Methods for altering the glycosylation profile of a recombinant glycoprotein produced in cell culture

Abstract

The present invention relates to a method for altering the glycosylation profile of a recombinant glycoprotein produced in cell culture, comprising culturing eukaryotic cells expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, wherein the glycosylation profile of the produced recombinant glycoprotein is altered to be more similar to the glycosylation profile of a reference glycoprotein than when the cell culture medium is not supplemented.

Description

Field of the Invention

[0001] The present invention relates to a method for altering the glycosylation profile of a recombinant glycoprotein produced in cell culture, comprising culturing eukaryotic cells expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, wherein the glycosylation profile of the produced recombinant glycoprotein is altered to be more similar to the glycosylation profile of a reference glycoprotein than when the cell culture medium is not supplemented. Background of the Invention

[0003] Glycoproteins are essential for the normal function of all branches of the immune system, including the innate and adaptive immune systems. For example, glycoproteins participate in the recognition, binding, signal transduction and elimination of threats via complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) (Rudd et al., Science, 291, 2001, 2370-2376; and Jefferis et al., Immunol Rev, 163, 1998, 59-76).

[0004] Antibodies expressed by mammals carry a single glycan on the Asn residue of their heavy chain (HC). The presence and composition of the glycan structure affect the receptor binding and effector function of the antibody (Rudd et al., Science, 291, 2001, 2370-2376; and Jefferis et al., Immunol Rev, 163, 1998, 59-76). For example, changes in the glycan composition of the Fc region (Fcγ) of IgG differentially affect the binding affinity of the Fc region and the Fc binding receptor (FcR). There are three types of FcγR, namely FcγRI, FcγRII and FcγRIII (Jefferis et al., Immunol Rev, 163, 1998, 59-76; Ravetch & Bolland, Annu Rev Immunol, 19, 2001, 275-290). The different affinities of antibodies and Fcγ receptors determine the fate of the host's immune response to a specific antigen, and may also be responsible for activation, inhibition, antibody efficacy / half-life, tolerance, and autoimmune responses (Ravetch & Bolland, Annu Rev Immunol, 19, 2001, 275-290). The affinity of antibodies for different types of receptors varies depending on the presence and composition of the polysaccharides they carry, thus highlighting the importance of oligosaccharide / protein interactions to the biological functions of antibodies (Raju et al., Glycobiology, 10, 2000, 477-486; Jefferis et al., Immunol Rev, 163, 1998, 59-76).

[0005] After expression of proteins in eukaryotic (e.g., mammalian) host cells, the proteins undergo post-translational modifications, typically including the enzymatic addition of sugar residues, generally referred to as "glycosylation." Glycosylation of polypeptides is typically either N-linked or O-linked. The attachment of the carbohydrate moiety to the side chain of an asparagine residue is referred to as N-linked glycosylation. The tripeptide sequences asparagine (Asn)-X-serine (Ser) and asparagine (Asn)-X-threonine (Thr) are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain (where X is any amino acid except proline). The attachment of one of N-acetylgalactosamine, galactose, fucose, N-acetylglucosamine, or xylose to a hydroxyamino acid (most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be involved) is referred to as O-linked glycosylation. The glycosylation patterns of proteins produced by mammals can be subdivided into several groups, including complex, high mannose and mixed structures, as well as glycoside-linked oligosaccharides (The Plasma Proteins: Structure, Function and Genetic Control, Putnam, FW ed., 2nd ed., Vol. 4, Academic Press, New York, 1984, especially pp. 271-315.).

[0006] In the past few decades, a lot of research has focused on the production of therapeutic recombinant glycoproteins, such as monoclonal antibodies. Although one approach in the literature is to use a culture medium containing serum or hydrolysate, chemically defined culture media have also been developed to eliminate the problem of batch-to-batch variability of complex components (Luo and Chen, Biotechnology and Bioengineering, 97 (6): 1654-1659 (2007)). A better understanding of cell culture has allowed a shift to chemically defined culture media without affecting growth, viability, titer, etc. Optimized chemically defined processes with titers as high as 7.5-10 g / L have been reported (Huang et al., Biotechnology Progress 26 (5): 1400-1410 (2010); Ma et al., Biotechnology Progress 25 (5): 1353-1363 (2009); Yu et al., Biotechnology and Bioengineering, 108 (5): 1078-1088 (2011)). Typically, high titer chemically defined processes are fed-batch processes with culture times of 11-18 days.

[0007] Many reports have demonstrated that the processing of N-glycans associated with recombinant proteins is specific to each mammalian cell (James et al., Bio / Technology, 13:592-596 (1995); Lifely et al., Glycobiology, 5:813-822 (1995)). These differences are important for the production of therapeutic glycoproteins, not only because they directly affect the antigenicity, in vivo clearance and stability of recombinant glycoproteins (Jenkins et al., Nature Biotechnol. 14:975-981 (1996)), but also because any given approved therapeutic glycoprotein must meet strict regulatory standards. Therefore, it is important not only to be able to characterize the glycans associated with therapeutic recombinant glycoproteins to predict the consequences of safety and efficacy in vivo, but also to be able to understand the cellular controls that support glycan processing in potential host cells (Grabenhorst et al., Glycoconjug. J., 16:81-97 (1999); James and Baker, Encyclopedia of bioprocess technology: Fermentation, biocatalysis and bioseparation. New York: John Wiley & Sons. p. 1336-1349 (1999)).

[0008] Just as the selection of eukaryotic cells for the production of therapeutic recombinant glycoproteins (such as monoclonal antibodies), glycosylation is also highly dependent on cell culture media and other production process parameters (Ho et al., BioProcess International; 14(4):30-8 (2016)). In particular, it has been shown that the composition of the growth and feed media (including the concentrations of ammonia, glutamine, glucose and metal ions) can affect antibody glycosylation (Hossler et al., Glycobiol. 19(9):936-946 (2009)). Therefore, strategies have been developed to control the glycosylation pattern and / or spectrum of recombinant glycoproteins by supplementing the growth medium. For example, WO2017079165 discloses supplementing a fucose source, such as fucose, to a culture of cells engineered to lack GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase or GDP-D-mannose-4,6-dehydratase activity to produce glycoproteins with specific fucosylation levels. Fucose supplementation (used alone (WO2017120359; WO2017120347) or in combination with nicotinamide (WO2017134667)) has been used to reduce afucosylated glycoforms, i.e., increase fucosylation of antibodies. Manganese supplementation (used alone (WO2017021871; WO2015011660; WO2013114245), in combination with copper (WO2016089919), or in combination with copper, iron and zinc (WO2015128314)) has been used to increase afucosylated glycoforms, i.e., reduce fucosylation. Manganese has also been used to control the galactosylation profile of the therapeutic monoclonal antibody Adalimumab (WO2012149197).

[0009] Therefore, there is a need in the art to identify methods that can predictably alter the glycosylation profile of a recombinant glycoprotein of interest to more closely resemble a reference recombinant glycoprotein that meets all safety, efficacy, and regulatory standards.

[0010] Brief description of the invention

[0011] The inventors have discovered that the glycosylation profile of a recombinant glycoprotein produced in cell culture can be altered by a method comprising culturing eukaryotic cells expressing the recombinant glycoprotein in a cell culture medium supplemented with fucose, manganese and taurine. The glycosylation profile is altered by the method of the invention such that the glycosylation profile of the produced recombinant glycoprotein is altered to be more similar to the glycosylation profile of a reference glycoprotein than when the cell culture is not supplemented.

[0012] The objects of the invention are solved by the subject-matter of the independent claims. Preferred embodiments are evident from the dependent claims.

[0013] Thus, in one embodiment, the invention provides a method of altering the glycosylation profile of a recombinant glycoprotein produced in cell culture, comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, wherein the glycosylation profile of the produced recombinant glycoprotein is altered to be more similar to the glycosylation profile of a reference glycoprotein than when the cell culture medium is not supplemented.

[0014] In one embodiment, the altered glycosylation profile comprises one or more of a fucosylation profile and / or a galactosylation profile. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein and / or the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0015] In one embodiment, the fucose concentration in the culture medium is increased by supplementation to 0.4 mM to 1.6 mM, preferably 0.4 mM to 1.2 mM, more preferably 0.6 mM to 1 mM and preferably 0.8 mM.

[0016] In one embodiment, the manganese concentration in the culture medium is increased by supplementation by 0.02 μM to 0.1 μM, preferably 0.04 μM to 0.08 μM, more preferably 0.06 μM to 0.08 μM, and most preferably 0.068 μM ± 0.01 μM.

[0017] In one embodiment, the final taurine concentration in the culture medium after supplementation is 12.5 mM to 50 mM, preferably 15 mM to 35 mM, more preferably 20 mM to 30 mM, and preferably 25 mM.

[0018] In one embodiment, the method further comprises the step of isolating the produced recombinant glycoprotein from the cell culture.

[0019] In one embodiment, the eukaryotic cell is a Chinese Hamster Ovary (CHO) cell.

[0020] In one embodiment, the recombinant glycoprotein is produced on a large scale.

[0021] In one embodiment, the recombinant glycoprotein is an IgG-type immunoglobulin.

[0022] In one embodiment, the recombinant glycoprotein is a monoclonal antibody, optionally a therapeutic monoclonal antibody.

[0023] In one embodiment, the cells are cultured for 14 days.

[0024] In one embodiment, the medium is supplemented every other day starting from the 3rd day of culturing, and optionally until the 13th day of culturing.

[0025] In one embodiment, manganese is supplemented with manganese chloride (MnCl 2 ), and the MnCl 2 supplementation of the medium is performed every other day starting from the 5th day of culturing, and optionally until the 9th day of culturing.

[0026] In one embodiment, supplementation of the culture medium is performed during production of the cell culture.

[0027] In one embodiment, the cell culture is performed at 37°C.

[0028] In one embodiment, the cell culture is performed at pH 7.05 ± 0.05.

[0029] In one embodiment, the cell culture is a fed-batch culture.

[0030] In one embodiment, the glycoprotein is a VEGF antagonist, preferably an anti-VEGF antibody. In this embodiment, the altered fucosylation profile is preferably at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of a reference VEGF antagonist (preferably an anti-VEGF antibody) and / or the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of a reference VEGF antagonist (preferably an anti-VEGF antibody).

[0031] In one embodiment, the glycoprotein is an anti-CD20 antibody. In this embodiment, the altered fucosylation profile is preferably at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference anti-CD20 antibody and / or the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference anti-CD20 antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : Effect of guanosine, fucose and mannose on the total major afucosylated (MAF) glycoforms of the anti-VEGF antibodies of the invention. Results are from day 12 of culture. Black bars: control batches without medium additives. Grey bars: batches fed with feed medium supplemented with guanosine at two different concentrations (20 mM and 60 mM); spotted bars: batches fed with feed medium supplemented with fucose at two different concentrations (10 mM and 20 mM); diagonal striped bars: batches fed with feed medium supplemented with mannose at two different concentrations (20 mM and 60 mM).

[0034] Figure 2 : Effect of different fucose concentrations on the total major afucosylated (MAF) glycoforms of the anti-VEGF antibodies of the invention. Results are from day 12 of culture. Black bars: control batches where the feed medium was not supplemented with fucose. Grey bars: batches fed with feed medium supplemented with 10 mM fucose; spotted bars: batches fed with feed medium supplemented with 20 mM fucose; diagonal striped bars: batches fed with feed medium supplemented with 40 mM fucose. Black line: range of total major afucosylated (MAF) glycoforms of the reference anti-VEGF antibodies of the invention.

[0035] Figure 3 : Effect of different taurine concentrations on the total major afucosylated (MAF) glycoforms of the anti-VEGF antibodies of the invention. Results are from day 14 of culture. Black bars: control batches cultured with medium not supplemented with taurine. Grey bars: batches cultured with medium supplemented with 12.5 mM taurine; spotted bars: batches cultured with medium supplemented with 25 mM taurine; diagonal striped bars: batches cultured with medium supplemented with 50 mM taurine. Black line: range of total major afucosylated (MAF) glycoforms of the reference anti-VEGF antibodies of the invention.

[0036] Figure 4 : Effect of taurine, fucose and manganese on the total major afucosylated (MAF) glycoforms of the anti-VEGF antibodies of the invention. Results are from day 14 of culture. Black bars: control batch fed with feed medium supplemented with 1.7 μM manganese chloride but not with fucose and taurine; grey bars: batch cultured with medium supplemented with 25 mM taurine and fed with feed medium supplemented with 1.7 μM manganese chloride but not with fucose-supplemented feed medium; spotted bars: batch cultured with medium supplemented with 25 mM taurine and fed with feed medium supplemented with 20 mM fucose and 1.7 μM manganese chloride. Black line: range of total major afucosylated (MAF) glycoforms of the reference anti-VEGF antibodies of the invention.

[0037] Figure 5: Total major afucosylation profile (MAF) of the anti-VEGF antibodies produced according to the invention. The results are from daily sample analysis from day 7 to day 13 of culture, or from day 7 to day 14 of culture, respectively. Black line: control batch not fed with taurine and fucose but fed with feeding medium supplemented with manganese; dash-dotted line: batch fed with feeding medium supplemented with fucose and manganese; dashed line: batch cultured with medium supplemented with taurine and fed with feeding medium supplemented with manganese; dotted line: batch cultured with medium supplemented with taurine and fed with feeding medium supplemented with manganese and fucose. Grey line: range of total major afucosylated glycoforms of the reference anti-VEGF antibody according to the invention.

[0038] Figure 6 : Non-galactosylated glycoforms (G0) profile of the anti-VEGF antibody produced according to the invention. Results are from daily sample analysis from day 7 to day 13 of culture, or from day 7 to day 14 of culture, respectively. Black line: control batch not fed with taurine and fucose but fed with feeding medium supplemented with manganese; dash-dotted line: batch fed with feeding medium supplemented with fucose and manganese; dashed line: batch cultured with medium supplemented with taurine and fed with feeding medium supplemented with manganese; dotted line: batch cultured with medium supplemented with taurine and fed with feeding medium supplemented with manganese and fucose. Grey line: range of non-galactosylated (G0) glycoforms of the reference anti-VEGF antibody according to the invention. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention as illustratively described below may suitably be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein.

[0041] The present invention will be described with respect to particular embodiments but the invention is not limited thereto but only by the claims.

[0042] Where the term "comprising" is used in the present description and claims, other elements are not excluded. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If hereinafter a group is defined as comprising at least a certain number of embodiments, this is meant to also include a group which preferably consists only of these embodiments.

[0043] Where an indefinite or definite article is used when referring to a singular noun, for example "a", "an" or "the", this includes a plural of that noun unless otherwise stated.

[0044] As used herein, the term "polypeptide" or "protein" refers to a continuous chain of amino acids linked together via peptide bonds. The term is used to refer to amino acid chains of any length, but one of ordinary skill in the art will understand that the term is not limited to long chains and can refer to a minimal chain comprising two amino acids linked together via peptide bonds. If a single polypeptide is a discrete functional unit and does require permanent physical association with other polypeptides to form a discrete functional unit, the terms "polypeptide" and "protein" as used herein are used interchangeably. If a discrete functional unit consists of more than one polypeptide physically associated with each other, the term "protein" as used herein refers to multiple polypeptides that are physically coupled and function together as a discrete unit.

[0045] The term "glycoprotein" is used herein to refer to a polypeptide or protein coupled to at least one carbohydrate moiety (e.g., a polysaccharide or oligosaccharide) attached to the protein via an oxygen-containing or nitrogen-containing side chain of an amino acid residue, such as a serine or threonine residue ("O-linked") or an asparagine residue ("N-linked"). "Glycoprotein" is used herein in the broadest sense to include full-length glycoproteins, genetically engineered glycoproteins, recombinant glycoproteins, chimeric glycoproteins, humanized glycoproteins, fully human glycoproteins, and fragments of such glycoproteins, such as peptides, as long as they remain functional and exhibit the desired biological activity. The "biological activity" of a glycoprotein refers to the ability of the glycoprotein to elicit a biological response, which can be measured in vitro or in vivo.

[0046] The term "recombinant glycoprotein" refers to all glycoproteins prepared, expressed, formed or isolated by recombinant means, such as glycoproteins isolated from transgenic host cells (such as, for example, NS0 or CHO cells), or from animals that are transgenic for the glycoprotein gene, or glycoproteins expressed using a recombinant expression vector transfected into a host cell (such as, for example, SP 2 / 0 mouse myeloma cells).

[0047] The term "glycan" refers to a polysaccharide or oligosaccharide, such as a polymer composed of monosaccharides. A glycan can be a homopolymer or heteropolymer of monosaccharide residues and can be linear or branched. After the protein is expressed in a eukaryotic (e.g., mammalian) host cell, the protein undergoes post-translational modification, typically including enzymatic addition of sugar residues, such as glycans. The addition of such sugar residues (e.g., glycans) is referred to herein as "glycosylation." "N-linked glycans" refer to glycans attached to the side chains of asparagine residues. "O-linked glycans" refer to glycans attached to hydroxyamino acids, most commonly serine or threonine, but may also involve 5-hydroxyproline or 5-hydroxylysine. Glycosylation includes, for example, galactosylation and / or fucosylation.

[0048] As used herein, the "glycosylation pattern" of a recombinant glycoprotein of interest refers to various physical properties of the polysaccharides or oligosaccharides of the glycoprotein, such as, for example, the quantity and quality of the various monosaccharides present, the degree of branching and / or linkage (e.g., N-linked or O-linked). The "glycosylation pattern" of a glycoprotein may also refer to the functional properties imparted by the oligosaccharides and polysaccharides of the glycoprotein. For example, the extent to which a glycoprotein can bind to FcγRIIIa and induce antibody-dependent cellular cytotoxicity (ADCC).

[0049] As used herein, the "glycosylation profile" or "degree of glycosylation" of a target recombinant glycoprotein refers to the amount of each monosaccharide present. The glycosylation profile may vary between different production batches of a glycoprotein of interest, for example, between a biosimilar therapeutic glycoprotein and its reference product glycoprotein. Although a certain degree of glycosylation profile change may be permitted within regulatory standards without the need to conduct new clinical trials to approve a therapeutic glycoprotein biosimilar, it is ideal to match the glycosylation profile of the reference glycoprotein as closely as possible, thereby also maintaining safety and efficacy at levels as close to the reference glycoprotein as possible. The glycosylation profile of a recombinant glycoprotein may include, for example, the fucosylation profile and / or the galactosylation profile of the recombinant glycoprotein.

[0050] The term "fucosylation" refers to the degree and distribution of fucose residues on polysaccharides and oligosaccharides (e.g., N-glycans, O-glycans, and glycolipids). The term "fucosylation profile" or "fucosylation degree" refers to the number of fucose residues on polysaccharides and oligosaccharides (e.g., N-glycans, O-glycans, and glycolipids). Therapeutic glycoproteins, such as antibodies or Fc fusion proteins, with non-fucosylated or "afucosylated" N-glycans exhibit significantly enhanced antibody-dependent cellular cytotoxicity (ADCC) due to enhanced FcγRIIIa binding ability, without any detectable changes in complement-dependent cytotoxicity (CDC) or antigen-binding ability. In certain cases, such as cancer treatment, non-fucosylated or "afucosylated" antibodies, i.e., antibodies with a low "fucosylation profile", are ideal because they can achieve therapeutic efficacy at low doses while inducing high cytotoxicity against tumor cells and triggering high effector function in natural killer (NK) cells through enhanced interaction with FcγRIIIa. In other cases, such as the treatment of inflammatory or autoimmune diseases, enhanced ADCC and binding to FcγRIIIa are undesirable, and therefore, therapeutic glycoproteins with higher levels of fucose residues in their N-glycans, i.e., higher fucosylation profiles, may be preferred. Fucosylation profiles may vary between different production batches of a glycoprotein of interest, e.g., between a biosimilar therapeutic glycoprotein and its reference product glycoprotein. Although some degree of fucosylation profile change may be permitted within regulatory standards without requiring new clinical trials for approval of a therapeutic glycoprotein biosimilar, it would be ideal to match the fucosylation profile of the reference glycoprotein as closely as possible, thereby also maintaining safety and efficacy at levels as close to the reference glycoprotein as possible.

[0051] "Galactosylation" refers to the type and distribution of galactose residues on polysaccharides and oligosaccharides (e.g., N-glycans, O-glycans, and glycolipids). The term "galactosylation profile" or "degree of galactosylation" refers to the number of galactose residues on polysaccharides and oligosaccharides (e.g., N-glycans, O-glycans, and glycolipids). "Galactose" refers to a group of monosaccharides, including open-chain and cyclic forms. An important disaccharide form of galactose is galactose-alpha-1,3-galactose (a-gal). The galactosylation profile may vary between different production batches of a glycoprotein of interest, for example, between a biosimilar therapeutic glycoprotein and its reference product glycoprotein. Although some degree of galactosylation profile variation may be permitted within regulatory standards without requiring new clinical trials for approval of a therapeutic glycoprotein biosimilar, it is ideal to match the galactosylation profile of the reference glycoprotein as closely as possible, thereby keeping safety and efficacy as close to the reference glycoprotein as possible.

[0052] The terms "altering the glycosylation profile", "altering the fucosylation profile" and "altering the galactosylation profile" refer to changes in the glycosylation profile, fucosylation profile and galactosylation profile, respectively, of a recombinant glycoprotein when produced under a first set of culture conditions (e.g., the presence of fucose, manganese and / or taurine in the cell culture medium) compared to the glycosylation profile, fucosylation profile and galactosylation profile of the same recombinant glycoprotein when produced under a second set of culture conditions (e.g., the absence of fucose, manganese and / or taurine in the cell culture medium). The glycosylation profile, fucosylation profile and / or galactosylation profile obtained under the first set of culture conditions may be increased or decreased relative to the profile obtained under the second set of culture conditions. Preferably, the glycosylation profile, fucosylation profile and / or galactosylation profile obtained by the first set of culture conditions is changed to be more similar to the respective glycosylation profile, fucosylation profile and / or galactosylation profile of the reference glycoprotein than the glycosylation profile, fucosylation profile and / or galactosylation profile obtained under the second set of culture conditions. More preferably, the glycosylation profile, fucosylation profile and / or galactosylation profile is changed to be at least 90%, still more preferably at least 95%, more preferably at least 96%, and most preferably at least 98% of the glycosylation profile, fucosylation profile and / or galactosylation profile of the reference glycoprotein, respectively.

[0053] As used herein, the terms "reference glycoprotein," "reference VEGF antagonist," "reference anti-VEGF antibody," or "reference anti-CD20 antibody" refer to the same glycoprotein, antagonist, or antibody of interest produced in a relevant cell culture, which may be expected to match or be more similar to the glycosylation profile, fucosylation profile, and / or galactosylation profile of the glycoprotein of interest. For example, the reference glycoprotein, antagonist, or antibody may have useful properties, such as stability, efficacy, safety, and / or other properties associated with a given glycoprotein, antagonist, or antibody. The reference glycoprotein, antagonist, or antibody may be, for example, a glycoprotein, antagonist, or antibody that has received regulatory approval for therapeutic use in a subject (e.g., a human subject). In this case, the glycoprotein produced in the method of altering the glycosylation profile, fucosylation profile, and / or galactosylation profile may be a biosimilar.

[0054] As used herein, the terms "culture", "cell culture" and "eukaryotic cell culture" refer to a population of eukaryotic cells maintained or grown in a cell culture medium under conditions suitable for the survival and / or growth of the cell population, either adherent or suspended. As used herein, these terms may refer to a combination of a population of mammalian cells and a cell culture medium in which the population is suspended. Cell culture may be, for example, a continuous culture, a batch culture, a fed-batch culture or other culture type.

[0055] As used herein, the terms "medium," "culture medium," "cell culture medium," "culture medium," "tissue culture medium," "tissue culture medium," and "growth medium" refer to solutions containing nutrients that support the growth of cultured eukaryotic cells. Typically, these solutions provide the essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required for minimal growth and / or survival of the cells. The solution may also contain components that enhance growth and / or survival beyond a minimum rate, including hormones and growth factors. The solution may be formulated to an optimal pH and salt concentration for cell survival and proliferation. The culture medium may also be a "defined medium" or "chemically defined medium," a serum-free medium that does not contain proteins, hydrolysates, or components of unknown composition. Defined media do not contain animal-derived components, and all components have a known chemical structure. Defined media may contain recombinant glycoproteins or proteins, such as, but not limited to, hormones, cytokines, interleukins, and other signaling molecules.

[0056] A cell culture medium is typically "serum-free" when the medium is substantially free of serum or fractions thereof from any mammalian source (e.g., substantially free of fetal bovine serum (FBS)). "Substantially free" means that the cell culture medium contains about 0-5% serum, preferably about 0-1% serum, and most preferably about 0-0.1% serum. Advantageously, a serum-free defined medium can be used, in which the identity and concentration of each component of the medium is known (i.e., undefined components, such as eukaryotic cell extracts, are not present in the medium).

[0057] Cell culture of the present invention can be carried out in any culture medium suitable for the specific cells to be cultured. In some embodiments, the culture medium contains, for example, inorganic salts, carbohydrates (e.g., sugars such as glucose, galactose, maltose or fructose), amino acids, vitamins (e.g., B vitamins (e.g., B12), vitamin A, vitamin E, riboflavin, thiamine and biotin), fatty acids and lipids (e.g., cholesterol and steroids), proteins and peptides (e.g., albumin, transferrin, fibronectin and fetuin), serum (e.g., compositions comprising albumin, growth factors and growth inhibitors such as fetal bovine serum, newborn calf serum and horse serum), trace elements (e.g., zinc, copper, selenium and tricarboxylic acid intermediates), hydrolyzates (hydrolyzed proteins derived from plant or animal sources), and combinations thereof. Commercially available culture media, such as 5x-concentrated DMEM / F12 (Invitrogen), CD OptiCHO feed (Invitrogen), CD Efficient Feed (Invitrogen), Cell Boost (HyClone), BalanCD CHO Feed (Irvine Scientific), BD Recharge (Becton Dickinson), Cellvento Feed (EMD Millipore), Ex-cell CHOZN Feed (Sigma-Aldrich), CHO Feed Bioreactor Supplement (Sigma-Aldrich), SheffCHO (Kerry), Zap-CHO (Invitria), ActiCHO (PAA / GE Healthcare), Ham's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM], Sigma) are exemplary cell culture media suitable for the methods of the present invention. In addition, any medium described in Ham and Wallace (1979) Meth. Enz., 58:44; Barnes and Sato (1980) Anal. Biochem. 102:255; US Pat. No. 4,767,704; 4,657,866; 4,927,762; 5,122,469 or 4,560,655; International Publication Nos. WO 90 / 03430 and WO 87 / 00195 can be used as a culture medium.Any of these media are supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), lipids (such as linoleic acid or other fatty acids) and suitable carriers thereof, and glucose or an equivalent energy source. In some embodiments, the culture medium is a serum-free medium, a protein-free medium, or a chemically defined medium. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art.

[0058] As used herein, the terms "basal medium preparation," "basal medium," or "basal medium" refer to any cell culture medium used to culture cells that has not been altered by the supplementation or selective removal of a particular component or components.

[0059] As used herein, the term "feed medium", "feed medium" or "feed" refers to any cell culture medium added after the start of cell culture. It is usually more concentrated than the cell culture medium used at the beginning of cell culture and is used to replenish nutrients that have been depleted or to provide supplements or additives to the cell culture.

[0060] The terms "supplementation", "supplementing" or "supplemented" refer to the addition of an additive or supplement to a cell culture medium (e.g., a basal medium or a feed medium). As used herein, the term "additive" or "supplement" refers to any supplementation of a basal medium or a feed medium for the purposes described in the present disclosure. An additive or supplement may include a single substance, such as taurine, manganese or fucose, or may include multiple substances, such as fucose and taurine, fucose and manganese, taurine and manganese, or fucose, manganese and taurine. The term "additive" or "supplement" refers to all added ingredients, even if they do not need to be added at the same time or in the same manner. For example, one or more ingredients of an additive or supplement may be added as a single pellet or two or more pellets from a stock solution, while other ingredients of the same additive or supplement may be added as part of a feed medium. The addition of supplements may also occur continuously or semi-continuously. In addition, any one or more ingredients in the additive or supplement may be present in the basal medium from the start of cell culture. Thus, supplementation may take place at the start of the culture, and / or after the start of the culture, for example during the growth phase and / or the production phase.

[0061] As used herein, the term "increase" or "increase" means that the concentration of the additive or supplement before supplementation with the supplement is increased by supplementing the supplement or additive to a higher concentration after supplementation. That is, the cell culture medium after supplementation with a defined amount has a higher concentration of the supplement or additive than before supplementation with the supplement or additive.

[0062] The term "growth phase" of a cell culture refers to an exponential cell growth phase (logarithmic phase), during which cells typically divide rapidly. The determination of the cell growth cycle of a cell culture can be determined for a particular cell envisioned without excessive experimentation. During the growth phase, cells are cultured in a cell culture medium containing essential additives, typically at about 25°C-40°C, preferably at 37°C, in a controlled atmosphere, to achieve optimal growth of a particular cell (e.g., a cell of a cell line). The cells are maintained in an exponential growth phase for a period of about one to five days, such as between two and four days, such as four days. For example, if the culture is maintained under the growth conditions of the present invention / as described above, the length of the growth phase will be a period of time sufficient to propagate a particular cell to a viable cell density in the range of about 20%-80% of the maximum possible viable cell density.

[0063] The "production phase" or "protein production phase" of a cell culture refers to the period of time when cell growth reaches a stationary phase. During the production phase, logarithmic cell growth has ended, and protein production is the primary activity of the cell culture. During this period of time, the culture medium is typically supplemented to support continued protein production and obtain the desired glycoprotein product. The production phase typically begins on day 5 of culture and continues until the end of culture, preferably until day 14 of culture.

[0064] As used herein, the term "cell viability" refers to the ability of cells in culture to survive under a given set of culture conditions or experimental changes. As used herein, the term also refers to the fraction of cells that survive at a particular time relative to the total number of live and dead cells in the culture at that time. As used herein, the term "cell density" or "viable cell density" refers to the number of viable cells present in a given volume of culture medium.

[0065] As used herein, the term "batch culture" refers to a method of culturing cells in which all components ultimately used to culture the cells, including the culture medium as well as the cells themselves, are provided at the beginning of the culture process. Batch culture is usually stopped at a certain point in time and the cells and / or components in the culture medium are collected and optionally purified.

[0066] As used herein, the term "fed-batch culture" refers to a method of culturing cells in which additional components are provided to the culture at some time after the start of the culture process. A fed-batch culture can be started using, for example, a basal medium. A culture medium that provides additional components to the culture at some time after the start of the culture process is a "feed medium" or "feed solution". Fed-batch cultures are typically stopped at a certain point in time and the cells and / or components in the culture medium are collected and optionally purified.

[0067] As used herein, the term "continuous culture" or "perfusion culture" refers to a method of culturing cells in which additional components are continuously or semi-continuously provided to the culture after the culture process begins. The components provided typically include nutritional supplements for the cells that have been depleted during the culture process. Typically, a portion of the cells and / or components in the culture medium are collected on a continuous or semi-continuous basis and optionally purified.

[0068] Cell culture is usually carried out in a bioreactor. As used herein, the term "bioreactor" refers to any container for the growth of mammalian cell cultures. Depending on the scale of culture, the bioreactor can be of any size as long as it can be used for the cultivation of mammalian cells. Typically, the bioreactor will be at least 500 milliliters and can be 1, 10, 50, 100, 250, 500, 1,000, 2,000, 2,500, 3,000, 5,000, 8,000, 10,000, 12,0000, 15,000, 20,000 liters or larger, or any volume therebetween. For example, the bioreactor will be 10 to 5,000 liters, 10 to 10,000 liters, 10 to 15,000 liters, 10 to 20,000 liters, 50 to 5,000 liters, 50 to 10,000 liters, 50 to 15,000 liters, 50 to 20,000 liters, 1,000 to 5,000 liters or 1,000 to 3,000 liters. The bioreactor can be a stirred tank bioreactor or a shake flask. The internal conditions of the bioreactor, including, for example, pH and temperature, are usually strictly controlled during the culture period. The bioreactor can be composed of any material suitable for keeping the mammalian cell culture suspended in the culture medium under the culture conditions of the present invention, including glass, plastic or metal. As used herein, the term "production bioreactor" refers to the final bioreactor used to produce the target glycoprotein or protein.

[0069] As used herein, the term "large-scale cell culture" or "large-scale production" refers to the culture of cells in a production bioreactor typically having a volume of at least 500 or 1,000 liters, preferably at least 5,000 or 8,000 liters, and most preferably 10,000 or 20,000 liters.

[0070] The terms "antibody" or "immunoglobulin" are used interchangeably herein and have the broadest meaning and include full-length antibodies, genetically engineered antibodies, recombinant antibodies, multivalent antibodies, monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, and fragments of such antibodies, so long as they retain biological function and exhibit the desired biological activity. The "biological activity" of an antibody refers to the ability of an antibody to bind to an antigen and produce a biological response, which can be measured in vitro or in vivo.

[0071] Naturally produced antibodies are molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains connected by disulfide bonds. From N- to C-terminal, each heavy chain has a variable domain (VH), also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three or four constant domains (CH1, CH2, CH3 and optional CH4). Similarly, from N- to C-terminal, each light chain has a variable domain (VL), also referred to as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. The light chain of an antibody, based on the amino acid sequence of its constant domain, can be determined as one of two types called kappa (κ) and lambda (λ).

[0072] A "full-length antibody" comprises a light chain (V L ) and heavy chain (V H ) antigen-binding variable region, light chain constant region (C L ) and heavy chain constant domain C H 1. C H 2 and C H 3.

[0073] The term "antibody fragment" or "antigen binding fragment" is used herein in the broadest sense and comprises a portion of a full-length antibody, preferably comprising its antigen binding region or variable region. Antibody fragments retain the original specificity of the parent immunoglobulin. Examples of antibody fragments include, for example, Fab, Fab', F(ab')2 and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Preferably, the antibody fragment is a Fab fragment.

[0074] A "monoclonal antibody" is an antibody specific for a single epitope of an antigen, ie, directed against a single determinant on the antigen. Methods for producing monoclonal antibodies are known to those skilled in the art.

[0075] The term "recombinant antibody" refers to all antibodies prepared, expressed, formed or isolated by recombinant means, such as antibodies isolated from transgenic host cells (such as, for example, NS0 or CHO cells), or from animals that are transgenic for immunoglobulin genes, or antibodies expressed using a recombinant expression vector transfected into a host cell (such as, for example, SP 2 / 0 mouse myeloma cells).

[0076] The antibody or immunoglobulin of the present invention is preferably an IgG molecule, such as an IgG1, IgG2, IgG3 or IgG4 molecule. More preferably, the immunoglobulin is IgG1. Even more preferably, the immunoglobulin is IgG1 wherein at least the Fc portion is human.

[0077] "Chimeric antibodies", e.g., mouse-human chimeric antibodies, are antibodies in which the Fc portion is human and the variable regions are of mouse origin.

[0078] In one embodiment of the invention, the chimeric antibody is rituximab or infliximab.

[0079] Rituximab is a chimeric anti-CD20 antibody which is described in detail in, for example, WO 94 / 11026.

[0080] Infliximab is a chimeric anti-TNFα antibody which is described in detail in, for example, WO 92 / 16553.

[0081] "Humanized antibodies" are human antibodies in which the antigen binding portion (complementarity determining region (CDR)) is derived from a non-human species (such as a mouse) and therefore has a different specificity than the parent immunoglobulin. The CDR protein sequences can be modified to increase their similarity to naturally occurring antibody variants in humans.

[0082] In one embodiment of the invention, the humanized antibody is trastuzumab or bevacizumab.

[0083] Trastuzumab is a humanized anti-HER2 antibody which is described in detail in, for example, WO 92 / 22653.

[0084] Bevacizumab is a humanized anti-VEGF antibody which is described in detail in, for example, WO 98 / 45331.

[0085] A "fully human antibody" is an antibody wherein all parts are derived from human sources.

[0086] In one embodiment of the invention, the human antibody is adalimumab or denosumab.

[0087] Adalimumab is a human anti-TNFα antibody which is described in detail in, for example, WO 97 / 29131.

[0088] Denosumab is a human anti-RANKL antibody which is described in detail in, for example, WO 03 / 002713.

[0089] In preferred embodiments, the antibody is rituximab or bevacizumab.

[0090] The term "therapeutic antibody" refers to an antibody used to treat a disease. Therapeutic antibodies can have a variety of mechanisms of action. Therapeutic antibodies can bind to and neutralize the normal function of a target associated with an antigen. For example, a monoclonal antibody that blocks the activity of a protein required for cancer cell survival can cause cell death. Another therapeutic monoclonal antibody can bind to and activate the normal function of a target associated with an antigen. For example, a monoclonal antibody can bind to a protein on a cell and trigger a cell apoptosis signal. There is also a monoclonal antibody that can bind to a target antigen that is expressed only on diseased tissues; combining a toxic payload (effective agent), such as a chemotherapeutic agent or a radiotherapeutic agent, with a monoclonal antibody can produce an agent for specifically delivering a toxic payload to diseased tissues, thereby reducing damage to healthy tissues. The "biologically functional fragment" of a therapeutic antibody will present at least one of the biological functions attributed to the complete antibody, if not part or all of which includes at least specific binding to the target antigen.

[0091] The term "osmolarity" is a measure of the osmotic pressure of dissolved solute particles in an aqueous solution. Solute particles include ions and non-ionized molecules. Osmolarity is expressed as the concentration of osmotically active particles (i.e., osmoles) dissolved in 1 kg of water (1 mOsm / kgH2O at 37°C is equivalent to an osmotic pressure of 2.5 kPA (19 mm Hg)). During cell culture and / or when additional solute particles are added (e.g., as a supplement), the osmolarity of the culture increases unless adjusted and / or stabilized. Therefore, the longer the cell culture period, the higher the osmolarity.

[0092] "Osmolarity" refers to the number of solute particles dissolved in 1 liter of solution. Thus, adding solutes, such as supplements, e.g., fucose, manganese, and / or taurine to a culture medium will increase its osmolarity. Typically, a given cell culture requires an optimal osmolarity range for the cell culture medium, and thus the osmolarity must be stably maintained within this optimal osmolarity range. As used herein, the abbreviation "mOsm" means "milliosmoles / kg H2O".

[0093] As used herein, the term "titer" refers to the total amount of recombinantly expressed glycoprotein produced by cell culture divided by a given amount of culture medium volume. The titer is usually expressed in milligrams of glycoprotein per milliliter of culture medium or in grams of glycoprotein per liter of culture medium.

[0094] As used herein, the terms "isolating," "isolate," "isolated," "purify," "purifying," and "purified" refer to the removal or separation of some or all of the cells, cell debris, cell culture medium, cell culture medium components, and other materials that constitute a recombinant glycoprotein in a cell culture in which the recombinant glycoprotein is produced. In general, it is often desirable to separate a glycoprotein produced in a cell culture. The glycoprotein can, for example, be secreted into the culture medium, and thus the cells and other solids can be removed, for example, by centrifugation or filtration, as a first step in the separation process. Alternatively, the expressed glycoprotein can be bound to the surface of cells of the cell culture. In this embodiment, the culture medium is removed and the cells expressing the glycoprotein are lysed as a first step in the purification process. Lysis of mammalian cells can be achieved by any means known to those of ordinary skill in the art, including physical disruption by glass beads and exposure to high pH conditions. Glycoproteins can be isolated and / or purified by standard methods, for example, chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), gel filtration, centrifugation, differential solubility, ethanol precipitation, or by any other technique useful for protein purification (see, e.g., Scopes, Protein Purification Principles and Practice, 2nd Edition, Springer-Verlag, New York, 1987; Higgins, SJ and Hames, BD (eds.), Protein Expression: A Practical Approach, Oxford Univ Press, 1999; and Deutscher, MP, Simon, MI, Abelson, JN (eds.), Guide to Protein Purification: Methods in Enzymology (Methods in Enzymology Series, Vol 182), Academic Press, 1997). In particular for immunoaffinity chromatography, glycoproteins can be isolated by binding to an affinity column comprising antibodies raised against the protein and immobilized on a fixed support. Alternatively, affinity tags (such as influenza coat sequences, polyhistidine or glutathione-S-transferase) can be attached to the glycoprotein by standard recombinant techniques to facilitate purification by passage through an appropriate affinity column. Protease inhibitors (such as phenylmethylsulfonyl fluoride (PMSF), leupeptin, pepstatin or aprotinin) can be added at any or all stages to reduce or eliminate degradation of the glycoprotein during isolation and / or purification.Protease inhibitors are particularly desirable when cells must be lysed to separate and purify the expressed glycoprotein. One of ordinary skill in the art will appreciate that the exact separation and / or purification technique will vary depending on the characteristics of the glycoprotein to be separated and / or purified, the characteristics of the cells expressing the glycoprotein, and the composition of the culture medium in which the cells are grown.

[0095] In cases where it is desired to adjust the glycosylation profile, for example, to reduce the amount of afucosylated glycoforms of a recombinant glycoprotein produced in a cell culture, for example, to reduce ADCC of a glycoprotein and / or to produce a glycoprotein with a fucosylation profile similar to a reference glycoprotein, the cell culture can be supplemented with fucose (see Example 2). However, the addition of fucose can also result in a reduction in the galactosylation profile of the produced glycoprotein, which in turn can result in an undesirable glycosylation and / or galactosylation profile because it is not similar to the reference glycoprotein in terms of glycosylation and / or galactosylation profile, and / or from the perspective of biological activity, safety and / or efficacy of the produced glycoprotein (see also Raju and Jordan. MAbs. May 1; 4(3): 385-391 (2012)).

[0096] As a countermeasure to the reduction of galactosylation, manganese can be further supplemented during the cell culture process. Although this additional supplementation of manganese changes the galactosylation profile of the produced glycoproteins, so that it does not cause the reduction observed with the supplementation of fucose alone, i.e. the galactosylation profile is similar / more similar to the galactosylation profile of the reference glycoprotein, it also leads to / increases the osmolarity of the culture. This increase in osmolarity, in addition to the relatively high osmolarity caused by the long cell culture (e.g. duration of 11-18 days, e.g. 13-15 days, e.g. 14 days), also increases the amount of glycoprotein produced, which in turn increases the formation of afucosylated glycoforms, i.e. it leads to a reduction in the fucosylation profile of the glycoproteins (see Example 3). This is of course the exact opposite of the effect achieved by the initial supplementation with fucose, and is contrary to previous observations in the art that higher osmolarity (regardless of the induced solute) leads to a decrease in afucosylated glycoforms, i.e. an increase in fucosylated glycoforms (Konno et al., Cytotechnology 64(3):249-65 (2012)).

[0097] In order to offset the increase in this undesirable non-fucosylated glycoform observed when the cell culture was supplemented with fucose and manganese, an attempt was made to further supplement taurine as an osmotic protectant (see Example 4). It was surprisingly found that taurine can not only be used to stabilize the molar osmotic pressure concentration of the cell culture, but also can further reduce the formation of the non-fucosylated glycoform of the glycoprotein produced, i.e., increase the fucosylation spectrum (see Example 5). Therefore, the cell culture medium supplemented with fucose, manganese and taurine can change the glycosylation spectrum of the glycoprotein produced in the cell culture, including fucosylation and / or galactosylation spectrum, so as to be more similar to the reference glycoprotein than when there is no supplementation. In contrast to the osmotic protectants commonly used in the art (such as betaine), the use of taurine also has the additional benefit of increasing production titer (WO2017024062).

[0098] Thus, in one embodiment, the invention provides a method for altering the glycosylation profile, including one or more of a fucosylation profile and / or a galactosylation profile, of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, and wherein the cell culture medium is supplemented with fucose, manganese and taurine, wherein the glycosylation profile, including one or more of a fucosylation profile and / or a galactosylation profile, of the produced recombinant glycoprotein is altered so as to be more similar to the glycosylation profile, including one or more of a fucosylation profile and / or a galactosylation profile, of a reference glycoprotein compared to when cultivated without said supplementation.

[0099] In one embodiment, the present invention provides a method for altering the fucosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, and wherein the fucosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the fucosylation profile of a reference glycoprotein than when cultivated without the supplementation. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein. Preferably, the altered fucosylation profile is at least 98% of the fucosylation profile of the reference glycoprotein.

[0100] In another embodiment, the present invention provides a method for altering the galactosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, and wherein the galactosylation profile of the recombinant glycoprotein produced is altered so that it is more similar to the galactosylation profile of a reference glycoprotein than when cultivated without the supplementation. Preferably, the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0101] In yet another preferred embodiment, the present invention provides a method for altering the fucosylation profile and galactosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, and wherein the fucosylation profile and galactosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the fucosylation profile and galactosylation profile of a reference glycoprotein than when cultivated without said supplementation. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein and the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0102] In one embodiment, the fucose concentration in the culture medium is increased by supplementation by 0.4 mM to 1.6 mM, e.g., 0.4, 0.44, 0.48, 0.52, 0.56, 0.6, 0.64, 0.68, 0.72, 0.76, 0.8, 0.84, 0.88, 0.92, 0.96, 1, 1.04, 1.08, 1.12, 1.16, 1.2, 1.24, 1.28, 1.32, 1.36, 1.4, 1.44, 1.48, 1.52, 1.56, or 1.6 mM. In a preferred embodiment, the fucose concentration in the culture medium is increased by 0.4 mM to 1.2 mM, such as 0.4, 0.44, 0.48, 0.52, 0.56, 0.6, 0.64, 0.68, 0.72, 0.76, 0.8, 0.84, 0.88, 0.92, 0.96, 1, 1.04, 1.08, 1.12, 1.16 or 1.2 mM. In a preferred embodiment, the fucose concentration in the culture medium is increased by 0.6 mM to 1 mM, such as 0.6, 0.64, 0.68, 0.72, 0.76, 0.8, 0.84, 0.88, 0.92, 0.96 or 1 mM. In a most preferred embodiment, the fucose concentration in the culture medium is increased by 0.8 mM.

[0103] Preferably, the supplementation of fucose is achieved by supplementation as part of a feed medium, wherein the fucose concentration in the feed medium is 10 mM to 40 mM, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 mM. In a preferred embodiment, the fucose concentration in the feed medium is 10 mM to 30 mM, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 mM. In a preferred embodiment, the fucose concentration in the feed medium is 15 mM to 25 mM, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 mM. In a most preferred embodiment, the fucose concentration in the feed medium is 20 mM.

[0104] In one embodiment, the present invention provides a method for altering the glycosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.4 mM to 1.6 mM, more preferably by 0.4 mM to 1.2 mM, even more preferably by 0.6 mM to 1 mM, and most preferably by 0.8 mM, and wherein the glycosylation profile of the produced recombinant glycoprotein is altered to be more similar to the glycosylation profile of a reference glycoprotein than when the cell culture medium is cultured without said supplement.

[0105] In one embodiment, the present invention provides a method for altering the fucosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.4 mM to 1.6 mM, more preferably by 0.4 mM to 1.2 mM, even more preferably by 0.6 mM to 1 mM, most preferably by 0.8 mM, and wherein the fucosylation profile of the produced recombinant glycoprotein is altered to be more similar to the fucosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein.

[0106] In another embodiment, the present invention provides a method for altering the galactosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.4 mM to 1.6 mM, more preferably by 0.4 mM to 1.2 mM, even more preferably by 0.6 mM to 1 mM, most preferably by 0.8 mM, and wherein the galactosylation profile of the produced recombinant glycoprotein is altered to be more similar to the galactosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0107] In yet another preferred embodiment, the present invention provides a method for altering the fucosylation profile and galactosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.4 mM to 1.6 mM, more preferably by 0.4 mM to 1.2 mM, even more preferably by 0.6 mM to 1 mM, and most preferably by 0.8 mM, and wherein the fucosylation profile and galactosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the fucosylation profile and galactosylation profile of a reference glycoprotein than when the cell culture medium is not supplemented. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein and the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0108] In one embodiment, the manganese concentration in the culture medium is increased by 0.02 μM to 0.1 μM, preferably by 0.04 μM to 0.08 μM, more preferably by 0.06 μM to 0.08 μM, and most preferably by 0.068 μM ± 0.01 μM.

[0109] Preferably, the manganese supplementation is achieved by supplementation as part of a feed medium, wherein the manganese concentration in the feed medium is 0.5 μM to 2.5 μM, preferably 1.0 μM to 2.0 μM, more preferably 1.5 μM to 2.0 μM and most preferably 1.7 μM ± 0.2 μM.

[0110] In one embodiment, the present invention provides a method for altering the glycosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the manganese concentration in the cell culture medium is increased by 0.02 μM to 0.1 μM, preferably by 0.04 μM to 0.08 μM, more preferably by 0.06 μM to 0.08 μM, and most preferably by 0.068 μM ± 0.01 μM, and wherein the glycosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the glycosylation profile of a reference glycoprotein than when cultured without said supplement.

[0111] In one embodiment, the present invention provides a method for altering the fucosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the manganese concentration in the cell culture medium is increased by 0.02 μM to 0.1 μM, preferably by 0.04 μM to 0.08 μM, more preferably by 0.06 μM to 0.08 μM, and most preferably by 0.068 μM ± 0.01 μM, and wherein the fucosylation profile of the produced recombinant glycoprotein is altered to be more similar to the fucosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein.

[0112] In another embodiment, the present invention provides a method for altering the galactosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the manganese concentration in the cell culture medium is increased by 0.02 μM to 0.1 μM, preferably by 0.04 μM to 0.08 μM, more preferably by 0.06 μM to 0.08 μM, and most preferably by 0.068 μM ± 0.01 μM, and wherein the galactosylation profile of the produced recombinant glycoprotein is altered to be more similar to the galactosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0113] In yet another preferred embodiment, the present invention provides a method for altering the fucosylation profile and galactosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the manganese concentration in the cell culture medium is increased by 0.02 μM to 0.1 μM, preferably by 0.04 μM to 0.08 μM, more preferably by 0.06 μM to 0.08 μM, and most preferably by 0.068 μM ± 0.01 μM, and wherein the fucosylation profile and galactosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the fucosylation profile and galactosylation profile of a reference glycoprotein than when the cell culture medium is not supplemented. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein and the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0114] In one embodiment, the present invention provides a method for altering the glycosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.4 mM to 1.6 mM, such that the manganese concentration in the cell culture medium is increased by 0.02 μM to 0.1 μM, preferably by 0.04 μM to 0.08 μM, more preferably by 0.06 μM to 0.08 μM, and most preferably by 0.068 μM ± 0.01 μM, and wherein the glycosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the glycosylation profile of a reference glycoprotein than when cultured without said supplementation.

[0115] In one embodiment, the present invention provides a method for altering the glycosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.8 mM, such that the manganese concentration in the cell culture medium is increased by 0.02 μM to 0.1 μM, preferably by 0.04 μM to 0.08 μM, more preferably by 0.06 μM to 0.08 μM, and most preferably by 0.068 μM ± 0.01 μM, and wherein the glycosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the glycosylation profile of a reference glycoprotein than when cultured without said supplementation.

[0116] In one embodiment, the present invention provides a method for altering the fucosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.4 mM to 1.6 mM, such that the manganese concentration in the cell culture medium is increased by 0.02 μM to 0.1 μM, preferably by 0.04 μM to 0.08 μM, more preferably by 0.06 μM to 0.08 μM, and most preferably by 0.068 μM ± 0.01 μM, and wherein the fucosylation profile of the produced recombinant glycoprotein is altered to be more similar to the fucosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein.

[0117] In one embodiment, the present invention provides a method for altering the fucosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.8 mM, such that the manganese concentration in the cell culture medium is increased by 0.02 μM to 0.1 μM, preferably by 0.04 μM to 0.08 μM, more preferably by 0.06 μM to 0.08 μM, and most preferably by 0.068 μM ± 0.01 μM, and wherein the fucosylation profile of the produced recombinant glycoprotein is altered to be more similar to the fucosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein.

[0118] In one embodiment, the present invention provides a method for altering the galactosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.4 to 1.6 mM, such that the manganese concentration in the cell culture medium is increased by 0.02 μM to 0.1 μM, preferably by 0.04 μM to 0.08 μM, more preferably by 0.06 μM to 0.08 μM, and most preferably by 0.068 μM ± 0.01 μM, and wherein the galactosylation profile of the produced recombinant glycoprotein is altered to be more similar to the galactosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0119] In one embodiment, the present invention provides a method for altering the galactosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.8 mM, such that the manganese concentration in the cell culture medium is increased by 0.02 μM to 0.1 μM, preferably by 0.04 μM to 0.08 μM, more preferably by 0.06 μM to 0.08 μM, and most preferably by 0.068 μM ± 0.01 μM, and wherein the galactosylation profile of the produced recombinant glycoprotein is altered to be more similar to the galactosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0120] In another preferred embodiment, the present invention provides a method for changing the fucosylation profile and galactosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.4 to 1.6 mM, the manganese concentration in the cell culture medium is increased by 0.02 μM to 0.1 μM, preferably by 0.04 μM to 0.08 μM, more preferably by 0.06 μM to 0.08 μM, and most preferably by 0.068 μM ± 0.01 μM, and wherein the fucosylation profile and galactosylation profile of the produced recombinant glycoprotein is changed so as to be more similar to the fucosylation profile and galactosylation profile of a reference glycoprotein compared to when the culture is not supplemented. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein and the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0121] In yet another preferred embodiment, the present invention provides a method for altering the fucosylation profile and galactosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.8 mM, such that the manganese concentration in the cell culture medium is increased by 0.02 μM to 0.1 μM, preferably by 0.04 μM to 0.08 μM, more preferably by 0.06 μM to 0.08 μM, and most preferably by 0.068 μM ± 0.01 μM, and wherein the fucosylation profile and galactosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the fucosylation profile and galactosylation profile of a reference glycoprotein than when the cell culture medium is not supplemented. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein and the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0122] In another preferred embodiment, the taurine concentration in the supplemented medium is between 12.5 mM and 50 mM, such as 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5 or 50 mM. In a preferred embodiment, the final taurine concentration in the supplemented medium is between 15 mM and 35 mM, such as 15, 17.5, 20, 22.5, 25, 27.5, 30, 32.5 or 35 mM. In a preferred embodiment, the final taurine concentration in the supplemented medium is between 20 mM and 30 mM, such as 20, 22.5, 25, 27.5 or 30 mM. In a most preferred embodiment, the final taurine concentration in the culture medium is 25 mM.

[0123] In one embodiment, the present invention provides a method for altering the glycosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the glycosylation profile of the produced recombinant glycoprotein is altered to be more similar to the glycosylation profile of a reference glycoprotein.

[0124] In one embodiment, the present invention provides a method for altering the fucosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, and the taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the fucosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the fucosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein.

[0125] In another embodiment, the present invention provides a method for altering the galactosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the galactosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the galactosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0126] In yet another preferred embodiment, the present invention provides a method for altering the fucosylation profile and galactosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the fucosylation profile and galactosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the fucosylation profile and galactosylation profile of a reference glycoprotein compared to when the cell culture is not supplemented. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein and the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0127] In one embodiment, the present invention provides a method for altering the glycosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.4 to 1.6 mM, and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, and most preferably 25 mM, and wherein the glycosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the glycosylation profile of a reference glycoprotein compared to when the cell culture medium is cultured without said supplementation.

[0128] In one embodiment, the present invention provides a method for altering the glycosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.8 mM, the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, and most preferably 25 mM, and wherein the glycosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the glycosylation profile of a reference glycoprotein compared to when the cell culture medium is cultured without said supplementation.

[0129] In one embodiment, the present invention provides a method for altering the fucosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.4 to 1.6 mM, the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the fucosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the fucosylation profile of a reference glycoprotein than when cultured without the supplementation. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein.

[0130] In one embodiment, the present invention provides a method for altering the fucosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.8 mM, the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the fucosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the fucosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein.

[0131] In one embodiment, the present invention provides a method for altering the galactosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.4 to 1.6 mM, the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the galactosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the galactosylation profile of a reference glycoprotein than when cultured without the supplementation. Preferably, the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0132] In another embodiment, the present invention provides a method for altering the galactosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.8 mM, the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the galactosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the galactosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0133] In yet another preferred embodiment, the present invention provides a method for altering the fucosylation profile and galactosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.4 to 1.6 mM, and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, and most preferably 25 mM, and wherein the fucosylation profile and galactosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the fucosylation profile and galactosylation profile of a reference glycoprotein compared to when the cell culture medium is not supplemented. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein and the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0134] In yet another preferred embodiment, the present invention provides a method for altering the fucosylation profile and galactosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the cell culture medium is increased by 0.8 mM, and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, and most preferably 25 mM, and wherein the fucosylation profile and galactosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the fucosylation profile and galactosylation profile of a reference glycoprotein compared to when the cell culture medium is not supplemented. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein and the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0135] In one embodiment, the present invention provides a method for altering the glycosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the manganese concentration in the cell culture medium is increased by 0.06 μM to 0.08 μM, and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, and most preferably 25 mM, and wherein the glycosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the glycosylation profile of a reference glycoprotein than when cultured without the supplementation.

[0136] In one embodiment, the present invention provides a method for altering the glycosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the manganese concentration in the cell culture medium is increased by 0.068 μM ± 0.01 μM, and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, and most preferably 25 mM, and wherein the glycosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the glycosylation profile of a reference glycoprotein than when the cell culture medium is cultured without the supplementation.

[0137] In one embodiment, the present invention provides a method for altering the fucosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the manganese concentration in the cell culture medium is increased by 0.06 μM to 0.08 μM, the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, and most preferably 25 mM, and wherein the fucosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the fucosylation profile of a reference glycoprotein than when cultured without the supplementation. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein.

[0138] In one embodiment, the present invention provides a method for altering the fucosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the manganese concentration in the cell culture medium is increased by 0.068 μM ± 0.01 μM, the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, and most preferably 25 mM, and wherein the fucosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the fucosylation profile of a reference glycoprotein than when cultured without the supplementation. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, and more preferably at least 98% of the fucosylation profile of the reference glycoprotein.

[0139] In another embodiment, the present invention provides a method for altering the galactosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the manganese concentration in the cell culture medium is increased by 0.06 μM to 0.08 μM, the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the galactosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the galactosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0140] In another embodiment, the present invention provides a method for altering the galactosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the manganese concentration in the cell culture medium is increased by 0.068 μM ± 0.01 μM, the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the galactosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the galactosylation profile of a reference glycoprotein than when cultured without the supplementation. Preferably, the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0141] In yet another preferred embodiment, the present invention provides a method for altering the fucosylation profile and galactosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the manganese concentration in the cell culture medium is increased by 0.06 μM to 0.08 μM, and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, and most preferably 25 mM, and wherein the fucosylation profile and galactosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the fucosylation profile and galactosylation profile of a reference glycoprotein compared to when the cell culture medium is not supplemented. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein and the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0142] In yet another preferred embodiment, the present invention provides a method for altering the fucosylation profile and galactosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the manganese concentration in the cell culture medium is increased by 0.068 μM ± 0.01 μM, and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, and most preferably 25 mM, and wherein the fucosylation profile and galactosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the fucosylation profile and galactosylation profile of a reference glycoprotein compared to when the cell culture medium is not supplemented. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein and the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0143] In one embodiment, the present invention provides a method for altering the glycosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the culture medium is increased by 0.4 to 1.6 mM, such that the manganese concentration in the cell culture medium is increased by 0.06 μM to 0.08 μM and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, and most preferably 25 mM, and wherein the glycosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the glycosylation profile of a reference glycoprotein than when cultured without said supplementation.

[0144] In one embodiment, the present invention provides a method for altering the glycosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the culture medium is increased by 0.8 mM, such that the manganese concentration in the cell culture medium is increased by 0.06 μM to 0.08 μM and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, and most preferably 25 mM, and wherein the glycosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the glycosylation profile of a reference glycoprotein than when cultured without said supplementation.

[0145] In one embodiment, the present invention provides a method for altering the glycosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the culture medium is increased by 0.4 to 1.6 mM, such that the manganese concentration in the cell culture medium is increased by 0.068 μM ± 0.01 μM and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, and most preferably 25 mM, and wherein the glycosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the glycosylation profile of a reference glycoprotein than when cultured without said supplementation.

[0146] In one embodiment, the present invention provides a method for altering the glycosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the culture medium is increased by 0.8 mM, the manganese concentration in the cell culture medium is increased by 0.068 μM ± 0.01 μM and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, and most preferably 25 mM, and wherein the glycosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the glycosylation profile of a reference glycoprotein than when cultured without said supplementation.

[0147] In one embodiment, the present invention provides a method for altering the fucosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the culture medium is increased by 0.4 to 1.6 mM, such that the manganese concentration in the cell culture medium is increased by 0.06 μM to 0.08 μM and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the fucosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the fucosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein.

[0148] In one embodiment, the present invention provides a method for altering the fucosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the culture medium is increased by 20 mM, such that the manganese concentration in the cell culture medium is increased by 0.06 μM to 0.08 μM and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the fucosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the fucosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein.

[0149] In one embodiment, the present invention provides a method for altering the fucosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the culture medium is increased by 0.4 to 1.6 mM, such that the manganese concentration in the cell culture medium is increased by 0.068 μM ± 0.01 μM and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the fucosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the fucosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein.

[0150] In one embodiment, the present invention provides a method for altering the fucosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the culture medium is increased by 0.8 mM, such that the manganese concentration in the cell culture medium is increased by 0.068 μM ± 0.01 μM and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the fucosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the fucosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein.

[0151] In another embodiment, the present invention provides a method for altering the galactosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the culture medium is increased by 0.4 to 1.6 mM, such that the manganese concentration in the cell culture medium is increased by 0.06 μM to 0.08 μM and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the galactosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the galactosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0152] In another embodiment, the present invention provides a method for altering the galactosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the culture medium is increased by 0.8 mM, such that the manganese concentration in the cell culture medium is increased by 0.06 μM to 0.08 μM and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the galactosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the galactosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0153] In another embodiment, the present invention provides a method for altering the galactosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the culture medium is increased by 0.4 to 1.6 mM, such that the manganese concentration in the cell culture medium is increased by 0.068 μM ± 0.01 μM and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the galactosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the galactosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0154] In another embodiment, the present invention provides a method for altering the galactosylation profile of a recombinant glycoprotein produced in a cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the culture medium is increased by 0.8 mM, such that the manganese concentration in the cell culture medium is increased by 0.068 μM ± 0.01 μM and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the galactosylation profile of the produced recombinant glycoprotein is altered so that it is more similar to the galactosylation profile of a reference glycoprotein than when cultured without said supplementation. Preferably, the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0155] In yet another preferred embodiment, the present invention provides a method for altering the fucosylation profile and galactosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the culture medium is increased by 0.4 to 1.6 mM, such that the manganese concentration in the cell culture medium is increased by 0.06 μM to 0.08 μM and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the fucosylation profile and galactosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the fucosylation profile and galactosylation profile of a reference glycoprotein compared to when the culture is not supplemented. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein and the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0156] In yet another preferred embodiment, the present invention provides a method for altering the fucosylation profile and galactosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the culture medium is increased by 0.8 mM, such that the manganese concentration in the cell culture medium is increased by 0.06 μM to 0.08 μM and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the fucosylation profile and galactosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the fucosylation profile and galactosylation profile of a reference glycoprotein than when the culture is not supplemented. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein and the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0157] In yet another preferred embodiment, the present invention provides a method for altering the fucosylation profile and galactosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the culture medium is increased by 0.4 to 1.4 mM, such that the manganese concentration in the cell culture medium is increased by 0.068 μM ± 0.01 μM and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the fucosylation profile and galactosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the fucosylation profile and galactosylation profile of a reference glycoprotein compared to when the culture is not supplemented. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein and the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0158] In yet another preferred embodiment, the present invention provides a method for altering the fucosylation profile and galactosylation profile of a recombinant glycoprotein produced in cell culture, the method comprising culturing a eukaryotic cell expressing the recombinant glycoprotein in a cell culture medium, wherein the cell culture medium is supplemented with fucose, manganese and taurine, such that the fucose concentration in the culture medium is increased by 0.8 mM, such that the manganese concentration in the cell culture medium is increased by 0.068 μM ± 0.01 μM and the final taurine concentration in the cell culture medium after supplementation is 12.5 mM to 50 mM, more preferably 15 mM to 35 mM, even more preferably 20 mM to 30 mM, most preferably 25 mM, and wherein the fucosylation profile and galactosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the fucosylation profile and galactosylation profile of a reference glycoprotein compared to when the culture is not supplemented. Preferably, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference glycoprotein and the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference glycoprotein.

[0159] In one embodiment, the method of any of the preceding embodiments further comprises the step of isolating the produced recombinant glycoprotein from the cell culture. In a specific embodiment, the isolating step comprises Protein A purification.

[0160] Any eukaryotic cell or cell type that is sensitive to cell culture and capable of expressing recombinant glycoproteins can be utilized according to the present invention and is particularly useful with any of the previous embodiments of the present invention. For example, plant cells, yeast cells, animal cells, insect cells, avian cells or mammalian cells can be utilized according to the present invention. Examples of mammalian cells that can be used according to the present invention include BALB / c mouse myeloma lines (NSO / 1, ECACC No: 85110503); human retinoblasts (CruCell, Leiden, The Netherlands); monkey kidney cell CV1 lines (COS-7, ATCC CRL 1651) transformed by SV40; human embryonic kidney lines (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36: 59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells ± DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216 (1980)), such as Chinese hamster ovary cell line M (CHO-M); mouse testicular Sertoli cells (TM4, Mather, Biol. Reprod., 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL5 1); TRI cells (Mather et al., Annals NY Acad. Sci., 383:44-68 (1982)); MRC5 cells; and FS4 cells.

[0161] Eukaryotic cells suitable for the present invention can be selected or engineered to produce high levels of glycoproteins. Often, cells are genetically engineered to produce high levels of glycoproteins, for example, by introducing genes encoding target recombinant glycoproteins and / or by introducing control elements for the expression of genes (whether endogenous or introduced) encoding target recombinant glycoproteins.

[0162] Eukaryotic cells may also or alternatively be selected or engineered to allow culture in serum-free medium.

[0163] In one embodiment, the eukaryotic cell comprises a vector comprising a polynucleotide encoding a protein that is subsequently glycosylated.

[0164] In another embodiment, commercially or non-commercially available hybridoma cell lines expressing at least one glycoprotein may be utilized according to the present invention. In a preferred embodiment, the eukaryotic cell is a Chinese hamster ovary (CHO) cell. In a preferred embodiment, the CHO cell comprises a vector comprising a polynucleotide encoding a protein that is subsequently glycosylated. For example, CHO-K1 (ATCC CCL-61), CHO-DUKX (ATCC CRL-9096), Lec1 ATCC CRL-1735, CHO-DG44 (ThermoFisher), CHO-M (Selexis), CHO-S (ThermoFisher), or CHO Pro-5 (ATCC CRL-1781) are suitable.

[0165] In a particularly preferred embodiment, the eukaryotic cell is a Selexis SURE CHO-M cell line (CHO-M). In a preferred embodiment, the CHO-M cell comprises a vector comprising a polynucleotide encoding a protein which is subsequently glycosylated.

[0166] In one embodiment, the recombinant glycoprotein whose glycosylation profile is altered by the method of any of the previous embodiments is produced on a large scale, large scale meaning a culture volume of at least 500 or 1,000 liters, preferably at least 5,000 or 8,000 liters and most preferably 10,000 or 20,000 liters.

[0167] In one embodiment, the recombinant glycoprotein whose glycosylation profile is altered by the method of any of the previous embodiments is produced on a small scale or laboratory scale, which scale means a culture volume of 1, 5, 10 or 100 liters.

[0168] In one embodiment, the recombinant glycoprotein whose glycosylation profile is altered by the method of any of the previous embodiments is an immunoglobulin of the IgG type. In a preferred embodiment, the recombinant glycoprotein is an antibody. In a more preferred embodiment, the recombinant glycoprotein is a monoclonal antibody. In a most preferred embodiment, the recombinant glycoprotein is a therapeutic monoclonal antibody.

[0169] The cell culture of any of the previous embodiments wherein the recombinant glycoprotein is produced can be carried out for 1 to 18 days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 days. In one embodiment, the cell culture is carried out for 4 to 18 days, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 days. In a preferred embodiment, the cell culture is carried out for 8 to 16 days, e.g., 8, 9, 10, 11, 12, 13, 14, 15 or 16 days. In a more preferred embodiment, the cell culture is carried out for 10 to 14 days, e.g., 10, 11, 12, 13 or 14 days. In a most preferred embodiment, the cell culture of any of the previous embodiments is carried out for 14 days.

[0170] The supplementation in the method of any of the previous embodiments can be carried out continuously or discontinuously from the beginning of cultivation to the end of cultivation. For example, one or more components of an additive or supplement can be added as a single pill or two or more pills from a storage solution, and other components of the same additive or supplement can be added as a part of a feed medium. In addition, any one or more components in the additive or supplement can be present in the basal medium from the beginning of cell culture. Therefore, supplementation can occur at the beginning of cultivation, and / or after the beginning of cultivation, for example, during the growth phase and / or production phase.

[0171] In one embodiment, the supplementation of the cell culture medium can be carried out from the beginning of the culture. In a preferred embodiment, the supplementation of the cell culture medium carried out from the beginning of the culture is supplemented with taurine. In a more preferred embodiment, the supplementation of the cell culture medium carried out from the beginning of the culture is that the final taurine concentration in the cell culture medium after the supplementation is 12.5 to 50 mM. In a most preferred embodiment, the supplementation of the cell culture medium carried out from the beginning of the culture is that the final taurine concentration in the cell culture medium after the supplementation is 25 mM.

[0172] In one embodiment, the supplementation of the culture medium is performed every other day starting from the 3rd day of the culture, preferably as part of the feed. In a preferred embodiment, the supplementation of the culture medium performed every other day starting from the 3rd day of the culture is supplemented with fucose. In a more preferred embodiment, the supplementation of the culture medium performed every other day starting from the 3rd day of the culture is supplemented with fucose, so that the fucose concentration in the cell culture medium is increased by 0.4 to 1.6 mM. In an even more preferred embodiment, the supplementation of the culture medium performed every other day starting from the 3rd day of the culture is supplemented with fucose, so that the fucose concentration in the cell culture medium is increased by 0.8 mM.

[0173] In a preferred embodiment, the supplementation of the culture medium is performed every other day from the 3rd day of the culture until the 13th day of the culture, preferably as part of the feed. In a more preferred embodiment, the supplementation of the culture medium performed every other day from the 3rd day of the culture until the 13th day of the culture is supplemented with fucose. In a more preferred embodiment, the supplementation of the culture medium performed every other day from the 3rd day of the culture until the 13th day of the culture is supplemented with fucose so that the fucose concentration in the cell culture medium is increased by 0.4 to 1.6 mM. In an even more preferred embodiment, the supplementation of the culture medium performed every other day from the 3rd day of the culture until the 13th day of the culture is supplemented with fucose so that the fucose concentration in the cell culture medium is increased by 0.8 mM.

[0174] In one embodiment, the medium supplementation in the method of any of the previous embodiments is performed every other day starting from the 5th day of the culture, preferably as part of the feed. In a preferred embodiment, the medium supplementation performed every other day starting from the 5th day of the culture is supplemented with manganese. In a more preferred embodiment, the medium supplementation performed every other day starting from the 5th day of the culture is supplemented with manganese, so that the manganese concentration in the cell culture medium is increased by 0.04 μM to 0.08 μM. In an even more preferred embodiment, the medium supplementation performed every other day starting from the 5th day of the culture is supplemented with manganese, so that the manganese concentration in the cell culture medium is increased by 0.068 μM ± 0.01 μM.

[0175] In a preferred embodiment, the supplementation of the culture medium is performed every other day from the 5th day of the culture until the 9th day of the culture, preferably as part of the feed. In a more preferred embodiment, the supplementation of the culture medium performed every other day from the 5th day of the culture is supplemented with manganese. In a more preferred embodiment, the supplementation of the culture medium performed every other day from the 5th day of the culture is supplemented with manganese, so that the manganese concentration in the cell culture medium is increased by 0.04 μM to 0.08 μM. In an even more preferred embodiment, the supplementation of the culture medium performed every other day from the 5th day of the culture is supplemented with manganese, so that the manganese concentration in the cell culture medium is increased by 0.068 μM ± 0.01 μM.

[0176] In a preferred embodiment, the supplementation of manganese to the culture medium in any of the previous embodiments is supplemented with manganese as manganese chloride (MnCl2). In a preferred embodiment, supplementation of manganese as manganese chloride is performed every other day from the 5th day of culture, optionally until the 9th day of culture, preferably as part of the feed. In a more preferred embodiment, supplementation of manganese as manganese chloride is performed every other day from the 5th day of culture, optionally until the 9th day of culture, so that the manganese concentration in the cell culture medium is increased by 0.04 μM to 0.08 μM. In an even more preferred embodiment, supplementation of manganese as manganese chloride is performed every other day from the 5th day of culture, optionally until the 9th day of culture, so that the manganese concentration in the cell culture medium is increased by 0.068 μM ± 0.01 μM.

[0177] In one embodiment, the medium supplementation in the method of any of the previous embodiments is supplemented with taurine from the beginning of the culture, supplemented with fucose every other day from the 3rd day of the culture, and supplemented with manganese every other day from the 5th day of the culture, optionally in the form of manganese chloride. In a preferred embodiment, the medium supplementation is supplemented with taurine from the beginning of the culture, supplemented with fucose every other day from the 3rd day of the culture until the 13th day of the culture, and supplemented with manganese every other day from the 5th day of the culture, optionally in the form of manganese chloride, until the 9th day of the culture, preferably as part of the feed, respectively. In another preferred embodiment, the medium supplementation is supplemented with taurine from the beginning of the culture, the taurine concentration in the cell culture medium after supplementation is 12.5 to 50mM, supplemented with fucose every other day from the 3rd day of the culture, so that the fucose concentration in the cell culture medium is increased by 0.4 to 1.6mM, and supplemented with manganese every other day from the 5th day of the culture, optionally in the form of manganese chloride, so that the manganese concentration in the cell culture medium is increased by 0.04μM to 0.08μM. In a more preferred embodiment, the medium is supplemented with taurine from the beginning of the culture, and the taurine concentration in the cell culture medium after supplementation is 12.5 to 50 mM, fucose is supplemented every other day from the third day of the culture until the 13th day of the culture, so that the fucose concentration in the cell culture medium is increased by 0.4 to 1.6 mM, and manganese is supplemented every other day from the fifth day of the culture, optionally in the form of manganese chloride, until the ninth day of the culture, so that the manganese concentration in the cell culture medium is increased by 0.04 μM to 0.08 μM. In another preferred embodiment, the medium is supplemented with taurine from the beginning of the culture, and the final taurine concentration in the cell culture medium after supplementation is 25 mM, fucose is supplemented every other day from the third day of the culture, so that the fucose concentration in the cell culture medium is increased by 0.8 mM, and manganese is supplemented every other day from the fifth day of the culture, optionally in the form of manganese chloride, so that the manganese concentration in the cell culture medium is increased by 0.068 μM ± 0.01 μM. In the most preferred embodiment, the medium is supplemented with taurine from the beginning of the culture, and the final taurine concentration in the cell culture medium after the supplementation is 25 mM, fucose is supplemented every other day starting from the 3rd day of the culture until the 13th day of the culture, so that the fucose concentration in the cell culture medium is increased by 0.8 mM, and manganese is supplemented every other day starting from the 5th day of the culture, optionally in the form of manganese chloride, until the 9th day, so that the manganese concentration in the cell culture medium is increased by 0.068 μM ± 0.01 μM.

[0178] In one embodiment, supplementation of the cell culture medium is performed during the production phase of the cell culture.

[0179] In one embodiment, the cell culture in the method of any of the previous embodiments is carried out between 35 and 40° C., e.g., at 35, 36, 37, 38, 39 or 40° C. or any temperature therein. In a preferred embodiment, the cell culture is carried out between 36 and 38° C., e.g., at 36, 37 or 38° C. or any temperature therein. In a most preferred embodiment, the cell culture is carried out at 37° C.

[0180] In one embodiment, the cell cultivation in the method of any of the preceding embodiments is performed at a pH between 6 and 9, such as at pH 6, 7, 8 or 9. In a preferred embodiment, the cell cultivation is performed at pH 7.05 ± 0.05.

[0181] In a particularly preferred embodiment, the cell culture of any of the preceding embodiments is a fed-batch culture.

[0182] In one embodiment, the glycoprotein in the method of any of the previous embodiments is a VEGF antagonist, preferably an anti-VEGF antibody (see Examples 1-5). In a preferred embodiment, the fucosylation profile of the VEGF antagonist (preferably an anti-VEGF antibody) is altered. In a more preferred embodiment, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, and more preferably at least 98% of the fucosylation profile of a reference VEGF antagonist (preferably an anti-VEGF antibody). In another preferred embodiment, the galactosylation profile of the VEGF antagonist (preferably an anti-VEGF antibody) is altered. In a more preferred embodiment, the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, and more preferably at least 98% of the galactosylation profile of a reference VEGF antagonist (preferably an anti-VEGF antibody). In another even more preferred embodiment, the fucosylation profile and galactosylation profile of the VEGF antagonist (preferably an anti-VEGF antibody) are altered. In a most preferred embodiment, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of a reference VEGF antagonist (preferably an anti-VEGF antibody) and the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of a reference VEGF antagonist (preferably an anti-VEGF antibody).

[0183] In one embodiment, the glycoprotein in the method of any of the previous embodiments is an anti-CD20 antibody (see Example 6). In a preferred embodiment, the fucosylation profile of the anti-CD20 antibody is altered. In a more preferred embodiment, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference anti-CD20 antibody. In another preferred embodiment, the galactosylation profile of the anti-CD20 antibody is altered. In a more preferred embodiment, the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference anti-CD20 antibody. In another even more preferred embodiment, the fucosylation profile and galactosylation profile of the anti-CD20 antibody are altered. In a most preferred embodiment, the altered fucosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of a reference anti-CD20 antibody and the altered galactosylation profile is at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of a reference anti-CD20 antibody.

[0184] Although the present invention has been described and illustrated in detail in the drawings and the foregoing description, such description and illustration are to be considered illustrative or exemplary rather than restrictive. The present invention is not limited to the disclosed embodiments. Other variations of the disclosed embodiments may be understood and effected by those skilled in the art in practicing the claimed invention by studying the drawings, the disclosure, and the dependent claims.

[0185] The detailed description is only exemplary in nature and is not intended to limit application and use. The following examples further illustrate the present invention, but do not limit the scope of the present invention. Those skilled in the art can make various changes and modifications based on the description of the present invention, and such changes and modifications are also included in the present invention. Example

[0186] 1. Effect of fucose source on fucosylation profile

[0187] In order to adjust the fucosylation profile of the anti-VEGF antibody to be more similar to the fucosylation profile of a reference anti-VEGF antibody that has received regulatory approval for human therapeutics, in this case to be higher than when the anti-VEGF antibody is produced without medium supplementation, a series of fed-batch culture experiments were performed to test the effects of medium and feed supplementation with different concentrations of guanosine, fucose and mannose. Mannose and guanosine play key roles in the de novo fucosylation pathway, with mannose serving as a substrate for the process and guanosine as a component of guanosine triphosphate, which is required for substrate activation in the enzymatic process, and fucose as a substrate for the so-called salvage synthesis pathway of fucosylation (Gramer et al., Biotechnol Bioeng 108: 1591-1602 (2011)).

[0188] Cell culture

[0189] cell

[0190] The Chinese hamster ovary cell line (CHO-M) derived from ordinary CHO K1 cells was purchased from Selexis (Selexis SA, Switzerland). CHO-M cells were adapted to grow in serum-free, chemically defined G11.2 basal medium (Irvine Scientific US). The CHO-M cell line was genetically engineered to express anti-VEGF antibodies by transfection with recombinant DNA encoding anti-VEGF antibodies.

[0191] The experiments were performed in laboratory-scale AMBR mini-shake flasks / benchtop bioreactors (Sartorius Stedim Biotech GmbH, Goettingen, Germany) with a working volume of 1-10 L or in 100 L process bioreactors. The production scale and maximum culture volume used in the examples was 5000 L. Culture cells

[0192] CHO-M cells were cultured under aerobic conditions for 12 days (dissolved oxygen (DO) levels were set to 40% and controlled with a mixture of air and pure oxygen). The pCO2 level was maintained below 60 mmHg by adjusting the aeration rate when necessary. The cells were cultured at 37°C. The pH was maintained at 7.05±0.05 using 12.3% (w / w) H3PO4 solution or 0.5 M NaHCO3 base solution throughout the culture period. To avoid glucose limitation and keep glucose levels in the target 10-30 mM range, 35% (w / w) glucose solution was added to the culture if necessary. An antifoaming agent was used in case of foaming. Relevant metabolites were measured every day.

[0193] Fed-batch culture

[0194] Fed-batch culture was performed by culturing cells initially in serum-free, chemically defined basal medium G11.2 (Irvine Scientific US) for 12 days. From day 3 of culture (post-seeding), concentrated feed solutions (Feed A and Feed B) were added to the culture medium in a shot-wise mode every other day.

[0195] The suppliers and catalog numbers of the culture media and additional supplements used in the Examples are summarized below.

[0196]

[0197] The detailed composition of basal medium and feed can be found in Table 1.

[0198] Table 1: Compounds of basal medium, feed A and feed B (day 3, 5, 7, 9, 11, 13*).

[0199]

[0200] On days 3, 5, 7, 9 and 11 of culture, feed A at 4% of the total culture volume after addition and feed B at 0.4% of the total culture volume after addition were added to the cell culture, wherein feed A was additionally supplemented with one of two different concentrations of guanosine (Sigma-Aldrich, catalog No.: G6264), fucose (L-(-)-fucose; Sigma-Aldrich, catalog No.: F2252) or mannose (D-(+)-mannose; Sigma-Aldrich, catalog No.: M6020): 20 or 60 mM guanosine, 10 or 20 mM fucose and 20 or 60 mM mannose.

[0201] purification

[0202] Protein A chromatography

[0203] For mass analysis, the obtained antibodies were affinity purified from the fermentation broth using protein A chromatography. This capture provides a particular selectivity for Fc-bearing molecules, thereby removing more than 99.5% of contaminants in a single step.

[0204] analyze

[0205] Viable cell density and viability

[0206] Trypan blue staining was used to detect TM The viable cell density and viability were determined using an automated cell counter (Invitrogen Carlsbad, CA, 2008).

[0207] glucose

[0208] Glucose concentration was measured by Cedex Bio HT analyzer (Roche, Mannheim, Germany).

[0209] pCO2

[0210] Dissolved carbon dioxide content (pCO2) was measured by ABL80 blood gas analyzer (Radiometer, Bronshoj, Denmark).

[0211] pH Measurement

[0212] For in situ pH meter recalibration, online pH measurements were performed using a S47 SevenMulti pH meter (Mettler Toledo, Zurich, Switzerland).

[0213] Osmolarity

[0214] Osmolarity was determined by an Advanced Model 2020 Multi-Sample Osmometer (Advanced Instruments, Norwood, MA).

[0215] Potency

[0216] Protein titers were determined by protein A affinity HPLC.

[0217] Glycosylation profile

[0218] The N-glycan profile of the antibodies of the present invention was determined as follows: After denaturation and deglycosylation of each antibody sample by on-column digestion with PNGase F at 37°C for 3 hours, the N-glycans were labeled with a fluorescent labeling reagent (2-AB). Excess dye was separated from the labeled N-glycans using a cleanup column (HILIC). The labeled purified N-glycans were then separated using hydrophilic interaction liquid chromatography (HILIC-UHPLC) on an amide stationary phase, and the relative amount of labeled glycans was determined using fluorescence detection. Specifically, with the aid of The antibody (200 mg, 350 ml) was buffer exchanged into ammonium formate buffer (10 mM, pH 8.6) using a centrifugal device (Pall, USA). N-glycoside-bound oligosaccharides were released by incubating 48-ml samples with 2 ml PNGase F (500,000 U / ml, New England Biolabs) at 45°C for 1 h. The released glycans were labeled with 2-AB at 65°C for 2 h ( Signal 2-AB labeling kit, ProZyme). Excess 2-AB was removed using a HyperSep-96 Diol column (Thermo) with a vacuum station. Labeled glycans were washed with 96% acetonitrile, eluted from the column and purified on a Dionex RSLCUltimate 3000RS or Waters ACQUITY The analysis was performed by HILIC-UHPLC using a Waters BEH Glycan Separation Technology column (2.1×150 mm, 1.7 mm) on the system. A 45-min acetonitrile gradient was applied and the fluorescence signal was detected at 420 nm (excitation at 330 nm). The software automatically integrates the peaks according to predefined parameters and calculates the relative glycan composition. HILIC and HILIC-UHPLC are described in more detail in Reusch et al., mAbs, 7(1), 2015, 167-179.

[0219] The amount of released glycans was calculated using the area % values ​​of the corresponding peaks. The four major glycans (G0F, G1F, G1'F, G2F) of the antibodies of the invention were evaluated compared to the respective reference antibodies. The acceptance criteria for anti-VEGF antibodies were: G0F: ≥73.4 area%, G1F: 4-12.9 area%, G1'F: 2.3-5.6 area%, G2F: 0.5-1.7 area%, total major afucosylation (MAF): 1.9-3.7 area%. The acceptance criteria for anti-CD20 antibodies were: G0F: 40-56 area%; G1F: 28-38 area%; G1'F: 9-13 area% and G2F: 5-12 area%.

[0220] result

[0221] Figure 1 Shown is the effect of different supplements on the total predominantly afucosylated form of anti-VEGF antibody after 12 days of culture.

[0222] Fed-batch culture experiments showed that the feeding supplemented with fucose resulted in the most significant reduction in the total major afucosylated forms (MAF) of the anti-VEGF antibody compared to the tested fucose substitutes guanosine and mannose. At the higher tested concentration of 60 mM, the feeding supplemented with guanosine appeared to be suboptimal in reducing the total MAF. This contrasts with the measurements after the feeding supplemented with fucose, where the reduction in total MAF was most effective at the lower tested fucose concentration of 10 mM. This was also true in the case of mannose, where the lower concentration of 20 mM was more effective in reducing the total major afucosylated forms compared to the feeding supplemented with 60 mM mannose. Therefore, fucose was selected for further experiments.

[0223] 2. Concentration-dependent effect of fucose on fucosylation profile

[0224] To further analyze the effect of fucose on the fucosylation profile of anti-VEGF antibodies, a series of fed-batch culture experiments were performed to test the effect of supplementing the feed medium with different concentrations of fucose.

[0225] Unless otherwise stated, cell culture, isolation and analysis were performed as in the above examples.

[0226] Fed-batch culture was carried out for 12 days. On the 3rd, 5th, 7th, 9th and 11th days of culture, the cell culture was supplemented with 4% of the total culture volume after addition of feed A and 0.4% of the total culture volume after addition of feed B, wherein feed A was additionally supplemented with fucose (L-(-)-fucose; Sigma-Aldrich, catalog number No.: F2252) at a concentration of 10 mM, 20 mM or 40 mM.

[0227] result

[0228] After addition of the corresponding fucose-containing feed, the total major afucosylated form of the anti-VEGF antibody decreased in a concentration-dependent manner as shown in Figure 2 shown.

[0229] Compared to the aforementioned reference anti-VEGF antibody, the biosimilarity of the anti-VEGF antibody was improved in terms of total major afucosylated forms (MAF) (i.e., fucosylation profile). Fed-batch culture experiments showed that the total major afucosylated forms of the anti-VEGF antibody measured on day 12 of culture were reduced by fucose feeding at all tested concentrations compared to control experiments in which the culture medium lacked fucose, which resulted in a total MAF amount more similar to that of the reference anti-VEGF antibody. This reduction in total MAF was concentration-dependent, as the most significant reduction was achieved when fucose was supplemented at a concentration of 40 mM. However, in order to avoid an increase in the cell culture osmolarity and the attendant adverse side effects, a feed supplemented with fucose at a concentration of 20 mM was selected for further experiments. Although feed supplementation with 20 mM fucose resulted in a fucosylation profile of the anti-VEGF antibody that was more similar to that of the reference anti-VEGF antibody, the extent of galactosylation of the anti-VEGF antibody was less than acceptable compared to the reference anti-VEGF antibody.

[0230] 3. Concentration-dependent effects of manganese on glycosylation profiles

[0231] In order to alter the galactosylation profile of the anti-VEGF antibody to be more similar to that of a reference anti-VEGF antibody, a series of fed-batch culture experiments were performed testing the effect of feeding at different times with different concentrations of manganese, a cofactor for β-1,4-galactosyltransferase.

[0232] Unless otherwise stated, cell culture, isolation and analysis were performed as in Example 1.

[0233] Fed-batch cultivation was carried out for 11 days. Manganese was supplemented to feed A in the form of manganese (II) chloride tetrahydrate (MnCl2*4H2O; Sigma-Aldrich, catalog number No.: M5005) at the concentrations shown in Table 2 on days 3, 5, 7 and 9 of the cultivation; days 5, 7 and 9; or day 9.

[0234] result

[0235] The results of the glycosylation profile analysis after feeding supplementation with manganese are shown in Table 2. The most favorable effect of manganese addition on galactosylation can be observed between days 5 and 9 of culture, during which cells can be characterized at the highest specific productivity. Therefore, on days 5, 7 and 9 of culture, in subsequent fed-batch experiments, feed A was supplemented with 1.7 μM manganese chloride. Although the galactosylation profile of the anti-VEGF antibody can be improved by supplementing the feed with manganese, it can also be seen that the addition of manganese leads to a significant increase in the level of afucosylated glycoforms (e.g., G0 increases with manganese and G0F decreases with manganese). Further supplementation of the culture medium must be considered to change the fucosylation profile of the anti-VEGF antibody to be more similar to the fucosylation profile of the reference anti-VEGF antibody.

[0236] Table 2: Effect of different MnCl2*4H2O supplementation strategies on the glycosylation profile of anti-VEGF antibodies. The concentration values ​​shown refer to the MnCl2*4H2O content of the feed A solution.

[0237]

[0238]

[0239] * Calculated from the mean and standard deviation of the glycosylation profiles of 9 commercially available anti-VEGF antibody lots.

[0240] 4. Effect of taurine supplementation on fucosylation profile

[0241] In order to cope with the undesirable increase in osmolarity and further inhibition of fucosylation when the cell culture time was extended to 14 days as planned in subsequent experiments, a series of fed-batch culture experiments were performed in which the cell culture medium was supplemented with an osmoprotectant. Taurine was selected as a possible osmoprotectant because it is known in the art that supplementation with taurine can also increase the titer of glycoproteins produced in cell culture.

[0242] Unless otherwise stated, cell culture, isolation and analysis were performed as in Example 1.

[0243] Fed-batch culture was carried out for 14 days. Taurine (Sigma-Aldrich, catalog number: T4571) was added to the culture medium at a concentration of 12.5 mM, 25 mM or 50 mM at the beginning of the culture (day 0 after inoculation). In addition, manganese was supplemented to feed A at a concentration of 1.7 μM in the form of manganese (II) chloride tetrahydrate (MnCl2*4H2O; Sigma-Aldrich, catalog number: M5005) on culture days 5, 7 and 9.

[0244] result

[0245] Figure 3 Shown is the concentration-dependent effect of basal medium supplementation with taurine on the total predominantly afucosylated form of the anti-VEGF antibody.

[0246] Surprisingly, a concentration-dependent decrease in the total major afucosylated forms of the anti-VEGF antibody was observed after supplementation of the basal medium with taurine. Fed-batch culture experiments showed that the amount of total major afucosylated forms (MAF) of the anti-VEGF antibody was reduced after 14 days of culture in all taurine-supplemented cultures compared to cultures in the basal medium lacking taurine. The high concentration taurine supplement (50 mM) showed a fucosylation profile that was closest to the corresponding range of the fucosylation profile of the reference anti-VEGF antibody. However, for the following experiments, a concentration of 25 mM taurine was selected for medium supplementation to avoid further increases in cell culture osmolarity and to maintain the role of taurine as an osmoprotectant.

[0247] 5. Effects of fucose, manganese, and taurine supplementation on glycosylation profiles

[0248] Next, the modifiability of the glycosylation profile of anti-VEGF antibodies by taurine supplementation of the basal medium in addition to feed supplementation with fucose and manganese was investigated.

[0249] Unless otherwise stated, cell culture, isolation and analysis were performed as in Example 1.

[0250] Cell culture

[0251] cell

[0252] The Chinese hamster ovary cell line (CHO-M) derived from ordinary CHO K1 cells was purchased from Selexis (Selexis SA, Switzerland). CHO-M cells were adapted to grow in serum-free, chemically defined G11.2 basal medium (Irvine Scientific US). The CHO-M cell line was genetically engineered to express anti-VEGF antibodies by transfection with recombinant DNA encoding anti-VEGF antibodies.

[0253] Fed-batch culture

[0254] At the start of the culture (day 0 after inoculation), fed-batch culture was performed for 14 days by culturing cells in serum-free, chemically defined basal medium G11.2 (Irvine Scientific US) supplemented with 25 mM taurine (Sigma-Aldrich, catalog number: T4571).

[0255] From the 3rd day of culture (after inoculation), feed A supplemented with 20 mM fucose at 4% of the total culture volume after addition and feed B at 0.4% of the total culture volume after addition were added to the medium separately in a shot-wise mode every other day. In addition, on the 5th, 7th and 9th days of culture (after inoculation), manganese was supplemented to feed A in the form of manganese (II) chloride tetrahydrate (MnCl2*4H2O; Sigma-Aldrich, catalog number: M5005) at a concentration of 1.7 μM in feed A.

[0256] The suppliers and catalog numbers of the media and additional supplements used in Example 5 are summarized below.

[0257]

[0258] The detailed composition of the basal medium and feed used in Example 5 is shown in Table 3.

[0259] Table 3: Compounds in basal medium, feed A (days 3, 11, 13), feed A (days 5, 7, 9) and feed B (days 3, 5, 7, 9, 11, 13) solutions

[0260]

[0261]

[0262] result

[0263] The effects of taurine, fucose and manganese on the total predominantly afucosylated form of the anti-VEGF antibody are shown in Figure 4 middle.

[0264] Fed-batch culture experiments showed that the amount of total major afucosylated forms (MAF) of anti-VEGF antibody after 14 days of culture could be reduced by adding 25 mM taurine to the basal medium compared to culture in basal medium lacking taurine and fucose. In addition, the total MAF amount of anti-VEGF antibody could be further reduced by subjecting the cell culture containing taurine to a 20 mM fucose feed. The synergistic effect of adding taurine and fucose to the cell culture resulted in a fucosylation profile that fit well within the corresponding range of the fucosylation profile of the reference anti-VEGF antibody.

[0265] Figure 5 Depicted are the fucosylation profiles based on daily analysis of fermentation broth samples starting from day 3 of culture (post-inoculation).

[0266] The analysis showed that feeding supplementation with 20 mM fucose, combined with feeding supplementation with 1.7 μM manganese and medium supplementation with 25 mM taurine combined with feeding supplementation with 1.7 μM manganese could alone reduce the total major afucosylated glycoforms (MAF) ( Figure 5 However, the lowest MAF spectrum was observed only when all three components were supplemented together ( Figure 5 The MAF profile is completely within the range of the total MAF profile, i.e. at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the fucosylation profile of the reference anti-VEGF antibody.

[0267] Similarly, the non-galactosylated glycoform (G0) profiles of each sample analyzed daily are shown in Figure 6 middle.

[0268] The experiments showed that feed supplementation with 20 mM fucose, combined with feed supplementation with 1.7 μM manganese and medium supplementation with 25 mM taurine combined with feed supplementation with 1.7 μM manganese could individually reduce the amount of non-galactosylated glycoforms (G0) ( Figure 6 However, the lowest G0 profile was observed only when all three components were supplemented in the medium and feed, respectively, which was within the range of the galactosylation profile, i.e. at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 98% of the galactosylation profile of the reference anti-VEGF antibody. Figure 6 In summary, supplementation of the culture with a combination of taurine, manganese and fucose can improve the similarity of the anti-VEGF antibody to the reference anti-VEGF antibody in terms of the non-galactosylated glycoform (G0) profile, i.e. the galactosylation profile.

[0269] 6. Effects of fucose, manganese, and taurine supplementation on glycosylation profiles

[0270] In similar fed-batch experiments with anti-CD20 antibodies, the same effects on the alteration of the glycosylation profile following supplementation of the culture medium with fucose, manganese and taurine could be achieved.

[0271] Unless otherwise stated, cell culture, feeding regimens, separations and analyses were performed as described in Example 5.

[0272] Fed-batch culture was performed by culturing cells for 12 days in serum-free, chemically defined basal medium G11.2 (Irvine Scientific US) supplemented with 25 mM taurine (Sigma-Aldrich, catalog number: T4571) at the start of the culture (day 0 after inoculation).

[0273] From the 3rd day of culture (after inoculation), feed A supplemented with 20 mM fucose at 4% of the total culture volume after addition and feed B supplemented with 0.4% of the total culture volume after addition were added to the medium separately in a shot-wise mode every other day. In addition, manganese was supplemented to feed A at a concentration of 1.7 μM in feed A in the form of manganese (II) chloride tetrahydrate (MnCl2*4H2O; Sigma-Aldrich, catalog number: M5005) on the 5th, 7th and 9th days of culture (after inoculation).

[0274] result

[0275] Table 4 shows the effect of different feeding strategies on the glycosylation profile of anti-CD20 antibodies based on daily analysis of fermentation broth samples starting from day 3 of culture (post-inoculation).

[0276] Glycoanalysis revealed that culturing in a medium supplemented with taurine for 3 or 4 days and supplementing with a fucose-supplemented feed had no effect on the fucosylation and galactosylation profiles of the anti-CD20 antibody. Only when a fucose- and manganese-supplemented feed was added to the cell culture starting from the 5th day of culture, in other words, when all three components, fucose, manganese and taurine, were present in the cell culture, the fucosylation and galactosylation profiles of the anti-CD20 antibody could be altered to be more similar to the corresponding fucosylation and galactosylation profiles of the reference anti-CD20 antibody.

[0277] Table 4: Effects of fucose, manganese and taurine supplementation on the glycosylation profile of anti-CD20 antibodies

[0278]

[0279] ** Calculated from the mean and standard deviation of the glycosylation profiles of 13 commercially available anti-CD20 antibody lots.

Claims

1. A method for altering the glycosylation profile of a recombinant glycoprotein produced in cell culture, comprising: Chinese hamster ovary (CHO) cells expressing the recombinant glycoprotein were cultured in a cell culture medium supplemented with fucose, manganese and taurine. wherein the glycosylation profile of the produced recombinant glycoprotein is altered so as to be more similar to the glycosylation profile of a reference glycoprotein compared to when cultured without said supplementation, such that the fucosylation profile of the produced recombinant glycoprotein is at least 90% of the fucosylation profile of the reference glycoprotein and / or such that the altered galactosylation profile is at least 90% of the galactosylation profile of the reference glycoprotein, wherein the recombinant glycoprotein is an anti-VEGF antibody or an anti-CD20 antibody, wherein the reference anti-VEGF antibody had the following glycosylation profile: G0F: ≥73.4 area%, G1F: 4-12.9 area%, G1'F: 2.3-5.6 area%, G2F: 0.5-1.7 area%, total major afucosylation (MAF): 1.9-3.7 area%; Wherein the reference anti-CD20 antibodies have the following glycosylation profiles: G0F: 40-56 area %; G1F: 28-38 area %; G1'F: 9-13 area % and G2F: 5-12 area %; wherein the final concentration of fucose in the supplemented medium is 0.4 mM to 1.6 mM, wherein the final concentration of manganese in the supplemented medium is 0.02 μM to 0.1 μM, The final taurine concentration in the supplemented culture medium is 12.5 mM to 50 mM.

2. The method of claim 1, wherein the altered fucosylation profile is at least 95% of the fucosylation profile of a reference glycoprotein, and / or the altered galactosylation profile is at least 95% of the galactosylation profile of a reference glycoprotein.

3. The method of claim 1, wherein the altered fucosylation profile is at least 96% of the fucosylation profile of a reference glycoprotein, and / or the altered galactosylation profile is at least 96% of the galactosylation profile of a reference glycoprotein.

4. The method of claim 1, wherein the altered fucosylation profile is at least 98% of the fucosylation profile of a reference glycoprotein, and / or the altered galactosylation profile is at least 98% of the galactosylation profile of a reference glycoprotein. The method according to claim 1 , wherein the final fucose concentration in the culture medium after supplementation is 0.4 mM to 1.2 mM. The method according to claim 1 , wherein the final fucose concentration in the culture medium after supplementation is 0.6 mM to 1 mM.

7. The method according to claim 1, wherein the final manganese concentration in the culture medium after supplementation is 0.04 μM to 0.08 μM.

8. The method according to claim 1, wherein the final manganese concentration in the culture medium after supplementation is 0.06 μM to 0.08 μM.

9. The method of claim 1, wherein the final taurine concentration in the culture medium after supplementation is 15 mM to 35 mM.

10. The method of claim 1, wherein the final taurine concentration in the culture medium after supplementation is 20 mM to 30 mM.

11. The method according to claim 1, wherein the method further comprises the step of isolating the produced recombinant glycoprotein from the cell culture.

12. The method of claim 1, wherein the recombinant glycoprotein is produced on a large scale.

13. The method according to any one of claims 1 to 12, wherein the cell culture is performed for 14 days.

14. The method according to any one of claims 1 to 12, wherein the culture medium is supplemented every other day starting from the 3rd day of culturing. The method according to claim 14 , wherein the supplementation of the culture medium is performed until the 13th day of culture.

16. The method according to any one of claims 1 to 12, wherein manganese is supplemented in the form of MnCl2 and wherein the culture medium is supplemented with MnCl2 every other day starting from the 5th day of culturing. The method according to claim 16 , wherein the supplementation of the culture medium is performed until the 9th day of culture.

18. The method according to any one of claims 1 to 12, wherein supplementation of the culture medium is performed during a production phase of the cell culture.

19. The method according to any one of claims 1 to 12, wherein the cell culture is carried out at 37°C.

20. The method according to any one of claims 1-12, wherein the cell culture is carried out at pH 7.05±0.

05.

21. The method according to any one of claims 1 to 12, wherein the cell culture is a fed-batch culture.

22. The method of any one of claims 1-12, wherein the glycoprotein is an anti-VEGF antibody.

23. The method according to any one of the preceding claims 1-12, wherein the glycoprotein is an anti-CD20 antibody.

Images