patent No. US2016/0215040), antibodies to Zika virus, antibodies to the lymphocyte activation gene 3 (for example, antibodies to LAG3, or antibodies to CD223), antibodies to nerve growth factor (for example, antibodies to NGF, as described in 2016/0017029 and US Pat. 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/CD20 bispecific antibody (as described in US Pat. US 2014/0088295 Al and US 20150266966 A1), an anti-CD3/mucin 16 bispecific antibody (for example, an anti-CD3/Muc16 bispecific antibody) and an anti-CD3/prostate specific membrane antigen bispecific antibody (for example, an anti-CD3/PSMA bispecific antibody). In some embodiments, the protein of interest is selected from the group consisting of alirocumab, sarilumab, fasinumab, nesvacumab, dupilumab, alarmumab, evinacumab, and rinucumab. All publications referred to throughout this disclosure are incorporated herein by reference in their entirety.

In some embodiments, the protein of interest is a recombinant protein that contains an Fc fragment and another domain (eg, an Fc fusion protein). In some embodiments, an Fc fusion protein is a receptor Fc fusion protein that contains one or more Fc fragment-linked receptor domains. In some embodiments, the Fc fragment contains a hinge region followed by an IgG CH2 and CH3 domain. According to some options

In an embodiment, the Fc receptor fusion protein comprises two or more distinct receptor chains that bind to either a single ligand or multiple ligands. For example, the Fc fusion protein is a TRAP protein, such as, for example, an IL-1 trap protein (eg, rilonacept, which contains the RAcP ligand-binding region of IL-1 fused to the extracellular R1 region of IL-1 fused to an hIgG1 Fc; see U.S. Patent No. 6,927,004, which is incorporated herein by reference in its entirety), or a VEGF decoy protein (e.g., aflibercept, which contains domain 2 of the Ig Flt1 VEGF receptor fused to domain 3 of the Ig Flk1 VEGF receptor fused with Fc hIgG1, see US Pat. Nos. 7,087,411 and 7,279,159). In other embodiments, the Fc fusion protein is a ScFv-Fc fusion protein that contains one or more of antigen-binding domains, such as a heavy chain variable fragment and a light chain variable fragment of an Fc-bound antibody.

The present invention is not limited to any particular cell type for protein production. Examples of cell types suitable 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 cells transfected with a virus to produce virus products. The cells may contain a recombinant heterologous polynucleotide construct that encodes a protein of interest. Such a construct may be an episome, and it may be an element that is physically integrated into the cell's genome. Cells can also form a protein of interest, which protein is not encoded on the heterologous polypeptide construct. In other words, in nature, a cell may encode a protein of interest, such as a B cell that produces an antibody. The cells may also be primary cells, such as chick embryo cells, or primary cell lines. Examples of suitable 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, chick 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 a CHO cell, such as the CHO-K1, CHO DUX B-11, CHO DG-44, Veggie-CHO, GS-CHO, S-CHO, or CHO 1ec mutant lines.

According to one embodiment, the cell, which is a CHO cell, expresses the protein ectopically. In accordance with one embodiment, the protein comprises an immunoglobulin heavy chain region, such as a CH1-, CH2-, or CH3 region. In accordance with one embodiment, the protein comprises the CH2 and CH3 region of a human or rodent immunoglobulin. In accordance with one embodiment, the protein comprises the CH1, CH2, and CH3 region of a human or rodent immunoglobulin. According to one embodiment, the protein comprises a hinge region and a CH1-, CH2- and CH3-region. In accordance with one embodiment, the protein comprises an immunoglobulin heavy chain variable domain. In accordance with one embodiment, the protein comprises an immunoglobulin light chain variable domain. In accordance with 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 human/rodent (eg, human/mouse, human/rat, or human/hamster) chimeric antibody.

The product formation phase can be carried out in any culture volume, from mixing flasks or bags for the Wave bioreactor to 1 liter bioreactors and large volume industrial bioreactors. Similarly, the growth phase of a seed fermenter system can be performed in any culture volume, from mixing flasks or bags for the Wave bioreactor to one liter or larger bioreactors. The high volume process can be carried out in a volume of from about 100 to 20,000 liters or more. One or more means may be used to control protein formation, such as temperature shift or chemical induction. The growth phase may take place at a higher temperature than the product formation phase. For example, the growth phase may occur at a first temperature of about 35 to 38°C and the product formation phase may occur at a second temperature of about 29 to 37°C, optionally from about 30 to 36°C, or from about 30 up to 34°С. In addition, chemical inducers of protein formation such as caffeine, butyrate, tamoxifen, estrogen, tetracycline, doxycycline, and hexamethylene bisacetamide (HMBA) may be added in addition to, before or after the temperature shift. If the inductors are added after the temperature shift, then they can be added one hour to five days after the temperature shift, for example, one to two days after the temperature shift. Culture of cells to form a product can be carried out as a continuous fed-batch culture system, as in a chemostat (see C. Altamirano et al., 2001, supra), or according to a fed-batch method (Huang, 2010, supra).

The present invention is useful for increasing protein production as a result of

- 17 041694 for cell culture. The cell lines used in the present invention can be genetically engineered to express a polypeptide of commercial or scientific interest. Genetic engineering of a cell line involves transfecting, transforming, or transducing cells with a recombinant polynucleotide molecule, or otherwise altering (e.g., by homologous recombination and gene activation, or fusion of a recombinant cell with a non-recombinant cell) so as 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, for example, various techniques are given in Current Protocols in Molecular Biology. Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); Kaufman, R. J., 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, Virginia) and from commercial suppliers. Examples of cell lines widely used in the industry include VERO, BHK, HeLa, CVI (including Cos), MDCK, 293, 3T3 cell lines, myeloma cell lines (e.g., NSO, NSI), PC12, WI38 and Chinese hamster ovary (CHO) cells. CHO cells are widely used to produce complex recombinant 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 Endocrinol 6:231-239 Wood et al (1990) J Immunol 145:3011-3016). Reductase deficient (DHFR) mutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77:4216-4220), DXBI 1 and DG-44 are desirable CHO host cell lines because DHFR is effectively selectable. and the amplification of the gene expression system in these cells ensures the expression of the recombinant protein at a high level (Kaufman RJ. (1990), Meth Enzymol 185:537-566). In addition, these cells are easy to work with as adherent or suspension cultures and are characterized by relatively high genetic stability. CHO cells and the proteins expressed recombinantly by them have been described in detail and have been approved for use in clinical research and commercial production by regulatory authorities. In some embodiments, 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 U.S. Patent Nos. 7455988 B2, 7435553 B2 and 7105348 B2.

The present invention is not limited in scope to the specific embodiments described herein, which are provided as illustrations of particular aspects or embodiments of the present invention. Functionally equivalent methods and components are within the scope of the present invention. Various modifications of the present 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 present invention.

Examples

Example 1.

Elevated antibody titers as a result of the addition of taurine.

Example 1A.

High throughput shake flask cultivation.

250 ml shake flasks were seeded with a culture for seeding a cell line producing a monoclonal antibody (Ab1) derived from CHO-Ki. The introduced cells were grown at 35.5°C for seventeen days and fed with glucose and other additional nutrients as needed. Cells were grown in chemically defined (hydrolyzate-free and serum-free) basic medium.

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

Table 2. Mean antibody titers over 17 days (g/l) and approximate increase in titers (%) from baseline

Flask Medium Additive Title AI 1a Without addition* 7.3 g/l 1b Taurine 7.9 g/l ^l 8%

*Baseline control for % titer increase; flask 1b vs. titer in unsupplemented medium (flask 1a) ^l The difference in final titer between the unsupplemented and supplemented cultures is statistically significant (p<0.05)

Titer values were calculated from protein harvested on day 17 and were statistically significant (p<0.05) compared to baseline. Taurine-supplemented cultures had an overall 8% increase in final protein titer compared to no-supplemented cultures.

Example 1B.

Desktop bioreactors.

In a similar example, but when producing a larger volume, 2 L bioreactors were seeded from a culture to seed a cell line producing monoclonal antibodies (Ab2, Ab3 or Ab4) derived from Cho-Κι. The inoculated cultures were grown at 35.5° C., 40.4% DO set point, and 22 cc bubbling air volume. see within 14 days. The processes for Ab2 and Ab3 had pH targets of 7.0±0.15 while the process for Ab4 had pH targets of 7.13±0.27. Glucose, antifoam, and base feed were fed to the bioreactors as needed. Cultures were grown in medium without addition (bioreactor 2a, 3a, 4a) or grown in medium supplemented with approximately 1 mM taurine (Ab2 and Ab3) or in medium supplemented with approximately 3 mM taurine (Ab4) added on day 0 of product formation. (bioreactors 2b, 3b and 4b respectively).

The output (titer) of antibodies was 6.4 g/l in the case of cells forming Ab2, while cells grown with taurine, gave 8 g/l of protein. The 24% increase in titer compared to cells grown without the addition of taurine was statistically significant (p<0.05). The final titers generated for Ab3-forming cultures and Ab4-forming cultures were also significantly higher (p<0.05) at 14 days (11% and 20%, respectively) compared to cultures without taurine addition. See table. 3.

Table 3. Mean antibody titers at 14 days (g/l) and approximate increase in titers (%) from baseline

Medium Additive Bioreactor No. Tiger AL2 Bioreactor No. Tiger ALZ Bioreactor No. Titer AL4 Without addition* 2a 6.4 g/l Behind 6.6 g/l 4a 4.4 g/l Taurine 2b 8r^ 24% 3b 7.3 g^ ^11% 4b 5.3 g^ 20%

*Non-supplemented medium is a baseline control for % titer increase, where % titer increase in bioreactors 2b, 3b, or 4b% is compared to the titer in unsupplemented medium (bioreactors 2a, 3a, or 4a, respectively) ^L Differences in final titer between cultures with and without addition are statistically significant (p<0.05)

The trend with respect to protein titer was plotted for the Ab3 producing culture and a significant increase in protein titer as a result of the addition of taurine was observed on each day of culture from day 6 onwards.

Table 4. Increase in antibody titer (g/l) as a result of the addition of taurine at characteristic time points

Biorector Medium Additive Titer of AL3 on the 6th day Titer of AL3 on the 9th day Titer of AL3 on the 14th day Behind Without addition* 1.7 g/l 4.1 g/l 6.6 g/l 3b Taurine 1.9 g/l 12% 5.3 g/l ^L 29% 7.3 g/l ^L eleven%

*Approximate increase (%) compared to unsupplemented medium harvested on the same day.

A significant increase in titer could be observed in the producer culture already on the 6th day (12% increase compared to the same culture without the addition of taurine). See also drawing. The maximum difference in such dynamics was observed on the 9th day (significant (p<0.05) increase by 29% in bioreactor 3b compared to 3a), and a significant (p<0.05) increase in titer by 11% was observed in last day (14th) of cultivation.

The benefits of protein formation with the addition of taurine were observed with different volumes (example 1A and 1B) and different cell lines (example 1B).

Example 2

Consistent productivity with varying taurine concentrations in high throughput shake flask culture.

The stability of the protein titer was studied by changing the amount of taurine added to the culture on day 0 of product formation. 250 ml shake flasks were seeded with a culture for seeding a cell line producing a monoclonal antibody (Ab1) derived from CHO-K1. The introduced cells were grown at 35.5° C. for fourteen days and fed with glucose and other additional nutrients as needed. Cells were grown in chemically defined (hydrolyzate-free and serum-free) basic medium.

- 19 041694

Each culture did not contain taurine (no addition) or contained taurine in concentrations

0.1 mM, 0.3 mM, 0.5 mM, 0.7 mM, 1 mM, 3 mM, 5 mM, 7.5 mM or 10 mM.

Table 5. Average antibody titers at 14 days (g/l) for cultures supplemented with 0.1 to 10 mM taurine

shake flask Medium Additive Title AM 5a Without addition* 6.5 g/l 5b 0.1 mM taurine# 6.7 g/l 3% 5s 0.3 mM taurine ^L 6.8 g/l 5% 5d 0.5 mM taurine ^l 6.9 g/l 6% 5e 0.7 mM taurine ^l 7.0 g/l 8% 5f 1 mM taurine ^l 7.0 g/l 8% 5g 5 mM taurine ^l 7.1 g/l 9% 5h 7.5 mM taurine ^l 7.1 g/l 9% 5i 10 mM taurine ^l 7.1 g/l 9%

*Non-added medium represents baseline control for % titer increase #Difference in final titer is statistically significant compared to no-supplement control (p<0.1) ^L Difference in final titer is statistically significant compared to no-supplement control (p <0.05)

It was shown that changing the amount of taurine supplementation consistently resulted in high titers when taurine was added in the range of at least 0.1 to 10 mM. The end titers in the case of conditions with the addition of taurine were significantly different from the conditions without addition. When 0.1 mM taurine was added, p<0.1 was noted, while when 0.3 to 10 mM taurine was added, p<0.05 was noted.

Example 3

Investigation of the change in taurine feeding schedules during high-throughput cultivation in a shake flask.

Example 3A.

Addition of taurine during the expansion phase of the seed fermenter system.

The benefits of adding taurine to the culture during the growth phase of the seed fermenter system were determined in a high throughput shake flask culture model. In flask 6a (Table 6), Ab1-forming CHO cells were thawed in chemically determined (hydrolyzate-free and serum-free) basic medium supplemented with 1 mM taurine. The taurine concentration of the main medium was maintained at 1 mM throughout the growth phase. During product formation, the basic culture medium was supplemented with 1 mM taurine on day 0. Glucose and basal nutrient replenishment were fed as needed during the 17 day product formation.

Flask 6b cells were grown in taurine-free (no addition) chemically defined (hydrolysate-free and serum-free) basic medium throughout the growth phase of the seed fermenter system. On day 0 of product formation, the basic culture medium was supplemented with 1 mM taurine. During 17 days of product formation, glucose and basic nutrient supplements were fed as needed.

Table 6. Average antibody titers over 17 days for cultures supplemented with 1 mM taurine during various phases of the process

shake flask Addition of 1 mM taurine Title AM 6a Growing phases of the seed fermenter system and product formation 7.7 g/l 6b Product formation phase only 7.8 g/l

Differences in the final titer (day 17) are not statistically significant (p>0.1).

End-of-day (day 17) titers for both conditions (taurine addition at product formation alone or together in the seed fermenter system and at product formation) are similar. The resulting titers are not statistically significant (p>0.1). The benefit of adding taurine in the growth phase of the seed fermenter system is similar to adding taurine in the product formation phase.

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Example 3B.

Changing taurine feeding schedules during the product formation phase.

To determine if changing the standard taurine feeding schedules had any effect on protein titer in the case of taurine supplemented cultures, additional experiments were performed in similar cultures in shake flasks in which Ab1 producing CHO cells were grown. Cells were subjected to different culture conditions similar to Example 2 where the feeding schedule was similar before adding a basic glucose/nutrient feed as needed.

Ab1-forming cultures were supplemented with a total of 5 mM taurine. Similar productivity (7.1 g/l, 6.8 g/l and 7.0 g/l) was observed when changing the taurine feeding schedules. The titer values compared in this experiment did not differ statistically (p>0.1) (see Table 7).

Table 7. Mean antibody titers at 14 days (g/l) for cultures supplemented with 5 mM taurine when changing schedules

Taurine supplement schedule AI titer on the 14th day 5 mM on day 0 7.1 g/l 1 mM on days 0, 3, 5, 7, 10 (total 5 mM) 6.8 g/l 1 mM on day 0; 2 mM on days 7, 10 (total 5 mM) 7.0 g/l

Differences between values on day 14 are not statistically significant (p>0.1).

The taurine feeding schedules neither had any negative effect nor changed the result when taurine supplementation was preferred for product yield. Thus, the addition of taurine can be performed once on day 0, or performed on subsequent days of the product phase, or can be performed at several intervals during the product phase.

Example 4

Measurement of ammonia by-product in high throughput shake flask culture.

The content of ammonia plug product was measured after 14 days of cultivation, carried out analogously to example 2, for cultures with the addition of taurine CHO cells forming Ab1. Cells were subjected to various culture conditions, similar to those described above, where a basic glucose/nutrient feed was added as needed.

Table 8. Average content and reduction (%) of ammonia over 17 days (mM)

Medium Additive Ammonia in Ab 1 Without addition* 2.56 nM Taurine 1.73 mm ^L -32%

* Baseline control for % ammonia reduction; condition with the addition of taurine compared with the content of ammonia in the medium without the addition of ^L The decrease in ammonia concentration is statistically significant (p<0.1)

In cells forming Ab1, the addition of taurine in the medium maintained normal long-term cultivation, while the content of the ammonia product was reduced by 32%. The decrease in ammonia concentration as a result of the addition of taurine is statistically significant (p<0.1).

The present invention can be carried out in other specific embodiments.

Claims (41)

CLAIM 1. A method for cultivating recombinant mammalian cells expressing an antigen-binding protein, comprising the steps: (a) propagating or maintaining the cells in a chemically defined cell culture medium during the growth phase; and (b) adding a chemically defined cell culture medium containing 0.1 to 10 mM L-taurine during the product formation phase, whereby adding L-taurine increases the titer of antigen-binding protein by at least 3% compared to cells expressing antigen-binding protein in cell culture medium containing less than 0.1 mM L-taurine. 2. Process according to claim 1, wherein the addition of taurine from (b) is provided 1 to 5 times during the product formation phase. 3. The method of claim 1, wherein the addition of taurine from (b) is provided every day during the product formation phase. 4. The method of claim 1, further comprising adding 0.1 to 10 mM L-taurine to the culture in a chemically defined cell culture medium during the growth phase. 5. The method of claim 1, wherein the chemically determined cell culture medium is serum-free and contains no hydrolysate. 6. The method of claim 1, wherein ornithine and putrescine are added to the chemically defined cell culture medium in step (b). 7. The method of claim 5, wherein the recombinant mammalian cell is selected from the group consisting of CHO, COS, retinal, Vero, CV1, kidney, HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL60, lymphocyte, epidermal, CV-1, U937, 3T3, L cells, C127 cells, SP2/0, NS-0, MMT cells, PER.C6® cells, stem cell, tumor cell and a cell line derived from the aforementioned cell. 8. The method of claim 7 wherein the recombinant mammalian cell is a CHO cell. 9. The method according to claim 7 or 8, wherein the CHO cell is a CHO-Ki, CHO DXB-11, CHO DG-44, Veggie-CHO, GS-CHO, S-CHO, or CHO lec mutant cell. 10. The method of claim 7, wherein the COS cell is a COS 7 cell, the kidney cell is a HEK293, 293 EBNA, MSR 293, MDCK, HaK, or BHK21 cell, the lymphocyte is a Jurkat or Daudi cell, or the epidermal cell is an A431 cell. 11. The method of claim 1 wherein the antigen binding protein comprises an Fc domain. 12. The method of claim 1, wherein the antigen binding protein is selected from the group consisting of an Fc fusion protein, a receptor Fc fusion protein (TRAP), an antibody, an antibody fragment, and a ScFv-Fc fusion protein. 13. The method of claim 1 wherein the antigen binding protein is selected from the group consisting of anti-PD1 antibody, anti-PDL-1 antibody, anti-Dll4 antibody, anti-ANG2 antibody, anti-AngPtl3 antibody, anti-PDGFR antibody, anti-Erb3 antibody, anti-PRLR antibody , TNF antibodies, EGFR antibodies, PCSK9 antibodies, GDF8 antibodies, GCGR antibodies, VEGF antibodies, IL1R antibodies, IL4R antibodies, IL6R antibodies, IL1 antibodies, IL2 antibodies, IL3 antibodies, antibodies anti-IL4, anti-IL5, anti-IL6, anti-IL7, anti-RSV, anti-NGF, anti-CD3, anti-CD20, anti-CD19, anti-CD28, anti-CD48, bispecific anti-CD3/CD20, an anti-CD3/MUC16 bispecific antibody; and an anti-CD3/PSMA bispecific antibody. 14. The method of claim 1 wherein the antigen binding protein is selected from the group consisting of alirocumab, sarilumab, fasinumab, nesvacumab, dupilumab, alarmumab, evinacumab and rinucumab. 15. The method of claim 12 wherein the TRAP protein is aflibercept. 16. The method of claim 1, wherein the recombinant mammalian cell comprises a stably integrated nucleic acid encoding an antigen-binding protein. 17. A method for producing a recombinant antigen-binding protein, comprising the steps: (a) introducing into a mammalian cell or cells a nucleic acid containing a nucleotide sequence encoding an antigen-binding protein; (b) selecting the cell(s) that express(es) the antigen-binding protein; (c) culturing the selected cell(s) in a cell culture medium containing 0.1 to 10 mM L-taurine; and (d) obtaining an antigen-binding protein in a cell where the antigen-binding protein is secreted into the medium, wherein the addition of L-taurine increases the titer of the antigen-binding protein by at least 3% compared to cells expressing the antigen-binding protein in a cell culture medium that contains less than 0 .1 mM L-taurine. 18. The method of claim 17, wherein the cell(s) of step (c) are capable of highly productive production of the antigen-binding protein. 19. The method of claim 17, wherein the cell is a CHO cell, a HEK293 cell, or a BHK cell. 20. The method of claim 19 wherein the CHO cell is a CHO-Ki, CHO DXB-11, CHO DG-44, Veggie-CHO, GS-CHO, S-CHO, or CHO 1ec mutant cell. 21. The method of claim 17 wherein the antigen binding protein comprises an Fc domain. 22. The method of claim 17, wherein the antigen binding protein is selected from the group consisting of an Fc fusion protein, an Fc receptor fusion protein (TRAP), an antibody, and an antibody fragment. 23. The method of claim 17 wherein the antigen binding protein is selected from the group consisting of anti-PD1 antibody, anti-PDL-1 antibody, anti-Dll4 antibody, anti-ANG2 antibody, anti-AngPtl3 antibody, anti-PDGFR antibody, anti-Erb3 antibody, anti-PRLR antibody , TNF antibodies, EGFR antibodies, PCSK9 antibodies, GDF8 antibodies, GCGR antibodies, VEGF antibodies, IL1R antibodies, IL4R antibodies, IL6R antibodies, IL1 antibodies, IL2 antibodies, IL3 antibodies, antibodies anti-IL4, anti-IL5, anti-IL6, anti-IL7, anti-RSV, anti-NGF, anti-CD3, anti-CD20, anti-CD19, anti-CD28, anti-CD48, bispecific anti-CD3/CD20, an anti-CD3/MUC16 bispecific antibody; and an anti-CD3/PSMA bispecific antibody. - 22 041694 24. The method of claim 17, wherein the antigen binding protein is selected from the group consisting of alirocumab, sarilumab, fasinumab, nesvacumab, dupilumab, alarmumab, evinacumab, and rinucumab. 25. The method of claim 22 wherein the TRAP protein is aflibercept. 26. The method of claim 17 wherein the mammalian cell comprises a stably integrated nucleic acid encoding an antigen binding protein. 27. A method for producing an antigen-binding protein in a cell culture medium with the addition of taurine, comprising the steps: (a) introducing into a mammalian cell a nucleic acid containing a sequence encoding an antigen-binding protein; (b) selecting a cell or cells expressing the antigen-binding protein; (c) culturing the selected cell(s) in a cell culture medium; (d) adding 0.1 to 10 mM taurine to the cell culture medium; (e) maintaining the cell culture under the conditions of addition of taurine from (d) for at least 6 days, wherein the addition of L-taurine increases the titer of the antigen-binding protein by at least 3% compared to cells expressing the antigen-binding protein in the cell culture medium , which contains less than 0.1 mm L-taurine, where the antigen-binding protein is secreted into the environment; and (f) harvesting the antigen binding protein. 28. The method of claim 27, wherein the assembled antigen binding protein is characterized by a protein titer that increases by at least 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or at least 20% after at least 6 days compared to cells expressing the antigen binding protein after at least 6 days in cell culture medium that contains less than 0.1 mM L-taurine. 29. The method of claim 27, wherein the selected cell(s) contains a stably integrated nucleic acid encoding an antigen-binding protein. 30. The method of claim 27, wherein the antigen binding protein is selected from the group consisting of an Fc fusion protein, an Fc receptor fusion protein (TRAP), an antibody, and an antibody fragment. 31. The method of claim 27 wherein the antigen binding protein is selected from the group consisting of anti-PD1 antibody, anti-PDL-1 antibody, anti-Dll4 antibody, anti-ANG2 antibody, anti-AngPtl3 antibody, anti-PDGFR antibody, anti-Erb3 antibody, anti-PRLR antibody , TNF antibodies, EGFR antibodies, PCSK9 antibodies, GDF8 antibodies, GCGR antibodies, VEGF antibodies, IL1R antibodies, IL4R antibodies, IL6R antibodies, IL1 antibodies, IL2 antibodies, IL3 antibodies, antibodies anti-IL4, anti-IL5, anti-IL6, anti-IL7, anti-RSV, anti-NGF, anti-CD3, anti-CD20, anti-CD19, anti-CD28, anti-CD48, bispecific anti-CD3/CD20, an anti-CD3/MUC16 bispecific antibody; and an anti-CD3/PSMA bispecific antibody. 32. The method of claim 27 wherein the antigen binding protein is selected from the group consisting of alirocumab, sarilumab, fasinumab, nesvacumab, dupilumab, alarmumab, evinacumab and rinucumab. 33. The method of claim 30 wherein the TRAP protein is aflibercept. 34. The method according to claim 27, where the method can increase the protein yield by at least 0.1 g/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 by at least 2.2 g/l, at least 2.4 g/l, or at least 2.5 g/l compared to the same method of preparation in a cell culture medium that contains less than 0.1 mM taurine . 35. The method of claim 1, wherein the addition of L-taurine reduces the level of ammonia by-product by at least 4% compared to cells expressing the antigen binding protein in a cell culture medium that contains less than 0.1 mM L-taurine. 36. The method of claim 1 wherein the titer of the antigen-binding protein cultured in a medium containing 0.1 to 10 mM L-taurine supplement is at least 1.8 g/l. 37. The method of claim 17, wherein the addition of L-taurine reduces the level of ammonia by-product by at least 4% compared to cells expressing the antigen binding protein in cell culture medium that contains less than 0.1 mM L-taurine. 38. The method of claim 17 wherein the titer of the antigen-binding protein cultured in a medium containing 0.1 to 10 mM L-taurine supplement is at least 1.8 g/L. 39. The method of claim 27, wherein the addition of L-taurine reduces the level of ammonia by-product by at least 4% compared to cells expressing the antigen-binding protein in a cell culture medium that contains less than 0.1 mM L-taurine. 40. The method of claim 27, wherein the titer of the antigen-binding protein cultured in a medium containing 0.1 to 10 mM L-taurine supplement is at least 1.8 g/l. 41. The method of claim 27, wherein the mammalian cell comprises a stably integrated nucleic acid encoding an antigen binding protein.