Total defucosylated glycoforms of antibodies produced in cell cultures

Abstract

The present invention relates to total defucosylated glycoforms of antibodies produced in cell cultures. Provided herein are methods of producing an antibody composition comprising a desired or predetermined or preselected level of a total defucosylated (TAF) glycoform. In an exemplary embodiment, the method comprises maintaining a glycosylation-capable cell in a cell culture medium comprising a specific concentration of fucose and / or glucose as described herein, the specific concentration depending on the level of TAF glycoforms desired. Also provided herein are related compositions comprising glycosylated proteins and TAF glycoforms thereof. Cell culture media are also provided.

Description

[0001] This application is a divisional application of an invention application filed on March 26, 2019, with Chinese national application number 201980021972.X, and invention name “Total defucosylated glycoforms of antibodies produced in cell culture”. Technical Field

[0002] The present application relates to post-translational modifications of antibodies, in particular to glycosylation of antibodies. Background Art

[0003] Glycosylation is one of the most common but important post-translational modifications because it plays a role in a variety of cellular functions, including, for example, protein folding, quality control, molecular transport and sorting, and cell surface receptor interactions. Glycosylation affects the therapeutic efficacy of recombinant protein drugs because it affects the biological activity, pharmacokinetics, immunogenicity, solubility, and in vivo clearance of therapeutic glycoproteins. In particular, the Fc glycoform profile is an important product quality attribute of recombinant antibodies because they directly affect the clinical efficacy and pharmacokinetics of antibodies.

[0004] It has been found that the high mannose (HM) glycoform content affects the pharmacokinetic properties of certain therapeutic antibodies (Goetze et al., (2011) Glycobiology 21, 949-59; Yu et al., (2012) MAbs 4, 475-87). HM glycoforms not only affect the serum clearance rate of antibodies, but also such glycoforms other than defucosylated (afuco) glycoforms can also affect antibody effector function or antibody-mediated target cell killing (also known as antibody-dependent cellular cytotoxicity (ADCC)).

[0005] Many factors affect the glycan structure and thus the final glycosylation form (glycoform) of a protein (glycoprotein). For example, the cell line expressing the antibody, the cell culture medium, the feed medium composition and the timing of the feed during cell culture can affect the generation of the glycoform of the protein.

[0006] Although research groups have proposed many methods to influence the levels of specific glycoforms in antibodies, the biopharmaceutical industry still needs simple and effective methods to manipulate and control the levels of total defucosylated (TAF) glycoforms during recombinant production of therapeutic antibodies. Summary of the invention

[0007] For the first time, data are described that demonstrate that the concentration of fucose and / or glucose in a cell culture medium containing cells producing a recombinant glycosylated protein (e.g., an antibody or antibody binding protein) affects the level of TAF glycoforms of the recombinant glycosylated protein produced. However, larger changes in the level of TAF glycoforms of a recombinant glycosylated protein (e.g., an antibody or antibody binding protein) can be achieved by manipulating the concentration of fucose in a cell culture medium containing cells producing a recombinant glycosylated protein (e.g., an antibody or antibody binding protein), and smaller changes in the level of TAF glycoforms can be achieved by changing the concentration of glucose in the cell culture medium as described herein. In addition, the data show that while the glucose concentration of the cell culture medium affects the levels of high mannose glycans and defucosylated glycans, the fucose concentration of the cell culture medium affects the level of defucosylated glycans without affecting the level of high mannose glycans. The finding that each of these sugars (chemical formulas differing by only one oxygen atom) resulted in a different effect on the level of TAF glycoforms was unexpected. Without being bound by a particular theory, maintaining cells that produce recombinant glycosylated proteins (e.g., antibodies or antibody binding proteins) in a cell culture medium comprising fucose and / or glucose at concentrations as taught herein allows for the production of recombinant glycosylated protein (e.g., antibody or antibody binding protein) compositions having desired or predetermined or preselected levels of TAF glycoforms (e.g., high mannose glycans and defucosylated glycans). Accordingly, the present disclosure relates to methods of producing recombinant glycosylated protein compositions (e.g., antibody compositions or antibody binding protein compositions) comprising desired or predetermined or preselected levels of TAF glycoforms.

[0008] The present disclosure provides a method for producing a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition). In an exemplary embodiment, the method comprises maintaining a glycosylation-competent cell in a cell culture medium comprising a specific concentration of fucose and / or glucose as described herein, the specific concentration depending on the level of the desired TAF glycoform.

[0009] In exemplary embodiments, the level of TAF glycoforms in the recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) is less than or about 10%, and in exemplary aspects, the method comprises maintaining the glycosylation-competent cells in a cell culture medium comprising fucose, wherein the fucose is present in the culture medium at a concentration between about 0.17 g / L and about 1.0 g / L.

[0010] In exemplary embodiments, the level of TAF glycoforms in the recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) is less than or about 10%, and in exemplary aspects, the method comprises maintaining the glycosylation-competent cells in a cell culture medium comprising fucose, wherein the fucose is present in the culture medium at a concentration between about 0.1 g / L and about 1.0 g / L, and wherein the glycosylation-competent cells have not been genetically modified to alter the activity of an enzyme of the de novo pathway or the salvage pathway.

[0011] The present disclosure also provides a method of producing a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition), the method comprising maintaining a glycosylation-competent cell in a cell culture medium comprising fucose and glucose, wherein the fucose is present in the culture medium at a concentration of about 0.1 g / L to about 1.0 g / L, and glucose is added to the cell culture medium according to a glucose feeding regimen to achieve an average glucose concentration of about 10 g / L or less.

[0012] Provided herein are recombinant glycosylated protein compositions (e.g., antibody compositions or antibody-binding protein compositions) produced by the methods disclosed herein. In addition, related pharmaceutical compositions and cell culture media are provided. In exemplary aspects, the cell culture media comprises exogenous nucleic acids encoding antibodies (e.g., IgG antibodies) and a culture medium comprising fucose at a concentration of about 0.1 g / L to about 1.0 g / L or about 0.17 g / L to about 1.0 g / L. In some cases, cells with glycosylation capability are not genetically modified to change the activity of enzymes in the de novo pathway or salvage pathway. Optionally, cells with glycosylation capability are not genetically modified to knock out genes encoding GDP-keto-6-deoxymannose (deoxymannonse)-3,5-epimerase, 4-reductase. In some aspects, the culture medium further comprises glucose at a concentration of less than about 10 g / L, optionally less than about 9 g / L or about 6 g / L or lower (e.g., about 0.5 g / L to about 4 g / L). In exemplary cases, the pH of the culture medium is about 6.85 to about 7.05, such as about 6.90 to about 7.00. In some cases, the cell culture medium does not contain mannose. In certain aspects, the antibody is an IgG1 antibody. In exemplary aspects, the antibody is specific for a tumor-associated antigen, such as an antigen comprising SEQ ID NO: 3.

[0013] Further provided herein are methods for altering or regulating the level of TAF glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by a cell culture medium having glycosylation capability. In exemplary aspects, the method comprises (A) adding fucose to a cell culture medium containing cells having glycosylation capability to achieve a fucose concentration of about 0.1 g / L to about 1.0 g / L, thereby reducing the level of TAF glycans; (B) adding glucose to a cell culture medium containing cells having glycosylation capability to achieve a glucose concentration of less than about 10 g / L, thereby increasing the level of TAF; or (C) a combination of (A) and (B).

[0014] Also provided is a method for regulating the level of defucosylated glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by a cell having glycosylation capability. In an exemplary embodiment, the method comprises (A) adding fucose to a cell culture medium containing cells having glycosylation capability to achieve a fucose concentration of about 0.1 g / L to about 1.0 g / L, thereby reducing the level of defucosylated glycans; (B) adding glucose to a cell culture medium containing cells having glycosylation capability to achieve a glucose concentration of less than or about 10 g / L, thereby increasing the level of defucosylated glycans; or (C) a combination of (A) and (B).

[0015] The present disclosure further provides a method for regulating the level of high mannose (HM) glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by a cell having glycosylation capability. In an exemplary embodiment, the method comprises adding glucose to a cell culture medium comprising cells having glycosylation capability to achieve a glucose concentration of less than about 10 g / L, thereby increasing the level of HM glycans.

[0016] The present disclosure also provides a method for regulating the level of defucosylated glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by a cell having glycosylation capability, the method comprising reducing the pH of the cell culture medium by about 0.03 to about 1.2, thereby reducing the level of defucosylated glycans of the composition by about 0.5% to about 2%, or increasing the pH of the cell culture medium by about 0.03 to about 1.2, thereby increasing the level of defucosylated glycans of the composition by about 0.5% to about 2%.

[0017] The present disclosure provides a method for reducing the level of defucosylated glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by a glycosylation-competent cell by about 1% to about 2%, the method comprising reducing the pH of the cell culture medium by about 0.05 to about 1.2.

[0018] In addition, a method is provided for reducing the level of defucosylated glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by a cell having glycosylation capability by about 0.5% to about 1.1%, the method comprising lowering the pH of the cell culture medium by about 0.03-0.07.

[0019] The present disclosure further provides a method for increasing the level of defucosylated glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by a glycosylation-competent cell by about 1% to about 2%, the method comprising increasing the pH of the cell culture medium by about 0.05 to about 1.2.

[0020] Also provided is a method for increasing the level of defucosylated glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by a cell having glycosylation capability by about 0.5% to about 1.1%, the method comprising increasing the pH of the cell culture medium by about 0.03-0.07.

[0021] The present disclosure further provides a method for regulating the level of TAF glycans in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by a cell having glycosylation capability, the method comprising regulating, reducing or increasing the level of defucosylated glycans of the composition according to the method for regulating, reducing or increasing the level of defucosylated glycans disclosed herein.

[0022] The present disclosure provides a method for producing a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition), wherein the level of defucosylated glycans in the composition is about 6.2% to about 8.4%, the method comprising maintaining glycosylation competent cells in a cell culture medium having a pH above 7.05 and below 7.2, wherein: (A) the pH of the cell culture medium varies by less than 0.15 (optionally less than 0.10) during the culture period, or (B) the temperature of the cell culture medium does not vary by more than 2°C or the method does not include culturing the cells in a cell culture medium comprising manganese or betaine, or (D) a combination of two or three of (A), (B) and (C). BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1A is an illustration of the three types of N-glycans (oligomannose, complex, and hybrid) and the commonly used symbols for these sugars.

[0024] Figure 1B is a diagrammatic representation of an exemplary glycan structure.

[0025] Figure 2Figure 1 is a diagram of the salvage pathway and the de novo pathway of fucose metabolism. In the salvage pathway, free L-fucose is converted to GDP-fucose, while in the de novo pathway, GDP-fucose is synthesized via three reactions catalyzed by GMD and FX. GDP-fucose is then transported from the cytoplasm to the Golgi lumen by GDP-Fuc transferase and transferred to acceptor oligosaccharides and proteins. Another reaction product, GDP, is converted to guanosine 5-monophosphate (GMP) and inorganic phosphate (Pi) by lumenal nucleotide diphosphatase. The former is exported to the cytoplasm (via a reverse transport system coupled to the transport of GDP-fucose), while the latter is assumed to leave the Golgi lumen via the Golgi anion channel GOLAC. See, e.g., Nordeen et al. 2000; Hirschberg et al. 2001.

[0026] Figure 3 is a graph depicting (A) glucose concentration (g / L) of cell cultures using a control medium (line with open triangles), a first test medium (line with open circles), and a second test medium (line with open hexagons) during a cell culture run, (B) fucose concentration (g / L) of cell cultures using a second test medium (line with open squares) during a cell culture run, and (C) TAF glycan levels (%) of cell cultures using a control medium (dotted line with solid triangles), a first test medium (dotted line with solid circles), and a second test medium (dotted line with solid hexagons) during a cell culture run.

[0027] Figure 4 is a graph depicting (A) glucose concentration (g / L) of cell cultures using a control medium (line with open triangles), a first test medium (line with open circles), and a second test medium (line with open hexagons) during a cell culture run, and (B) high mannose (HM) glycan levels (%) of cell cultures using a control medium (dotted line with solid triangles), a first test medium (dotted line with solid circles), and a second test medium (dotted line with solid hexagons) during a cell culture run.

[0028] Figure 5is a graph depicting (A) glucose concentration (g / L) of cell cultures using a control medium (line with open triangles), a first test medium (line with open circles), and a second test medium (line with open hexagons) during a cell culture run, and (B) defucosylated (afuc) glycan levels (%) of cell cultures using a control medium (dotted line with solid triangles), a first test medium (dotted line with solid circles), and a second test medium (dotted line with solid hexagons) during a cell culture run.

[0029] Figure 6 is a graph of TAF glycan levels (%) as a function of fucose concentration (g / L) in cell culture media containing 0X glucose, 1X glucose, or 2X glucose.

[0030] Figure 7 is a graph showing ADCC levels (expressed as % relative to a control antibody having the same amino acid sequence) as a function of fucose concentration (g / L) in cell culture medium containing 0X glucose, 1X glucose or 2X glucose.

[0031] Figure 8 is a diagram of a model illustrating the effects of glucose and fucose on TAF glycan levels (%). The minimum, maximum and average TAF are shown according to QTPP. The equation in the figure below shows the mathematical relationship between glucose, fucose and TAF.

[0032] Fig.9A is a series of graphs showing (i) TAF glycan levels (%) as a function of fucose concentration (g / L) in cell culture medium (upper left quadrant), or as a function of glucose concentration (g / L) in cell culture medium (upper right quadrant), and (ii) ADCC levels (expressed as % relative to a control antibody having the same amino acid sequence) as a function of fucose concentration (g / L) in cell culture medium (lower left quadrant), or as a function of glucose concentration (g / L) in cell culture medium (lower right quadrant). TAF glycan levels (%) ranged from 3.30684 to 3.73083, and ADCC levels ranged from 78.3092-90.4408. At 0.2 g / L fucose and 3.0 g / L glucose, TAF glycan levels (%) were 3.518836, and ADCC levels (%) were 84.37501.

[0033] Fig. 9BIt is a series of graphs showing the following aspects: (i) TAF glycan levels (%) in cell culture medium as a function of fucose concentration (g / L) (upper left quadrant), or as a function of glucose concentration (g / L) in cell culture medium (upper right quadrant), and (ii) ADCC levels (expressed as % relative to innovative antibodies or commercially available antibodies) in cell culture medium as a function of fucose concentration (g / L) (lower left quadrant) or as a function of glucose concentration (g / L) in cell culture medium (lower right quadrant). TAF glycan levels (%) ranged from 3.52301 to 4.0559, and ADCC levels ranged from 75.6911-90.9385. At 0 g / L fucose and 0.554 g / L glucose, TAF glycan levels (%) were 3.789458, and ADCC levels (%) were 83.3148.

[0034] Fig. 9C It is a series of graphs showing the following aspects: (i) TAF glycan levels (%) in cell culture medium as a function of fucose concentration (g / L) (upper left quadrant), or as a function of glucose concentration (g / L) in cell culture medium (upper right quadrant), and (ii) ADCC levels (expressed as % relative to innovative antibodies or commercially available antibodies) in cell culture medium as a function of fucose concentration (g / L) (lower left quadrant) or as a function of glucose concentration (g / L) in cell culture medium (lower right quadrant). The range of TAF glycan levels (%) was 2.9975-3.68468, and the range of ADCC levels was 79.2215-98.8836. At 0.492 g / L fucose and 6.0 g / L glucose, the TAF glycan levels (%) were 3.341092, and the ADCC levels (%) were 89.05256.

[0035] Fig.10 is a graph plotting the osmotic pressure versus fucose concentration.

[0036] Fig.11 is a graph plotting the fucose concentration in cell culture as a function of time (duration).

[0037] Fig.12 is a graph of %TAF and fucose feed, each plotted over time.

[0038] Fig.13 are a pair of graphs demonstrating that controlling glucose within the target range from day 6 or earlier produces equivalent TAF results.

[0039] Fig.14 is a graph in which the % defucosylation is plotted versus culture time. DETAILED DESCRIPTION

[0040] Many secreted proteins undergo post-translational glycosylation, a process whereby sugar moieties (e.g., glycans, sugars) are covalently attached to specific amino acids of proteins. In eukaryotic cells, two types of glycosylation reactions occur: (1) N-linked glycosylation, in which glycans are attached to asparagine at the recognition sequence Asn-X-Thr / Ser, where "X" is any amino acid except proline; and (2) O-linked glycosylation, in which glycans are attached to serine or threonine. Regardless of the type of glycosylation (N-linked or O-linked), there is microheterogeneity in protein glycoforms due to the wide range of glycan structures associated with each site (O or N).

[0041] All N-glycans have a common core sugar sequence: Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser / Thr(Man3GlcNAc2Asn), and are classified into one of three types: (A) high mannose (HM) or oligomannose (OM) types, which consist of two N-acetylglucosamine (GalNAc) moieties and a large number (e.g., 5, 6, 7, 8, or 9) of mannose (Man) residues; (B) complex types, which contain more than two GlcNAc moieties and any number of other sugar types; or (C) hybrid types, which contain Man residues on one side of the branch and GlcNAc at the base of the complex branch. Figure 1A (Taken from Stanley et al., Chapter 8: N-Glycans, Essentials of Glycobiology, 2nd ed., Cold Spring Harbor Laboratory Press; 2009) Three types of N-glycans are shown.

[0042] N-linked glycans generally contain one or more monosaccharides of galactose (Gal), N-acetylgalactosamine (GalNAc), galactosamine (GalN), glucose (GLc), N-acetylglucosamine (ClcNAc), glucosamine (GlcN), mannose (Man), N-acetylmannosamine (ManNAc), mannosamine (ManN), xylose (Xyl), N0 acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxynononic acid (2-keto-3-doxynononic acid, Kdn), fucose (Fuc), glucuronic acid (GLcA), iduronic acid (IdoA), galacturonic acid (Gal A), mannuronic acid (Man A). Common symbols for such sugars are shown in Figure 1A Exemplary glycans and their identities are shown in Figure 1B middle.

[0043] N-linked glycosylation begins in the endoplasmic reticulum (ER), where a set of complex reactions causes the attachment of a core glycan structure essentially formed by two GlcNAc residues and three Man residues. The glycan complex formed in the ER is modified in the Golgi under the action of an enzyme. If the enzyme is relatively difficult to access the sugar, it usually maintains the initial HM form. If the enzyme can access the sugar, many Man residues are cleaved and further modified to produce a complex N-glycan structure. For example, mannosidase-1 located in the cis-Golgi can cleave or hydrolyze HM glycans, while fucosyltransferase FUT-8 located in the intermediate Golgi fucosylates the glycans (Hanrue Imai-Nishiya (2007), BMC Biotechnology [BMC Biotechnology], 7: 84).

[0044] Therefore, the sugar composition and structural configuration of the glycan structure vary depending on the glycosylation machinery in the ER and Golgi, the accessibility of the machinery to the glycan structure, the order of action of each enzyme and the stage at which the protein is released from the glycosylation machinery, among other factors.

[0045] The disclosure provided herein relates to a method for producing an antibody composition comprising a desired or predetermined or preselected level of TAF glycoforms. In an exemplary embodiment, the method comprises maintaining a glycosylation-competent cell in a cell culture medium comprising a specific concentration of fucose and / or glucose as described herein, the specific concentration depending on the level of the desired TAF glycoform. In addition, the disclosure relates to a method for producing an antibody composition comprising a desired or predetermined or preselected level of defucosylated glycoforms, for example, the level of defucosylated glycans in the antibody composition is about 6.2% to about 8.4%. In an exemplary embodiment, the method comprises maintaining a glycosylation-competent cell in a cell culture medium having a pH above 7.05 and below 7.2, wherein: (A) during the culture period, the pH of the cell culture medium changes by less than 0.15 (optionally less than 0.10), or (B) the temperature of the cell culture medium changes by no more than 2°C, or the method does not include culturing the cell in a cell culture medium comprising manganese or betaine, or (D) a combination of two or three of (A), (B) and (C). Without being bound by a particular theory, it is believed that the methods of the present disclosure provide a means for tailoring compositions that contain specific amounts of specific glycoforms for a given antibody.

[0046] In an exemplary embodiment, the level of TAF glycans is regulated. As used herein, "total defucosylated glycans" or "TAF glycans" or "total defucosylated glycoforms" or "TAF glycoforms" refer to the total amount of high mannose (HM) glycans and defucosylated glycans. In an exemplary embodiment, the level of HM glycans is regulated. As used herein, the terms "high mannose glycans" or "HM glycans" or "high mannose glycoforms" or "HM glycoforms" or "HM" encompass glycans comprising 5, 6, 7, 8, or 9 mannose residues, abbreviated as Man5, Man6, Man7, Man8, and Man9, respectively. In an exemplary embodiment, the level of defucosylated glycans is regulated. As used herein, the term "defucosylated glycan" or "afuco glycan" or "defucosylated glycoform" or "Afuco" refers to a glycoform lacking core fucose (e.g., α1,6-linked fucose) on the GlcNAc residue involved in the amide bond to the Asn of the N-glycosylation site. Defucosylated glycoforms include, but are not limited to, A1G0, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5. Additional defucosylated glycans include, for example, A1G1a, G0[H3N4], G0[H4N4], G0[H5N4], FO-N[H3N3]. See, for example, Reusch and Tejada, Glycobiology 25(12):1325-1334 (2015). In exemplary aspects, the level of TAF and the amount of HM glycoforms and defucosylated glycoforms are determined by hydrophilic interaction liquid chromatography (HILIC), as further described in Example 1 herein. After enzymatic cleavage of the N-glycans, HILIC is performed to obtain a chromatogram with several peaks, each of which represents the average distribution (amount) of a different glycoform. For these purposes, % peak area = peak area / total peak area x 100%, and % total peak area = total area of ​​sample / total area of ​​standard x 100%. Calculations for the purpose of determining % TAF can be performed as follows:

[0047] % Defucosylated glycoforms = % A1G0 + % A2G0 + % A2G1a + % A2G1b + % A2G2 + % A1G1M5.

[0048] % High mannose glycoform = % Man5 (if detectable) + % Man6 (if detectable) + % Man7 (if detectable) + % Man8 (if detectable) + % Man9 (if detectable)

[0049] The present disclosure provides methods for producing recombinant glycosylated protein compositions. In an exemplary embodiment, the recombinant glycosylated protein composition is an antibody composition. In an exemplary embodiment, the method comprises maintaining a glycosylation-competent cell in a cell culture medium comprising a specific concentration of fucose and / or glucose as described herein, the specific concentration depending on the level of the desired TAF glycoform.

[0050] Fucose

[0051] In embodiments of the methods disclosed herein, fucose is present in the culture medium at a concentration of from about 0.1 g / L to about 2.0 g / L, optionally, from about 0.1 g / L to about 1.75 g / L, from about 0.1 g / L to about 1.5 g / L, or from about 0.1 g / L to about 1.2 g / L. In exemplary cases, fucose is present in the culture medium at a concentration of less than or about 1.2 g / L. In exemplary cases, fucose is present in the culture medium at a concentration of from about 0.1 g / L to about 1.0 g / L. In exemplary cases, the culture medium comprises fucose at a concentration of from about 0.17 g / L to about 2.0 g / L, from about 0.17 g / L to about 1.75 g / L, from about 0.17 g / L to about 1.5 g / L, or from about 0.17 g / L to about 1.2 g / L. In exemplary aspects, fucose is present in the culture medium at a concentration of from about 0.17 g / L to about 1.2 g / L. In exemplary cases, fucose is present in the culture medium at a concentration of from about 0.17 g / L to about 1.0 g / L. In exemplary cases, the culture medium comprises fucose at a concentration of from about 0.2 g / L to about 2.0 g / L, from about 0.2 g / L to about 1.75 g / L, from about 0.2 g / L to about 1.5 g / L, or from about 0.2 g / L to about 1.2 g / L. In exemplary cases, fucose is present in the culture medium at a concentration of from about 0.2 g / L to about 1.0 g / L. In exemplary cases, fucose is present in the culture medium at a concentration of less than or about 1.0 g / L. For example, in some cases, the fucose concentration of the culture medium is about 0.10 g / L, about 0.11 g / L, about 0.12 g / L, about 0.13 g / L, about 0.14 g / L, about 0.15 g / L, about 0.16 g / L, about 0.17 g / L, about 0.18 g / L, about 0.19 g / L, or about 0.20 g / L. In some cases, the fucose concentration of the culture medium is about 0.3 g / L, about 0.4 g / L, about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, or about 0.9 g / L. In exemplary aspects, the fucose concentration is no more than or about 1.0 g / L, no more than or about 0.9 g / L, no more than or about 0.8 g / L, or no more than or about 0.7 g / L. In exemplary cases, fucose is present in the culture medium at a concentration of less than or about 0.75 g / L, or about 0.25 g / L to about 0.75 g / L, such as about 0.4 g / L to about 0.5 g / L, or about 0.6 g / L. In exemplary aspects, fucose is present in the culture medium at a concentration of less than about 0.6 g / L, such as about 0.2 g / L to about 0.5 g / L.

[0052] In exemplary aspects, a method of producing a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) comprises maintaining cells capable of glycosylation in two different cell culture media. In exemplary aspects, a method of producing a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) comprises maintaining cells capable of glycosylation in a first cell culture medium for an initial period of time, and then maintaining these cells capable of glycosylation in a second cell culture medium, optionally wherein the first cell culture medium does not contain fucose at a concentration of about 0.1 g / L to about 1.0 g / L, and the second cell culture medium contains fucose at a concentration of about 0.1 g / L to about 1.0 g / L and the second cell culture medium contains fucose at, for example, one of the above concentrations. In exemplary cases, the initial period of time begins when the cells are inoculated into a bioreactor containing a cell culture medium (e.g., a cell culture medium). In some aspects, the initial period of time is about 1 day to about 3 days, for example, about 24 hours to about 72 hours. In exemplary aspects, the initial time period is greater than about 3 days (about 72 hours), but less than about 10 days (about 240 hours) or less than about 156 hours. In exemplary aspects, the initial time period is about 3, about 4, about 5, about 6, about 7, about 8 or about 9 days. In exemplary aspects, the method includes adding fucose to the culture medium after the initial time period. In some aspects, fucose is added to the first culture medium to obtain a second cell culture medium. For example, in various aspects, the method includes adding fucose after about 1 day to about 3 days, about 3 days but less than about 10 days, or about 3, about 4, about 5, about 6, about 7, about 8, or about 9 days. In exemplary aspects, the method includes adding fucose to the cell culture medium (e.g., the first cell culture medium) on the 6th, 7th, 8th or 9th day after the cell culture is inoculated. In exemplary aspects, fucose is added to a final concentration of greater than about 0.1 g / L, greater than about 0.17 g / L, or greater than about 0.2 g / L and less than about 2.0 g / L. In exemplary aspects, the first cell culture medium does not comprise fucose. In exemplary aspects, the first cell culture medium comprises fucose, but at an undetectable or unmeasurable concentration, or at a concentration substantially lower than the fucose concentration of the second cell culture medium, e.g., substantially less than 0.1 g / L, less than about 0.17 g / L, or less than about 0.2 g / L.

[0053] In alternative aspects, the method comprises maintaining the glycosylation-competent cells in a cell culture medium comprising fucose (e.g., at a concentration greater than about 0.1 g / L, greater than about 0.17 g / L, or greater than about 0.2 g / L and less than about 2.0 g / L) for the entire duration that the glycosylation-competent cells are maintained in cell culture or for a majority of the culture period. In some aspects, a method of producing a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) comprises inoculating the glycosylation-competent cells in a bioreactor comprising a cell culture medium containing fucose, and maintaining the cells in the cell culture medium at a concentration of fucose that is substantially the same throughout the cell culture period.

[0054] In an exemplary embodiment, the concentration of fucose fluctuates little during the cell culture process. In an exemplary embodiment, the fucose concentration fluctuates by about 0.2 g / L or less during the period of maintaining cells with glycosylation capability in a cell culture medium comprising fucose. In an exemplary aspect, the fucose concentration fluctuates by about 0.1 g / L or less during the period of maintaining cells with glycosylation capability in a cell culture medium comprising fucose. In an exemplary aspect, when fucose is added to the cell culture medium (e.g., after the initial cell culture period), fucose is added to the culture medium no more than once or twice during the cell culture period.

[0055] glucose

[0056] In exemplary embodiments of the methods disclosed herein, glucose is present in the culture medium. In exemplary aspects, glucose is present in the culture medium at a concentration of less than or about 10 g / L, less than or about 9.0 g / L, or less than or about 6.0 g / L. In exemplary aspects, glucose is present in the culture medium at a concentration from about 0.5 g / L to about 4.0 g / L. In exemplary aspects, glucose is present in the culture medium at a concentration from about X g / L to about Y g / L. g / L, wherein X is about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, or 3.9, and Y is about 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, or 3.9. 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0, provided that X is less than Y.

[0057] In exemplary aspects, the method includes maintaining the glucose concentration in the cell culture medium for a time period equivalent to the cell culture period. In exemplary cases, maintaining the concentration of glucose in the cell culture medium includes sampling the cell culture medium regularly (e.g., every hour, every two hours, every 3, 4, 5 or 6 hours once, once a day, twice a day, three times a day or 4 times a day, etc.), measuring the glucose concentration of the sampled cell culture medium, if the glucose concentration of the sampled cell culture medium is lower than the required glucose concentration to be maintained, then glucose is added to the cell culture. In exemplary aspects, maintaining the glucose concentration in the cell culture medium includes measuring the glucose concentration of the cell culture medium via a glucose receptor. In exemplary aspects, measuring the glucose concentration at regular intervals (e.g., every hour, every two hours, every 3, 4, 5 or 6 hours once, once a day, twice a day, three times a day or 4 times a day, etc.) via a glucose sensor, and if determining the glucose concentration via a glucose receptor to be lower than the required glucose concentration to be maintained, then glucose is added to the cell culture. In alternative aspects, the method includes maintaining cells with glycosylation ability in a cell culture medium comprising glucose, but only maintaining the concentration of glucose in the cell culture medium after the initial time period. In an exemplary embodiment, the initial time period is about 1 day to about 3 days (about 24 hours to about 72 hours). In some cases, the initial time period is less than or about 6 days, optionally, wherein the initial time period is 3 days or 4 days or 5 days after cell culture inoculation. In an exemplary aspect, the method includes maintaining cells with glycosylation capability in a cell culture medium comprising glucose and maintaining the concentration of glucose in the cell culture medium on the sixth day after inoculation and thereafter. In an exemplary aspect, the concentration of glucose is maintained for at least about 4 days or about 5 days after the initial time period, or optionally for at least about 6 days after the initial time period.

[0058] In exemplary aspects, for the initial time period, the cell culture medium comprises an initial glucose concentration. For example, in various aspects, the initial glucose concentration is about 1.0 g / L to about 15 g / L, about 1.0 to about 12 g / L, or about 1.0 g / L to about 10 g / L. The initial glucose concentration, in some aspects, is about 1.0 g / L, about 1.5 g / L, about 2.0 g / L, about 2.5 g / L, about 3.0 g / L, about 3.5 g / L, about 4.0 g / L, about 4.5 g / L, about 5.0 g / L, about 5.5 g / L, about 6.0 g / L, about 6.5 g / L, about 7.0 g / L, about 7.5 g / L, about 8.0 g / L, about 8.5 g / L, about 9.0 g / L, about 9.5 g / L, about 10.0 g / L, about 10.5 g / L, about 11.0 g / L, about 11.5 g / L, or about 12.0 g / L. In some aspects, the initial glucose concentration is about 12 g / L ± 1 g / L or about 9 g / L ± 1 g / L or about 6 g / L ± 1 g / L. In some aspects, the initial glucose concentration is less than about 5.0 g / L or less than about 4.0 g / L. In exemplary aspects, the initial glucose concentration is the glucose concentration of the cell culture medium used during the initial time period. In exemplary aspects, the initial glucose concentration is the glucose concentration of the cell culture medium maintained during the initial time period.

[0059] In exemplary aspects, the initial glucose concentration is the same as the glucose concentration maintained after the initial period. In alternative aspects, the initial glucose concentration is different from the glucose concentration maintained after the initial period. In exemplary aspects, the method includes adding glucose to the cell culture medium after the initial period, and maintaining the glucose at a different concentration relative to the initial glucose concentration. In exemplary aspects, the method includes adding glucose to the cell culture medium after the initial period to maintain the glucose at a different concentration relative to the initial glucose concentration, wherein the step of adding glucose reaches a glucose concentration of about 10 g / L or less (e.g., about 9 g / L or less, about 6 g / L or less, about 0.5 g / L to about 4 g / L).

[0060] In exemplary aspects, the method includes adding glucose to the cell culture medium according to the glucose feeding scheme. In some aspects, the glucose feeding scheme starts after the initial time period. For example, in some aspects, the initial time period is at least 3 days or 4 days, and the glucose feeding scheme starts from about 4 to about 6 days (for example, about 4 days, about 5 days, about 6 days after the cell culture inoculation) after the cell culture inoculation. In exemplary cases, the glucose feeding scheme reaches an average glucose concentration of about 10g / L or lower (for example, about 9g / L or lower, about 6g / L or lower, about 0.5g / L to about 4g / L). The term "average glucose concentration" refers to the average concentration of glucose in the cell culture medium determined by a glucose receptor through a period of time (for example, 1 to 2 days). In exemplary cases, the glucose feeding scheme reaches an average glucose concentration based on the fucose concentration of the cell culture medium. In some aspects, average glucose concentration is calculated based on formula I:

[0061] T=3.354-1.388F+0.111G+[F-0.4375]x[1.9527(F-0.4375)]

[0062] (Formula I)

[0063] wherein T is the target total defucosylated (TAF) glycan % and is about 2.5% to about 6%, about 2.75% to about 5.5%, or about 3% to about 5%, F is the concentration of fucose in the culture medium (g / L), and G is the average glucose concentration (g / L).

[0064] In exemplary cases, (i) the fucose concentration is about 0.2±0.1 g / L and the average glucose concentration is about 2 to about 4 g / L; (ii) the fucose concentration is about 0.5±0.1 g / L and the average glucose concentration is about 3 to about 6 g / L; or (iii) the fucose concentration is about 0.75±0.1 g / L and the average glucose concentration is about 4.5 to about 9 g / L.

[0065] TAF, HM and defucosylated glycan levels

[0066] In exemplary embodiments, the methods disclosed herein produce a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition), wherein the level of TAF glycans in the composition is less than or about 10%. In exemplary aspects, the level of TAF glycans in the composition is less than or about 9%, less than or about 8%, less than or about 7%, less than or about 6%, less than or about 5%. In exemplary aspects, the level of TAF glycans in the composition is greater than or about 4%, for example, between about 4% and about 10%. In some aspects, the level of TAF glycans in the composition is about 2% to about 6% or about 2.5% to about 5%. In some aspects, the level of TAF glycans is about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5%, about 5.5%, or about 6.0%. In exemplary aspects, the level of TAF glycans is about 2% to about 5% or about 2% to about 4%.

[0067] In exemplary aspects of the method of producing a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition), fucose is present in the culture medium at a concentration between about 0.1 g / L and about 1.0 g / L, or between about 0.17 g / L and about 1.0 g / L, and the level of TAF glycans in the composition is less than about 10%.

[0068] In exemplary embodiments, the methods disclosed herein produce a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition), wherein the level of high mannose glycans in the antibody composition is less than or about 3.5%, e.g., less than or about 3.25%, less than or about 3.0%, less than or about 2.5%, less than or about 2.0%. In exemplary aspects, the level of high mannose glycans in the antibody composition is about 0.7% to about 3.0%, optionally, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, or about 3.0%.

[0069] In exemplary embodiments, the methods disclosed herein produce a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition), wherein the level of defucosylated glycans in the antibody composition is less than or about 3.5%, for example, less than or about 3.25%, less than or about 3.0%, less than or about 2.5%, less than or about 2.0%. In exemplary aspects, the level of defucosylated glycans in the antibody composition is about 0.8% to about 2.8%, optionally, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, or about 2.8%.

[0070] Glycoform measurement methods

[0071] A variety of methods are known in the art for assessing the glycoforms present in a composition containing a glycoprotein or for determining, detecting or measuring the glycoform profile of a particular sample containing a glycoprotein. Suitable methods include, but are not limited to, cation MALDI-TOF analysis, anion MALDI-TOF analysis, weak anion exchange (WAX) chromatography, normal phase chromatography (NP-HPLC), exoglycosidase digestion, Bio-Gel P-4 chromatography, anion exchange chromatography, and one-dimensional nuclear magnetic resonance spectroscopy and combinations thereof. See, e.g., Mattu et al., JBC 273:2260-2272 (1998); Field et al., Biochem J [Journal of Biological Chemistry] 299 (Part 1): 261-275 (1994); Yoo et al., MAbs 2 (3): 320-334 (2010); Wuhrer M. et al., Journal of Chromatography B [Journal of Chromatography Series B], 2005, Vol. 825, No. 2, pp. 124-133; Ruhaak LR, Anal Bioanal Chem [Analysis and Bioanalytical Chemistry], 2010, Vol. 397: 3457-3481 and Geoffrey, RG et al. Analytical Biochemistry [Biological Analytical Chemistry] 1996, Vol. 240, pp. 210-226. In addition, the examples described herein describe suitable methods for evaluating glycoforms present in a composition containing a glycoprotein.

[0072] In connection with the present disclosure, the cell culture can be maintained according to any set of conditions suitable for recombinant glycosylated protein production. For example, in some aspects, the cell culture is maintained at a specific pH, temperature, cell density, culture volume, dissolved oxygen level, pressure, osmotic pressure, etc. In an exemplary aspect, the cell culture before inoculation is shaken (e.g., at 70 rpm) at 5% CO2 in a CO2 incubator under standard humidification conditions. In an exemplary aspect, the cell culture is maintained at about 10% CO2 in 1.5 L of culture medium. 6 The cell culture was inoculated at a seeding density of 10 cells / mL.

[0073] In exemplary aspects, the methods of the disclosure include maintaining the glycosylation competent cells at a pH of about 6.85 to about 7.05, e.g., about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, about 6.90, about 6.91, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.00, about 7.01, about 7.02, about 7.03, about 7.04, or about 7.05 in a cell culture medium. In some aspects, the cell culture medium has a pH of about 6.9 to about 7.0.

[0074] In exemplary aspects, the method includes maintaining the cell culture at a temperature between 30° C. and 40° C. In exemplary embodiments, the temperature is between about 32° C. and about 38° C. or between about 35° C. and about 38° C.

[0075] In exemplary aspects, the method includes maintaining osmotic pressure between about 200mOsm / kg and about 500mOsm / kg. In exemplary aspects, the method includes maintaining osmotic pressure between about 225mOsm / kg and about 400mOsm / kg or about 225mOsm / kg and about 375mOsm / kg. In exemplary aspects, the method includes maintaining osmotic pressure between about 225mOsm / kg and about 350mOsm / kg. In various aspects, osmotic pressure (mOsm / kg) is maintained at about 200, 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475 or about 500.

[0076] In exemplary aspects, the method includes maintaining the dissolved oxygen (DO) level of the cell culture at about 20% to about 60% oxygen saturation during the initial cell culture period. In an exemplary example, the method includes maintaining the DO level of the cell culture at about 30% to about 50% (e.g., about 35% to about 45%) oxygen saturation during the initial cell culture period. In an exemplary example, the method includes maintaining the DO level of the cell culture at about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% oxygen saturation during the initial cell culture period. In exemplary aspects, the DO level is about 35mm Hg to about 85mmHg or about 40mm Hg to about 80mmHg or about 45mm Hg to about 75mm Hg.

[0077] The cell culture is maintained in any one or more culture media. In an exemplary aspect, the cell culture is maintained in a culture medium suitable for cell growth and / or one or more feed media according to any suitable feeding scheme is provided for the cell culture. In an exemplary aspect, the method includes maintaining the cell culture in a culture medium comprising glucose, lactate, ammonia, glutamine and / or glutamate. In an exemplary aspect, the method includes maintaining the cell culture in a culture medium comprising manganese at a concentration lower than about 1 μM during the initial cell culture period. In an exemplary aspect, the method includes maintaining the cell culture in a culture medium comprising about 0.25 μM to about 1 μM manganese. In an exemplary aspect, the method includes maintaining the cell culture in a culture medium comprising a negligible amount of manganese. In an exemplary aspect, the method includes maintaining the cell culture in a culture medium comprising copper at a concentration lower than or about 50 ppb during the initial cell culture period. In an exemplary aspect, the method includes maintaining the cell culture in a culture medium comprising copper at a concentration lower than or about 40 ppb during the initial cell culture period. In exemplary aspects, the method comprises maintaining the cell culture in a medium comprising copper at a concentration of less than or about 30 ppb during an initial cell culture period. In exemplary aspects, the method comprises maintaining the cell culture in a medium comprising copper at a concentration of less than or about 20 ppb during an initial cell culture period. In exemplary aspects, the medium comprises copper at a concentration of greater than or about 5 ppb or greater than or about 10 ppb. In exemplary aspects, the cell culture medium comprises mannose. In exemplary aspects, the cell culture medium does not comprise mannose.

[0078] In an exemplary embodiment, the type of cell culture is a fed-batch culture or a continuous perfusion culture. However, the methods of the present disclosure are advantageously not limited to any particular type of cell culture.

[0079] cell

[0080] The present disclosure relates to a method for producing a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition), the method comprising maintaining a cell capable of glycosylation in a cell culture medium. In exemplary aspects, the cell capable of glycosylation is a eukaryotic cell, including but not limited to a yeast cell, a filamentous fungal cell, a protozoan cell, an algae cell, an insect cell, or a mammalian cell. Such host cells are described in the art. See, e.g., Frenzel et al., Front Immunol [Immunology Frontier] 4: 217 (2013). In exemplary aspects, the eukaryotic cell is a mammalian cell. In exemplary aspects, the eukaryotic cell is a non-human mammalian cell. In some aspects, the cells are Chinese hamster ovary (CHO) cells and derivatives thereof (e.g., CHO-K1, CHO pro-3), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), cells engineered to lack dihydrofolate reductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or derivatives thereof (e.g., HEK293T, HEK293-EBNA), green African monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human osteosarcoma bone epithelial cells U2-OS, adenocarcinoma human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells P19, mouse embryonic fibroblasts NIH 3T3, mouse fibroblast L929, mouse neuroblastoma N2a, human breast cancer MCF-7, retinoblastoma Y79, human retinoblastoma SO-Rb50, human hepatoma Hep G2, mouse B myeloma J558L or baby hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2):257-263 (2013)).

[0081] Cells without glycosylation ability can also be converted into cells with glycosylation ability, for example, by transfecting them with genes encoding relevant enzymes necessary for glycosylation. Exemplary enzymes include, but are not limited to, oligosaccharyl transferases, glycosidases, glucosidase I, glucosidase II, calnexin / calreticulin, glycosyltransferases, mannosidases, GlcNAc transferases, galactosyltransferases, and sialyltransferases.

[0082] In an exemplary embodiment, the glycosylation-competent cell has not been genetically modified to alter the activity of enzymes in the de novo pathway or the salvage pathway. The two fucose metabolic pathways are shown in Figure 2In an exemplary embodiment, the cell with glycosylation capability is not genetically modified to change the activity of any one or more of the following: fucosyltransferase (FUT, such as FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9), fucokinase, GDP-fucose pyrophosphorylase, GDP-D-mannose-4,6-dehydratase (GMD), and GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase (FX). In an exemplary embodiment, the cell with glycosylation capability is not genetically modified to knock out the gene encoding FX.

[0083] In an exemplary embodiment, the glycosylation-competent cell is not genetically modified to alter the activity of β(1,4)-N-acetylglucosamine transferase III (GNTIII) or GDP-6-deoxy-D-lyso-4-hexulose reductase (RMD). In an exemplary aspect, the glycosylation-competent cell is not genetically modified to overexpress GNTIII or RMD.

[0084] Recombinant glycosylated protein

[0085] In an exemplary embodiment, the recombinant glycosylated protein comprises an amino acid sequence comprising one or more N-glycosylation consensus sequences of the following formula:

[0086] Asn-Xaa1-Xaa2

[0087] wherein Xaa1 is any amino acid except Pro, and Xaa2 is Ser or Thr.

[0088] In an exemplary embodiment, the recombinant glycosylated protein comprises a crystallizable fragment (Fc) polypeptide. As used herein, the term "Fc polypeptide" includes polypeptides in the form of natural and mutant proteins derived from the Fc region of an antibody. Such polypeptides in the form of truncated hinge regions that promote dimerization are also included. Fusion proteins comprising Fc portions (and oligomers formed therefrom) provide the advantage of being easily purified by affinity chromatography on protein A or protein G columns. In an exemplary embodiment, the recombinant glycosylated protein comprises the Fc of IgG (e.g., human IgG). In an exemplary aspect, the recombinant glycosylated protein comprises the Fc of IgG1 or IgG2. In an exemplary aspect, the recombinant glycosylated protein is an antibody, an antibody protein product, a peptibody, or an Fc fusion protein.

[0089] In an exemplary aspect, the recombinant glycosylated protein is an antibody. As used herein, the term "antibody" refers to a protein having a conventional immunoglobulin format, comprising a heavy chain and a light chain, and comprising a variable region and a constant region. For example, an antibody can be an IgG, which is a "Y-shaped" structure of two pairs of identical polypeptide chains, each pair having a "light" chain (usually having a molecular weight of about 25 kDa) and a "heavy" chain (usually having a molecular weight of about 50-70 kDa). Antibodies have a variable region and a constant region. In the IgG form, the variable region is generally about 100-110 or more amino acids, contains three complementary determining regions (CDRs), is primarily responsible for antigen recognition, and is very different from other antibodies that bind to different antigens. See, e.g., Janeway et al., "Structure of the Antibody Molecule and the Immunoglobulin Genes," Immunobiology: The Immune System in Health and Disease, 4th ed. Elsevier Science Ltd. / Garland Publishing, (1999).

[0090] In brief, in the antibody framework, the CDRs are embedded in a framework in the heavy and light chain variable regions, where these CDRs constitute the regions primarily responsible for antigen binding and recognition. The variable region comprises at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Maryland; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883), within a framework region (designated framework regions 1-4, i.e., FR1, FR2, FR3, and FR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra).

[0091] Human light chains are divided into κ and λ light chains. Heavy chains are divided into μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. IgG has several subclasses, including (but not limited to) IgG1, IgG2, IgG3 and IgG4. IgM has subclasses, including (but not limited to) IgM1 and IgM2. Embodiments of the present disclosure include all such antibody classes or isotypes. The light chain constant region may be, for example, a κ or λ type light chain constant region, such as a human κ or λ type light chain constant region. The heavy chain constant region may be, for example, an α, δ, ε, γ or μ type heavy chain constant region, such as a human α, δ, ε, γ or μ type heavy chain constant region. Therefore, in an exemplary embodiment, the antibody is an antibody of isotype IgA, IgD, IgE, IgG or IgM, including any one of IgG1, IgG2, IgG3 or IgG4.

[0092] In various aspects, the antibody may be a monoclonal antibody or a polyclonal antibody. In some aspects, the antibody comprises a sequence substantially similar to a naturally occurring antibody produced by a mammal, such as a mouse, rat, rabbit, goat, horse, chicken, hamster, pig, human, etc. In this regard, the antibody may be considered a mammalian antibody, such as a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody, a horse antibody, a chicken antibody, a hamster antibody, a pig antibody, a human antibody, etc. In some aspects, the recombinant glycosylated protein is a human antibody. In some aspects, the recombinant glycosylated protein is a chimeric antibody or a humanized antibody. The term "chimeric antibody" is used herein to refer to an antibody containing a constant domain from one species and a variable domain from a second species, or more generally, an antibody containing an amino acid sequence segment from at least two species. The term "humanization" when used with respect to an antibody refers to an antibody having at least a CDR region from a non-human source, which is engineered to have a structure and immune function more similar to a real human antibody than the original source antibody. For example, humanization can involve grafting CDRs from non-human antibodies (eg, mouse antibodies) into human antibodies. Humanization can also involve selecting amino acid substitutions to make non-human sequences look more like human sequences.

[0093] In various aspects, antibodies are cleaved into fragments by enzymes such as papain and pepsin. Papain cleaves antibodies to produce two Fab fragments and a single Fc fragment. Pepsin cleaves antibodies to produce F(ab')2 fragments and pFc' fragments. In exemplary aspects, recombinant glycosylated proteins are antibody fragments that retain at least one glycosylation site, such as Fab, Fc, F(ab')2 or pFc'. With regard to the methods disclosed herein, antibodies may lack certain parts of antibodies and may be antibody fragments. In various aspects, antibody fragments contain glycosylation sites. In some aspects, the fragment is a "glycosylated Fc fragment" that contains at least a portion of an antibody Fc region that is post-translationally glycosylated in a eukaryotic cell.

[0094] The architecture of antibodies has been developed to produce an increasing number of alternative antibody forms spanning a molecular weight range of at least or about 12-150 kDa and a range of valencies (n) from monomers (n=1), dimers (n=2), and trimers (n=3) to tetramers (n=4) and potentially higher; such alternative antibody forms are referred to herein as "antibody protein products" or "antibody binding proteins."

[0095] The antibody protein product can be an antigen-binding form based on antibody fragments that retain full antigen binding ability, such as scFv, Fab, and VHH / VH. The smallest antigen-binding fragment that retains its complete antigen-binding site is the Fv fragment, which is composed entirely of the variable (V) region. A soluble, flexible amino acid peptide linker is used to connect the V region to the scFv (single-chain fragment variable) fragment to stabilize the molecule, or to add the constant (C) domain to the V region to produce a Fab fragment [antigen-binding fragment]. Both scFv and Fab are widely used fragments that can be easily produced in prokaryotic hosts. Other antibody protein products include scFv (ds-scFv) stabilized by disulfide bonds, single-chain Fab (scFab), and dimer and multimer antibody forms, such as bifunctional antibodies, trifunctional antibodies, and tetrafunctional antibodies, or mini-antibodies (miniAbs) composed of different forms of scFv connected to an oligomerization domain. The smallest fragment is VHH / VH of Camelidae heavy chain Ab and single domain Ab (sdAb). The building blocks most commonly used to generate novel antibody formats are single-chain variable (V) domain antibody fragments (scFv), which contain V domains (VH and VL domains) from heavy and light chains connected by a peptide linker of about 15 amino acid residues. Peptibodies or peptide-Fc fusions are yet another antibody protein product. The structure of a peptibody consists of a biologically active peptide grafted onto an Fc domain. Peptibodies are well described in the art. See, e.g., Shimamoto et al., mAbs 4(5):586-591 (2012).

[0096] Other antibody protein products include single-chain antibodies (SCAs), bifunctional antibodies, trifunctional antibodies; tetrafunctional antibodies; bispecific or trispecific antibodies, etc. Bispecific antibodies can be divided into five categories: BsIgG, additional IgG, BsAb fragments, bispecific fusion proteins and BsAb conjugates. See, for example, Spiess et al., Molecular Immunology 67 (2) Part A: 97-106 (2015).

[0097] In exemplary aspects, the recombinant glycosylated protein comprises any of these antibody protein products (e.g., scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g., bifunctional antibody, trifunctional antibody, tetrafunctional antibody), miniAb, peptibody, VHH / VH of camelid heavy chain antibody, sdAb, diabody; trifunctional antibody; tetrafunctional antibody; bispecific or trispecific antibody, BsIgG, additional IgG, BsAb fragment, bispecific fusion protein and BsAb conjugate) and comprises one or more N-glycosylation consensus sequences, optionally, one or more Fc polypeptides. In various aspects, the antibody protein product comprises a glycosylation site. In exemplary aspects, the antibody protein product can be a glycosylated Fc fragment conjugated to an antibody binding fragment ("glycosylated Fc fragment antibody product").

[0098] The recombinant glycosylated protein can be an antibody protein product in monomeric form or in polymeric, oligomeric or multimeric form. In certain embodiments where the antibody comprises two or more different antigen binding region fragments, the antibody is considered bispecific, trispecific or multispecific or bivalent, trivalent or multivalent, depending on the number of different antigenic determinants recognized and bound by the antibody.

[0099] Advantageously, these methods are not limited to the antigen specificity of the antibody. Thus, the antibody has any binding specificity for almost any antigen. In exemplary aspects, the antibody binds to a hormone, a growth factor, a cytokine, a cell surface receptor, or any ligand thereof. In exemplary aspects, the antibody binds to a protein expressed on the cell surface of an immune cell. In exemplary aspects, the antibody binds to a differentiation cluster molecule selected from the group consisting of: CD1a, CD1b, CD1c, CD1d, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11A, CD11B, CD11C, CDw12, CD13, CD14, CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61, CD62, CD63, CD64, CD65, CD66, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD77, CD78, CD79, CD80, CD81, CD82, CD83, CD84, CD85 D33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD 47. CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD 79α, CD79β, CD80, CD81, CD82, CD83, CDw84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99 , CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDw108, CD109, CD114, CD115, CD116, CD117, CD118, CD119, CD120 a, CD120b, CD121a, CDw121b, CD122, CD123, CD124, CD125, CD126, CD127, CDw128, CD129, CD130, CDw131, CD132, CD134, CD135, CDw136,CDw137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CD145, CD146, CD147, CD148, CD150, CD151, CD152, CD153, CD154, CD155, CD156, CD157, CD158a, CD158b, CD161, CD162, CD163, CD164, CD165, CD166, and CD182.

[0100] In exemplary aspects, the antibody is one of those described in: U.S. Pat. No. 7947809 and U.S. Patent Application Publication No. 20090041784 (glucagon receptor); U.S. Pat. No. 7939070, U.S. Pat. No. 7833527, U.S. Pat. No. 7767206, and U.S. Pat. No. 7786284 (IL-17 receptor A); U.S. Pat. No. 7872106 and U.S. Pat. No. 7592429 (sclerostin); U.S. Pat. No. 7871611, U.S. Pat. No. 7815907, U.S. Pat. No. 7037498, U.S. Pat. No. 7700742, and U.S. Patent Application Publication No. 20100255538 (IGF-1 receptor ); U.S. Patent No. 7868140 (B7RP1); U.S. Patent No. 7807159 and U.S. Patent Application Publication No. 20110091455 (myostatin); U.S. Patent No. 7736644, U.S. Patent No. 7628986, U.S. Patent No. 7524496 and U.S. Patent Application Publication No. 20100111979 (deletion mutant of epidermal growth factor receptor); U.S. Patent No. 7728110 (SARS coronavirus); U.S. Patent No. 7718776 and U.S. Patent Application Publication No. 20100209435 (OPGL); U.S. Patent No. 7658924 and U.S. Patent No. 7521053 (angiopoietin-2); U.S. U.S. Patent No. 7601818, U.S. Patent No. 7795413, U.S. Patent Application Publication No. 20090155274, U.S. Patent Application Publication No. 20110040076 (NGF); U.S. Patent No. 7579186 (TGF-β type II receptor); U.S. Patent No. 7541438 (connective tissue growth factor); U.S. Patent No. 7438910 (IL1-R1); U.S. Patent No. 7423128 (properdin); U.S. Patent No. 7411057, U.S. Patent No. 7824679, U.S. Patent No. 7109003, U.S. Patent No. 6682736, U.S. Patent No. 7132281 and U.S. Patent No. 7807797 (CT LA-4); U.S. Patent No. 7084257, U.S. Patent No. 7790859, U.S. Patent No. 7335743, U.S. Patent No. 7084257, and U.S. Patent Application Publication No. 20110045537 (interferon-γ); U.S. Patent No. 7932372 (MAdCAM); U.S. Patent No. 7906625, U.S. Patent Application Publication No. 20080292639, and U.S. Patent Application Publication No. 20110044986 (amyloid); U.S. Patent No. 7815907 and U.S. Patent No. 7700742 (insulin-like growth factor I); U.S. Patent No. 7566772 and U.S. Patent No. 7964193 (interleukin-1β);U.S. Patent No. 7563442, U.S. Patent No. 7288251, U.S. Patent No. 7338660, U.S. Patent No. 7626012, U.S. Patent No. 7618633, and U.S. Patent Application Publication No. 20100098694 (CD40); U.S. Patent No. 7498420 (c-Met); U.S. Patent No. 7326414, U.S. Patent No. 7592430, and U.S. Patent No. 7728113 (M-CSF); U.S. Patent No. 6924360, U.S. Patent No. 7067131, and U.S. Patent No. 7090844 (MUC18); U.S. Patent No. 6235883, U.S. Patent No. 7807798, and U.S. Patent Application Publication No. 201003 05307 (epidermal growth factor receptor); U.S. Patent No. 6716587, U.S. Patent No. 7872113, U.S. Patent No. 7465450, U.S. Patent No. 7186809, U.S. Patent No. 7317090 and U.S. Patent No. 7638606 (interleukin-4 receptor); U.S. Patent Application Publication No. 20110135657 (β-KLOTHO); U.S. Patent No. 7887799 and U.S. Patent No. 7879323 (fibroblast growth factor-like polypeptide); U.S. Patent No. 7867494 (IgE); U.S. Patent Application Publication No. 20100254975 (α-4β-7); U.S. Patent Application Publication No. 20100197005 and U.S. Patent No. No. 7537762 (activin receptor-like kinase 1); U.S. Patent No. 7585500 and U.S. Patent Application Publication No. 20100047253 (IL-13); U.S. Patent Application Publication No. 20090263383 and U.S. Patent No. 7449555 (CD148); U.S. Patent Application Publication No. 20090234106 (activin A); U.S. Patent Application Publication No. 20090226447 (angiopoietin-1 and angiopoietin-2); U.S. Patent Application Publication No. 20090191212 (angiopoietin-2); U.S. Patent Application Publication No. 20090155164 (C-FMS); U.S. Patent No. 7537762 (activin receptor-like kinase -1); U.S. Patent No. 7371381 (galanin); U.S. Patent Application Publication No. 20070196376 (insulin-like growth factor); U.S. Patent No. 7267960 and U.S. Patent No. 7741115 (LDCAM); U.S. Patent No. 7265212 (CD45RB); U.S. Patent No. 7709611, U.S. Patent Application Publication No. 20060127393 and U.S. Patent Application Publication No. 20100040619 (DKK1); U.S. Patent No. 7807795, U.S. Patent Application Publication No. 20030103978 and U.S. Patent No. 7923008 (osteoprotegerin); U.S. Patent Application Publication No. 20090208489 (OV064);U.S. Patent Application Publication No. 20080286284 (PSMA); U.S. Patent No. 7888482, U.S. Patent Application Publication No. 20110165171 and U.S. Patent Application Publication No. 20110059063 (PAR2); U.S. Patent Application Publication No. 20110150888 (hepaticidal peptide); U.S. Patent No. 7939640 (B7L-1); U.S. Patent No. 7915391 (c-Kit); U.S. Patent No. 7807796, U.S. Patent No. 7193058 and U.S. Patent No. 7807796 U.S. Patent No. 7427669 (ULBP); U.S. Patent No. 7786271, U.S. Patent No. 7304144 and U.S. Patent Application Publication No. 20090238823 (TSLP); U.S. Patent No. 7767793 (SIGIRR); U.S. Patent No. 7705130 (HER-3); U.S. Patent No. 7704501 (spinocerebellar ataxia-1-like polypeptide); U.S. Patent No. 7695948 and U.S. Patent No. 7199224 (TNF-α converting enzyme); U.S. Patent No. Application Publication No. 20090234106 (activin A); U.S. Patent Application Publication No. 20090214559 and U.S. Patent No. 7438910 (IL1-R1); U.S. Patent No. 7579186 (TGF-β type II receptor); U.S. Patent No. 7569387 (TNF receptor-like molecule); U.S. Patent No. 7541438 (connective tissue growth factor); U.S. Patent No. 7521048 (TRAIL receptor-2); U.S. Patent No. 6319499, U.S. Patent No. 7081 523 and U.S. Patent Application Publication No. 20080182976 (erythropoietin receptor); U.S. Patent Application Publication No. 20080166352 and U.S. Patent No. 7435796 (B7RP1); U.S. Patent No. 7423128 (properdin); U.S. Patent No. 7422742 and U.S. Patent No. 7141653 (interleukin-5); U.S. Patent No. 6740522 and U.S. Patent No. 7411050 (RANKL); U.S. Patent No. 7378091 (carbonic anhydrase IX (CA IX) tumor antigens); U.S. Patent No. 7318925 and U.S. Patent No. 7288253 (parathyroid hormone); U.S. Patent No. 7285269 (TNF); U.S. Patent No. 6692740 and U.S. Patent No. 7270817 (ACPL); U.S. Patent No. 7202343 (monocyte chemoattractant protein-1); U.S. Patent No. 7144731 (SCF); U.S. Patent No. 6355779 and U.S. Patent No. 7138500 (4-1BB); U.S. Patent No. 7135174 (PDGFD); U.S. Patent No. 6630143 and U.S. Patent No. 7045128 (Flt-3 ligand); U.S. Patent No. 6849450 (metalloproteinase inhibitors); U.S. Patent No. 6596852 (LERK-5);U.S. Patent No. 6232447 (LERK-6); U.S. Patent No. 6500429 (brain-derived neurotrophic factor); U.S. Patent No. 6184359 (epithelial-derived T-cell factor); U.S. Patent No. 6143874 (neurotrophic factor NNT-1); U.S. Patent Application Publication No. 20110027287 (proprotein convertase subtilisin KEXIN type 9 (PCSK9)); U.S. Patent Application Publication No. 20110014201 (IL-18 receptor); and U.S. Patent Application Publication No. 20090155164 (C-FMS). The above patents and published patent applications are incorporated herein by reference in their entirety for the purpose of their variable domain polypeptides, variable domain encoding nucleic acids, host cells, vectors, methods of preparing polypeptides encoding said variable domains, pharmaceutical compositions, and methods of treating diseases associated with the corresponding targets of variable domain-containing antigen binding proteins or antibodies. ;

[0101] In an exemplary embodiment, the antibody is one of the following: muromonab-CD3 (trade name Orthoclone products marketed under the trade names of products), rituximab (available under the trade name products marketed under the trade name products marketed under the trade name products marketed under the trade name products), infliximab (marketed under the trade name products), trastuzumab (marketed under the trade name Alemtuzumab (marketed under the trade name Adalimumab (marketed under the trade name Tositumomab-I131 (marketed under the trade name products sold on the market), efalizumab (available under the trade name products), cetuximab (marketed under the trade name Ibritumomab (marketed under the trade name Omalizumab (marketed under the trade name products marketed under the trade name of products sold on the market), natalizumab (available under the trade name products marketed under the trade name products marketed under the trade name products marketed under the trade name products marketed under the trade name products marketed under the trade name Canakinumab (marketed under the trade name products marketed under the trade name products sold on the market), Ustekinumab (marketed under the trade name products marketed under the trade name ofatumumab (marketed under the trade name products marketed under the trade name Belimumab (marketed under the trade name products sold under the trade names of products) and pertuzumab (available under the trade name Commercially available products). In an exemplary embodiment, the antibody is one of: an anti-TNFα antibody, such as adalimumab, infliximab, etanercept, golimumab, and bercezolizumab; an anti-IL1β antibody, such as canakinumab; an anti-IL12 / 23 (p40) antibody, such as ustekinumab and briakinumab; and an anti-IL2R antibody, such as daclizumab. In an exemplary aspect, the antibody binds to a tumor-associated antigen and is an anti-cancer antibody. Examples of suitable anti-cancer antibodies include, but are not limited to, anti-BAFF antibodies, such as belimumab; anti-CD20 antibodies, such as rituximab; anti-CD22 antibodies, such as epratuzumab; anti-CD25 antibodies, such as daclizumab; anti-CD30 antibodies, such as iratumumab; anti-CD33 antibodies, such as gemtuzumab; anti-CD52 antibodies, such as alemtuzumab; anti-CD152 antibodies, such as ipilimumab; anti-EGFR antibodies, such as cetuximab; anti-HER2 antibodies, such as trastuzumab and pertuzumab; anti-IL6 antibodies, such as siltuximab; and anti-VEGF antibodies, such as bevacizumab; anti-IL6 receptor antibodies, such as tocilizumab. In an exemplary aspect, the tumor-associated antigen is CD20 and the antibody is an anti-CD20 antibody. In an exemplary aspect, the tumor-associated antigen comprises SEQ ID NO: 3. In an exemplary case, the antibody comprises the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2. In an exemplary aspect, the antibody is an anti-CD20 antibody, for example, an anti-CD20 monoclonal antibody. In an alternative aspect, the IgG1 antibody is rituximab or a biosimilar thereof. The term rituximab refers to an IgG1κ chimeric mouse / human monoclonal antibody that binds to the CD20 antigen (see CAS No.: 174722-31-7; Drug Bank-DB00073; Kyoto Encyclopedia of Genes and Genomes, KEGG entry D02994). In an exemplary aspect, the antibody comprises a light chain comprising CDR1, CDR2, and CDR3 listed in Table A. In an exemplary aspect, the antibody comprises a heavy chain comprising CDR1, CDR2, and CDR3 listed in Table A. In various cases, the antibody comprises the VH and VL listed in Table A or comprises VH-IgG1 and VL-IgGκ sequences.

[0102] Table A: Rituximab amino acid sequence

[0103]

[0104]

[0105] LC, light chain; HC, heavy chain; VL, variable light chain; VH, variable heavy chain.

[0106] In exemplary aspects, the antibody is an anti-EGFR antibody, for example, an anti-HER2 monoclonal antibody. In exemplary aspects, the antibody is trastuzumab or a biosimilar thereof. The term trastuzumab refers to an IgG1κ humanized monoclonal antibody (see CAS No.: 180288-69-1; Drug Bank-DB00072; Kyoto Encyclopedia of Genes and Genomes (KEGG) entry D03257) that binds to HER2 / neu antigen. In exemplary aspects, the antibody comprises a light chain containing CDR1, CDR2, and CDR3 listed in Table B. In exemplary aspects, the antibody comprises a heavy chain containing CDR1, CDR2, and CDR3 listed in Table B. In different cases, the antibody comprises VH and VL listed in Table B or comprises VH-IgG1 and VL-IgGκ sequences.

[0107] Table B: Trastuzumab amino acid sequence

[0108]

[0109]

[0110] LC, light chain; HC, heavy chain; VL, variable light chain; VH, variable heavy chain.

[0111] Additional Steps

[0112] In various aspects, the methods disclosed herein include additional steps. For example, in some aspects, the method includes one or more upstream steps or downstream steps related to the production, purification and formulation of recombinant glycosylated proteins. In an exemplary embodiment, the method includes a step for generating a host cell expressing a recombinant glycosylated protein (e.g., an antibody or an antibody-binding protein). In some aspects, the host cell is a prokaryotic host cell, such as Escherichia coli (E. coli) or Bacillus subtilis (Bacillus subtilis), or in some aspects the host cell is a eukaryotic host cell, such as a yeast cell, a filamentous fungal cell, a protozoan cell, an insect cell or a mammalian cell (e.g., a CHO cell). Such host cells are described in the art. See, for example, Frenzel et al., Front Immunol [Immunology Frontier] 4: 217 (2013) and the "Cells [cell]" section herein. For example, in some cases, the method includes introducing into the host cell a vector comprising a nucleic acid comprising a nucleotide sequence encoding a recombinant glycosylated protein or a polypeptide chain thereof.

[0113] In exemplary embodiments, the methods disclosed herein include steps for isolating and / or purifying recombinant glycosylated proteins (e.g., recombinant antibodies) from a culture. In exemplary aspects, the method includes one or more chromatography steps, including, but not limited to, affinity chromatography (e.g., protein A affinity chromatography), ion exchange chromatography, and / or hydrophobic interaction chromatography. In exemplary aspects, the method includes steps for producing a crystalline biomolecule from a solution containing a recombinant glycosylated protein.

[0114] In various aspects, the methods of the present disclosure include one or more steps for preparing a composition, which in some aspects includes a pharmaceutical composition comprising a purified recombinant glycosylated protein. Such compositions are discussed below.

[0115] Composition

[0116] Compositions comprising recombinant glycosylated proteins are provided herein. In an exemplary embodiment, the composition is prepared by an inventive method for producing a recombinant glycosylated protein composition described herein. In an exemplary aspect, the recombinant glycosylated protein is an antibody. Therefore, antibody compositions are provided herein. In an exemplary embodiment, the antibody composition comprises different glycoforms of the antibody. In an exemplary embodiment, the antibody composition comprises a TAF glycoform, an HM glycoform and / or a defucosylated glycoform of the antibody. Compositions comprising antibody fragments or antibody protein products are also provided. In various aspects, the antibody fragment, the antibody protein product, the glycosylated Fc fragment or the glycosylated Fc fragment antibody product comprises a glycosylation site. In an exemplary embodiment, the antibody composition is produced by a method comprising maintaining a cell having glycosylation capability in a cell culture medium comprising fucose, wherein the fucose is present in the culture medium at a concentration between about 0.17 g / L and about 1.0 g / L. In an exemplary embodiment, the antibody composition is produced by a method comprising maintaining a glycosylation-competent cell in a cell culture medium comprising fucose, wherein the fucose is present in the culture medium at a concentration between about 0.1 g / L and about 1.0 g / L, and wherein the glycosylation-competent cell has not been genetically modified to alter the activity of an enzyme of a de novo pathway or a salvage pathway. In an exemplary embodiment, the antibody composition is produced by a method comprising maintaining a glycosylation-competent cell in a cell culture medium comprising fucose and glucose, wherein the fucose is present in the culture medium at a concentration of about 0.1 g / L to about 1.0 g / L, and glucose is added to the cell culture medium according to a glucose feeding regimen that achieves an average glucose concentration of about 10 g / L or less. In an exemplary embodiment, the antibody composition is produced after implementing a method for regulating the level of TAF glycans, defucosylated glycans, or high mannose glycans of an antibody composition produced by a glycosylation-competent cell. In an exemplary aspect, the antibody composition is produced after performing a method for modulating the level of TAF glycans, the method comprising (A) adding fucose to a cell culture medium comprising cells having glycosylation capability to achieve a fucose concentration of about 0.1 g / L to about 1.0 g / L, thereby reducing the level of TAF glycans; (B) adding glucose to a cell culture medium comprising cells having glycosylation capability to achieve a glucose concentration of less than about 10 g / L, thereby increasing the level of TAF glycans; or (C) both (A) and (B).In exemplary aspects, the antibody composition is produced after implementing a method for regulating the level of defucosylated glycans of an antibody composition produced by a cell having glycosylation capability, the method comprising (A) adding fucose to a cell culture medium comprising cells having glycosylation capability to achieve a fucose concentration of about 0.1 g / L to about 1.0 g / L, thereby reducing the level of defucosylated glycans; (B) adding glucose to a cell culture medium comprising cells having glycosylation capability to achieve a glucose concentration of less than or about 10 g / L, thereby increasing the level of defucosylated glycans; or (C) both (A) and (B). In exemplary cases, the antibody composition is produced after implementing a method for regulating the level of high mannose glycans of an antibody composition produced by a cell having glycosylation capability, the method comprising adding glucose to a cell culture medium comprising cells having glycosylation capability to achieve a glucose concentration of less than or about 10 g / L, thereby increasing the level of high mannose glycans.

[0117] In exemplary aspects, less than or about 50% (e.g., less than or about 40%, less than or about 30%, less than or about 25%, less than or about 20%, less than or about 15%) of the recombinant glycosylated proteins in the composition are TAF glycoforms. In exemplary aspects, less than about 10% (e.g., less than or about 9%, less than or about 8%, less than or about 7%, less than or about 6%, less than or about 5%, less than or about 4%, less than or about 3%, less than or about 2%) of the recombinant glycosylated proteins in the composition are TAF glycoforms. In exemplary aspects, about 4% to about 10% of the recombinant glycosylated proteins in the composition are TAF glycoforms. In exemplary aspects, about 2% to about 6% of the recombinant glycosylated proteins in the composition are TAF glycoforms. In exemplary aspects, about 2.5% to about 5% of the recombinant glycosylated proteins in the composition are TAF glycoforms. In exemplary aspects, less than or about 4% of the recombinant glycosylated proteins in the composition are TAF glycoforms. In exemplary aspects, less than or about 4% and greater than or about 2% of the recombinant glycosylated proteins in the composition are TAF glycoforms.

[0118] In exemplary aspects, the compositions of the present disclosure have a glycoform profile of less than or about 50% (e.g., less than or about 40%, less than or about 30%, less than or about 25%, less than or about 20%, less than or about 15%) TAF glycoforms. In exemplary aspects, the compositions of the present disclosure have a glycoform profile of less than or about 10% (e.g., less than or about 9%, less than or about 8%, less than or about 7%, less than or about 6%, less than or about 5%, less than or about 4%, less than or about 3%, less than or about 2%) TAF glycoforms. In exemplary aspects, the compositions of the present disclosure have a glycoform profile comprising about 4% to about 10% TAF glycoforms. In exemplary aspects, the compositions of the present disclosure have a glycoform profile of about 2% to about 6% TAF glycoforms. In exemplary aspects, the compositions of the present disclosure have a glycoform profile of about 2.5% to about 5% TAF glycoforms. In exemplary aspects, the compositions of the present disclosure have a glycoform profile of less than or about 4% TAF glycoforms. In exemplary aspects, the compositions of the disclosure have a glycoform profile of less than or about 4% and greater than or about 2% TAF glycoforms.

[0119] In exemplary aspects, less than or about 5% of the recombinant glycosylated proteins (e.g., antibodies or antibody binding proteins) in the composition are defucosylated glycoforms. In exemplary aspects, less than or about 4% of the recombinant glycosylated proteins (e.g., antibodies or antibody binding proteins) in the composition are defucosylated glycoforms. In exemplary aspects, less than or about 3.5% of the recombinant glycosylated proteins (e.g., antibodies or antibody binding proteins) in the composition are defucosylated glycoforms. In exemplary aspects, about 0.8% to about 2.8% of the recombinant glycosylated proteins in the composition are defucosylated glycoforms. In some aspects, the level of defucosylated glycans in the antibody composition is about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, or about 2.8%.

[0120] In exemplary aspects, less than or about 5% of the recombinant glycosylated proteins (e.g., antibodies or antibody binding proteins) in the composition are high mannose glycoforms. In exemplary aspects, less than or about 4% of the recombinant glycosylated proteins (e.g., antibodies or antibody binding proteins) in the composition are high mannose glycoforms. In exemplary aspects, less than or about 3.5% of the recombinant glycosylated proteins (e.g., antibodies or antibody binding proteins) in the composition are high mannose glycoforms. In exemplary aspects, about 0.7% to about 3.0% of the recombinant glycosylated proteins in the composition are high mannose glycoforms. In some aspects, the level of high mannose glycans in the antibody composition is about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, or about 3.0%.

[0121] In an exemplary embodiment, the composition is combined with a pharmaceutically acceptable carrier, diluent or excipient. Thus, provided herein are pharmaceutical compositions comprising a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) described herein and a pharmaceutically acceptable carrier, diluent or excipient. As used herein, the term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents.

[0122] Cell culture medium

[0123] Provided herein is a cell culture medium comprising: (a) a cell capable of glycosylation, the cell capable of glycosylation comprising an exogenous nucleotide sequence encoding an antibody; and (b) a culture medium comprising fucose at a concentration of about 0.1 g / L to about 1.0 g / L or about 0.17 g / L to about 1.0 g / L. The cell capable of glycosylation may be any cell described herein. In an exemplary case, the cell capable of glycosylation has not been genetically modified to alter the activity of an enzyme of a de novo pathway or a salvage pathway, and optionally, wherein the cell capable of glycosylation has not been genetically modified to knock out a gene encoding GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase. In an exemplary embodiment, the culture medium further comprises glucose. In some aspects, the culture medium comprises glucose at a concentration of less than about 10 g / L or less than about 9 g / L, such as about 6 g / L or less or about 0.5 g / L to about 4 g / L. In some aspects, the culture medium comprises fucose. In exemplary aspects, the pH of the culture medium is about 6.85 to about 7.05, optionally, about 6.90 to about 7.00. In exemplary cases, the cells with glycosylation capability have not been genetically modified to change the activity of enzymes of the de novo pathway or the salvage pathway. For example, the cells with glycosylation capability have not been genetically modified to knock out the gene encoding GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase. In exemplary aspects, the antibody is an IgG antibody, optionally, an IgG1 antibody. In exemplary aspects, the IgG1 antibody is specific for a tumor-associated antigen (e.g., CD20). In exemplary aspects, the cell culture medium does not contain mannose.

[0124] Adjustment method

[0125] Further provided herein are methods for altering or regulating the level of TAF glycans of a recombinant glycosylated protein (e.g., an antibody composition or an antibody binding protein composition) produced by a cell culture medium having glycosylation capability. In exemplary aspects, the method comprises (A) adding fucose to a cell culture medium containing cells having glycosylation capability to achieve a fucose concentration of about 0.1 g / L to about 1.0 g / L, thereby reducing the level of TAF glycans; (B) adding glucose to a cell culture medium containing cells having glycosylation capability to achieve a glucose concentration of less than about 10 g / L, thereby increasing the level of TAF glycans; or (C) both (A) and (B).

[0126] The present disclosure also provides a method for regulating the level of defucosylated glycans of a recombinant glycosylated protein (e.g., an antibody composition or an antibody-binding protein composition) produced by a cell having glycosylation capability. In an exemplary aspect, the method comprises (A) adding fucose to a cell culture medium containing cells having glycosylation capability to achieve a fucose concentration of about 0.1 g / L to about 1.0 g / L, thereby reducing the level of defucosylated glycans; or (B) adding glucose to a cell culture medium containing cells having glycosylation capability to achieve a glucose concentration of less than about 10 g / L, thereby increasing the level of defucosylated glycans; or (C) both (A) and (B).

[0127] Also provided are methods for regulating the level of high mannose glycans of a recombinant glycosylated protein (e.g., an antibody composition or an antibody binding protein composition) produced by a cell having glycosylation capability. In an exemplary embodiment, these methods include adding glucose to a cell culture medium containing cells having glycosylation capability to achieve a glucose concentration of less than about 10 g / L, thereby increasing the level of HM glycans.

[0128] Thus, in some exemplary embodiments, the methods of the present disclosure relate to increasing the level of TAF glycans, HM glycans, or defucosylated glycans of a protein (e.g., an antibody) produced by cells in a cell culture. In exemplary aspects, the level of the HM glycoform of the recombinant glycosylated protein is increased relative to a control cell culture. In exemplary aspects, the level of one or more of Man5, Man6, Man7, Man8, and / or Man9 of the recombinant glycosylated protein is increased relative to a control cell culture. In exemplary aspects, the level of defucosylated glycoforms of the recombinant glycosylated protein is increased relative to a control cell culture. In exemplary aspects, the level of one or more of A1G0, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5 of the recombinant glycosylated protein is increased relative to a control cell culture. In exemplary aspects, the level of one or more of A1G1a, G0[H3N4], G0[H4N4], G0[H5N4], and FO-N[H3N3] of the recombinant glycosylated protein is increased relative to a control cell culture. In some aspects, the increase is an increase relative to a control cell culture, as determined by hydrophilic interaction liquid chromatography (HILIC). In some aspects, the increase is an increase relative to a control cell culture, as determined by methods known to those skilled in the art.

[0129] In some aspects, relative to control cell cultures, the methods disclosed herein increase TAF, HM or afuco glycoform levels to any degree or level. For example, in some aspects, relative to control cell cultures, the increase provided by the methods disclosed herein is at least or about 1% to about 10% increase (e.g., at least or about 1% increase, at least or about 2% increase, at least or about 3% increase, at least or about 4% increase, at least or about 5% increase, at least or about 6% increase, at least or about 7% increase, at least or about 8% increase, at least or about 9% increase, at least or about 9.5% increase, at least or about 9.8% increase, at least or about 10% increase). In an exemplary embodiment, relative to control cell cultures, the increase provided by the methods disclosed herein exceeds 100%, for example 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or even 1000%. In an exemplary embodiment, the level of TAF, HM or afuco glycoform of the protein increases at least about 1.5 times relative to the control cell culture. In an exemplary embodiment, the level of TAF, HM or afuco glycoform of the protein increases at least about 2 times relative to the control cell culture. In an exemplary embodiment, the level of TAF, HM or afuco glycoform of the protein increases at least about 3 times relative to the control cell culture. In an exemplary embodiment, the level of TAF, HM or afuco glycoform of the protein increases at least about 4 times or about 5 times relative to the control cell culture.

[0130] In exemplary aspects, an increase in the level of a TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable as early as day 1 after the change in fucose and / or glucose concentration. In exemplary aspects, an increase in the level of a TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable as early as day 2 after the change. In exemplary aspects, an increase in the level of a TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable as early as day 3 after the change. In exemplary aspects, an increase in the level of a TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable as early as day 4 after the change. In exemplary aspects, an increase in the level of a TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable after about day 5 after the change. In exemplary aspects, an increase in the level of a TAF glycoform of a protein is observed or observable or detectable or detectable when the recombinant glycosylated protein is harvested from a cell culture.

[0131] In exemplary aspects, the level of TAF glycoforms of the recombinant glycosylated protein is observed to increase for longer than about day 4, day 5, or day 6 or more of cell culture. In exemplary aspects, the level of TAF glycoforms of the recombinant glycosylated protein is observed to increase for 7, 8, 9, 10, 11, or 12 days or more (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 6 months, or about 1 year) of cell culture (after seeding). In exemplary aspects, the level of TAF glycoforms of the protein is observed to increase when the protein is harvested from the cell culture.

[0132] In other aspects, the methods disclosed herein relate to reducing the level of TAF glycoforms of proteins produced by cells in cell culture. In exemplary aspects, the level of HM glycoforms of recombinant glycosylated proteins is reduced relative to control cell cultures. In exemplary aspects, the level of one or more of Man5, Man6, Man7, Man8 and / or Man9 of recombinant glycosylated proteins is reduced relative to control cell cultures. In exemplary aspects, the level of defucosylated glycoforms of recombinant glycosylated proteins is reduced relative to control cell cultures. In exemplary aspects, the level of one or more of A1G0, A2G0, A2G1a, A2G1b, A2G2 and A1G1M5 of recombinant glycosylated proteins is reduced relative to control cell cultures. In exemplary aspects, the level of one or more of A1G1a, G0[H3N4], G0[H4N4], G0[H5N4] and FO-N[H3N3] of recombinant glycosylated proteins is reduced relative to control cell cultures. In exemplary aspects, the method is a method of reducing the level of TAF glycoforms by about 1% to about 4%, and the method comprises maintaining glycosylation competent cells in a first cell culture medium and increasing the fucose concentration to about 0.1 g / L to about 1.0 g / L. In some aspects, the reduction is a reduction relative to a control cell culture, as determined by HILIC. In some aspects, the reduction is a reduction relative to a control cell culture, as determined by methods known to those skilled in the art.

[0133] In some aspects, the methods of the present disclosure reduce one or more levels of TAF, HM or afuco glycoforms to any degree or level relative to control cell cultures. For example, relative to the levels of control cell cultures, the reduction provided by the methods of the present disclosure is a reduction of at least or about 0.1% to about 1% (e.g., a reduction of at least or about 0.1%, a reduction of at least or about 0.2%, a reduction of at least or about 0.3%, a reduction of at least or about 0.4%, a reduction of at least or about 0.5%, a reduction of at least or about 0.6%, a reduction of at least or about 0.7%, a reduction of at least or about 0.8%, a reduction of at least or about 0.9%, a reduction of at least or about 0.95%, a reduction of at least or about 0.98%, a reduction of at least or about 1.0%). In exemplary embodiments, the reduction provided by the methods of the present disclosure is greater than about 100%, such as about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or even about 1000%, relative to the level of the control cell culture. In exemplary embodiments, the level of TAF, HM, or afuco glycoforms of the protein is reduced by at least or about 1.5 times relative to the control cell culture. In exemplary embodiments, the level of TAF, HM, or afuco glycoforms of the protein is reduced by at least about 2 times relative to the control cell culture. In exemplary embodiments, the level of TAF, HM, or afuco glycoforms of the protein is reduced by at least about 3 times relative to the control cell culture. In exemplary embodiments, the level of TAF, HM, or afuco glycoforms of the protein is reduced by at least about 4 times or at least about 5 times relative to the control cell culture.

[0134] In exemplary aspects, a reduction in the level of a TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable as early as about day 1 after inoculation. In exemplary aspects, a reduction in the level of a TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable as early as about day 2 after inoculation. In exemplary aspects, a reduction in the level of a TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable as early as about day 3 after inoculation. In exemplary aspects, a reduction in the level of a TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable as early as about day 4 after inoculation. In exemplary aspects, a reduction in the level of a TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable after about day 5 after inoculation. In exemplary aspects, a reduction in the level of a TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable at about the time the protein is harvested from the cell culture.

[0135] In exemplary aspects, the level of a TAF glycoform of a protein is observed to be reduced for longer than about day 4, about day 5, or about day 6 of cell culture or beyond the initial cell culture period. In exemplary aspects, the level of a TAF glycoform of a protein is observed to be reduced for about 7, about 8, about 9, about 10, about 11, or about 12 days or longer (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 6 months, or about 1 year) of cell culture (post-seeding). In exemplary aspects, the level of a TAF glycoform of a protein is observed to be reduced at about the time the protein is harvested from the cell culture.

[0136] With respect to the methods of the present disclosure, the regulation, increase or decrease achieved by such methods is relative to a "control" or "control cell culture". In exemplary aspects, the control is the level of the TAF glycoform of the protein when the steps of the present method are not performed. In exemplary aspects, the control is the level of the TAF glycoform of the protein when a known method of recombinant production is performed. In exemplary aspects, the control is the level of the TAF glycoform when a known glucose or fucose concentration is maintained during recombinant production. As used herein, the term "control cell culture" means a cell culture maintained in the same manner as a cell culture that performs the steps of the present method (e.g., cell culture of the disclosed method) except for the fucose / glucose concentration. In exemplary aspects, a control cell culture is a cell culture maintained under known operating or standard parameters (including control fucose / glucose concentration). As used herein, the term "control fucose concentration" or "control glucose concentration" may refer to a known operating fucose / glucose concentration, such as the fucose / glucose concentration of a cell culture maintained at a first time point or at a time point before performing the method of the present disclosure. In exemplary aspects, the control fucose / glucose concentration is the fucose / glucose concentration of a cell culture whose TAF level is known or determined.

[0137] In exemplary aspects of regulating the methods of the present disclosure, the glycosylation competent cell is not genetically modified to alter the activity of an enzyme of the de novo pathway or the salvage pathway. Optionally, the glycosylation competent cell is not genetically modified to knock out a gene encoding GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase.

[0138] In exemplary aspects, after performing the methods of the disclosure, the level of TAF glycans in the antibody composition is less than about 10%, eg, about 2% to about 6%, about 2% to about 5%, or about 2% to about 4%.

[0139] In exemplary aspects, after performing the methods of the disclosure, the level of high mannose glycans in the antibody composition is less than about 3.5%, optionally, about 0.7% to about 3.0%.

[0140] In exemplary aspects, the level of defucosylated glycans in the antibody composition after performing the methods of the disclosure is less than about 3.5%, optionally, about 0.8% to about 2.8%.

[0141] With respect to the conditioning methods described herein comprising adding fucose, the final fucose concentration of the cell culture medium is in various aspects from about 0.17 g / L to about 1.0 g / L, or from about 0.2 g / L to about 0.5 g / L. However, any fucose concentration described herein is contemplated.

[0142] With respect to the regulation methods described herein comprising adding glucose to a cell culture medium, in some aspects, glucose is added according to a glucose feeding regimen that achieves an average glucose concentration of about 10 g / L or less or about 9 g / L or less. In some aspects, the average glucose concentration is less than about 6.0 g / L, optionally, less than about 4.0 g / L. In some cases, the average glucose concentration is based on the fucose concentration in the cell culture medium. For example, the average glucose concentration can be calculated based on Formula I:

[0143] T=3.354-1.388F+0.111G+[F-0.4375]x[1.9527(F-0.4375)]

[0144] Formula I

[0145] Wherein T is the target TAF glycan % and is 2.5% to about 6%, about 2.75% to about 5.5%, or about 3% to about 5%, F is the concentration of fucose in the culture medium (g / L), and G is the average glucose concentration (g / L).

[0146] The present disclosure further provides methods for modulating (reducing or increasing) the level of defucosylated glycans of a recombinant glycosylated protein composition (eg, an antibody composition or an antibody binding protein composition) produced by a cell having glycosylation competence. In exemplary embodiments, the method comprises lowering the pH of a cell culture medium by about 0.03 to about 1.2 (e.g., 0.05 to about 1.0) to reduce the level of defucosylated glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) by about 0.5% to about 2% (e.g., 0.5% to about 1.5%, 0.5% to about 1.0%, 1.0% to about 2%, 1.5% to about 2.0%) or increasing the pH of a cell culture medium by about 0.03 to about 1.2 (e.g., 0.05 to about 1.0) to increase the level of defucosylated glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) by about 0.5% to about 2% (e.g., 0.5% to about 1.5%, 0.5% to about 1.0%, 1.0% to about 2%, 1.5% to about 2.0%). In exemplary aspects, the method comprises lowering the pH of the cell culture medium by about 0.05 to about 1.2 (e.g., 0.06, 0.07, 0.08, 0.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.20) to reduce the level of defucosylated glycans of the recombinant glycosylated protein composition (e.g., antibody composition or antibody binding protein composition) by about 1% to about 2% (e.g., 1.0% to 1.5%, 1.5% to 1.6%, 1.7% to 1.8%, 1.9% to 2.10%, 1.11% to 2.12%, 1.13% to 1.14%, 1.15% to 1.16%, 1.17% to 1.18%, 1.19% to 1.20). 2.0%), or increasing the pH of the cell culture medium by about 0.05 to about 1.2 (e.g., 0.06, 0.07, 0.08, 0.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.20) to increase the level of defucosylated glycans of the recombinant glycosylated protein composition (e.g., antibody composition or antibody binding protein composition) by about 1% to about 2% (e.g., 1.0% to 1.5%, 1.5% to 2.0%).In various cases, the method comprises lowering the pH of the cell culture medium by about 0.03 to about 0.07 (e.g., 0.03, 0.04, 0.05, 0.06, 0.07) to reduce the level of defucosylated glycans of the recombinant glycosylated protein composition (e.g., antibody composition or antibody binding protein composition) by about 0.5% to about 1.1% (e.g., 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 1.0%, 1.01%, 1.02%, 1.03%, 1.04%, 1.05%, 1.06%, 1.07%, 1.08%, 1.09%, 1.10%, 1.11%, 1.12%, 1.13%, 1.14%, 1.15%, 1.16%, 1.17%, 1.18%, 1.19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 50%, 51%, 52%, 5 1.10%) or increasing the pH of the cell culture medium by about 0.03 to about 0.07 (e.g., 0.03, 0.04, 0.05, 0.06, 0.07) to increase the level of defucosylated glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) by about 0.5% to about 1.1% (e.g., 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 1.0%, 1.01%, 1.02%, 1.03%, 1.04%, 1.05%, 1.06%, 1.07%, 1.08%, 1.09%, 1.10%).

[0147] Also provided herein is a method for reducing the level of defucosylated glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by a cell having glycosylation capability by about 1% to about 2%. In an exemplary embodiment, the method comprises reducing the pH of the cell culture medium by about 0.05 to about 1.2. Optionally, the method comprises reducing the pH by about 0.05 to about 0.07 to reduce about 1% of the defucosylated glycans; or reducing the pH by about 0.09 to about 1.2 to reduce more than about 1.5% of the defucosylated glycans. In various aspects, the method comprises culturing cells at a pH between about 7.10 and about 7.20, optionally between about 7.12 and about 7.19.

[0148] In addition, a method is provided for reducing the level of defucosylated glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) produced by a cell having glycosylation capability by about 0.5% to about 1.1%. In an exemplary embodiment, the method includes reducing the pH of the cell culture medium by about 0.03-0.07. In various aspects, the method includes reducing the pH by about 0.03 to about 0.06, reducing the defucosylated glycans by about 0.8%. In some aspects, the method includes reducing the pH by about 0.05 to about 0.07, reducing the defucosylated glycans by about 1%. In various cases, the method includes culturing cells at a pH between about 7.05 and about 7.15, optionally between about 7.07 and about 7.13.

[0149] The present disclosure provides a method for increasing the level of defucosylated glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by a cell having glycosylation capability by about 1% to about 2%. In an exemplary embodiment, the method comprises increasing the pH of the cell culture medium by about 0.05 to about 1.2. Optionally, the method comprises increasing the pH by about 0.05 to about 0.07 to reduce about 1% of the defucosylated glycans; or increasing the pH by about 0.09 to about 1.2 to reduce more than about 1.5% of the defucosylated glycans. In some aspects, the method comprises culturing cells at a pH between about 7.10 and about 7.20, optionally between about 7.12 and about 7.19.

[0150] The present disclosure also provides a method for increasing the level of defucosylated glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by a cell having glycosylation capability by about 0.5% to about 1.1%. In an exemplary embodiment, the method includes increasing the pH of the cell culture medium by about 0.03-0.07 or increasing the pH by about 0.03 to about 0.06 to reduce about 0.8% of the defucosylated glycans; or increasing the pH by about 0.05 to about 0.07 to reduce more than about 1% of the defucosylated glycans. In different cases, the method includes culturing cells at a pH between about 7.05 and about 7.15, optionally between about 7.07 and about 7.13.

[0151] In any of the foregoing methods, throughout the entire culture, the pH of the cell culture medium is greater than 7.0, optionally, greater than 7.05 and less than 7.2. In some aspects, the level of defucosylated glycans in the recombinant glycosylated protein composition (e.g., antibody composition or antibody binding protein composition) is less than about 10%, for example, about 6.2% to about 8.4%. In any of the foregoing methods, during the culture period, the temperature changes by less than 2°C. For example, in some aspects, the temperature of the culture changes by no more than 1.5°C or 1.0°C. In any of the foregoing methods, the cell culture medium does not contain any detectable amount of manganese or betaine. In some aspects, the cell culture medium contains about 0.10 g / L to about 1.0 g / L of fucose, optionally, about 0.17 to about 1.0 g / L of fucose. Optionally, fucose is present in the culture medium at a concentration of less than about 0.75 g / L, less than about 0.6 g / L, or about 0.2 g / L to about 0.5 g / L. The addition or presence of fucose in the culture medium can be according to any teaching provided herein. In exemplary aspects, the cells with glycosylation capability are not genetically modified to change the activity of enzymes of the de novo pathway or salvage pathway. In any of the foregoing methods, glucose is added to the cell culture medium according to a glucose feeding scheme reaching an average glucose concentration of about 10 g / L or less. Glucose can be added according to any teaching provided herein.

[0152] As discussed above, TAF glycans are the sum of HM glycans and defucosylated glycans. Therefore, the methods for regulating defucosylated glycans disclosed herein will regulate the level of TAF glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) under different circumstances. Therefore, methods for regulating the level of TAF glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) produced by a cell having glycosylation capability are provided herein. In an exemplary aspect, the method comprises regulating the level of defucosylated glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) according to the methods for regulating the level of defucosylated glycans disclosed herein. In an exemplary embodiment, the method of regulating TAF glycans comprises reducing the level of defucosylated glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) according to the presently disclosed method of reducing the level of defucosylated glycans or increasing the level of defucosylated glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) according to the presently disclosed method of increasing the level of defucosylated glycans.

[0153] In addition, the present disclosure provides a method for producing an antibody composition, wherein the level of defucosylated glycans in the antibody composition is about 6.2% to about 8.4%. In an exemplary embodiment, the method comprises maintaining a glycosylation-competent cell in a cell culture medium having a pH greater than 7.05 and less than 7.2,

[0154] Wherein, optionally:

[0155] (A) during the culture period, the pH of the cell culture medium varies by less than 0.15 (optionally less than 0.10) or

[0156] (B) The temperature of the cell culture medium does not vary by more than 2°C or

[0157] (C) the method does not involve culturing the cells in a cell culture medium comprising manganese or betaine;

[0158] (D) A combination of two or three of (A), (B) and (C).

[0159] In various aspects, during the culture period, the pH is maintained at a pH of about 7.07 to about 7.19 (e.g., 7.08, 7.09, 7.10, 7.11, 7.12, 7.13, 7.14, 7.15, 7.16, 7.17, 7.18, or 7.19), optionally wherein the pH is maintained at about 7.07 or higher and below 7.10, or about 7.10 or higher and below 7.15, or about 7.15 or up to about 7.19. In various aspects, the level of defucosylated glycans in the antibody composition is less than about 10%, optionally, about 6.2% to about 8.4%. In various cases, the temperature variation during the culture period is less than 2°C, optionally, the temperature variation of the culture does not exceed 1.5°C or 1.0°C. In various aspects, the cell culture medium does not contain any detectable amount of manganese or betaine. In exemplary aspects, the cell culture medium comprises about 0.10 g / L to about 1.0 g / L fucose (optionally, about 0.17 to about 1.0 g / L fucose), optionally, the fucose is present in the culture medium at a concentration of less than about 0.75 g / L, less than about 0.6 g / L, or about 0.2 g / L to about 0.5 g / L. In some aspects, the glycosylation-competent cells are not genetically modified to alter the activity of enzymes of the de novo pathway or the salvage pathway, and in some aspects, glucose is added to the cell culture medium according to a glucose feeding regimen that achieves an average glucose concentration of about 10 g / L or less.

[0160] Exemplary Embodiments

[0161] The present disclosure provides methods for producing recombinant glycosylated protein compositions (e.g., antibody compositions or antibody binding protein compositions). In an exemplary embodiment, the method comprises maintaining glycosylation competent cells in a cell culture medium comprising a specific concentration of fucose and / or glucose as described herein, the specific concentration depending on the level of the desired TAF glycoform. In an exemplary embodiment, the level of TAF glycoforms in the recombinant glycosylated protein composition (e.g., antibody composition or antibody binding protein composition) is less than or about 10%, and in an exemplary aspect, the method comprises maintaining glycosylation competent cells in a cell culture medium comprising fucose, wherein the fucose is present in the culture medium at a concentration between about 0.17 g / L and about 1.0 g / L. In an exemplary embodiment, the level of TAF glycoforms in the recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) is less than or about 10%, and in an exemplary aspect, the method comprises maintaining a glycosylation-competent cell in a cell culture medium comprising fucose, wherein the fucose is present in the culture medium at a concentration between about 0.1 g / L and about 1.0 g / L, and wherein the glycosylation-competent cell has not been genetically modified to alter the activity of an enzyme of a de novo pathway or a salvage pathway. The present disclosure also provides a method for producing a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition), the method comprising maintaining a glycosylation-competent cell in a cell culture medium comprising fucose and glucose, wherein the fucose is present in the culture medium at a concentration of about 0.1 g / L to about 1.0 g / L, and glucose is added to the cell culture medium according to a glucose feeding schedule that achieves an average glucose concentration of about 10 g / L or less. In an exemplary aspect, the fucose is present in the culture medium at a concentration of less than about 0.75 g / L, optionally less than about 0.6 g / L. In exemplary cases, fucose is present in the culture medium at a concentration of about 0.2 g / L to about 0.5 g / L. In some aspects, fucose is present in the culture medium for the entire duration that the cells with glycosylation capability are maintained in the cell culture. In exemplary cases, the method for producing a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) includes maintaining the cells with glycosylation capability in a first cell culture medium for an initial period of time, and then maintaining the cells with glycosylation capability in a second cell culture medium, wherein the first cell culture medium does not contain fucose at a concentration of about 0.1 g / L to about 1.0 g / L, and the second cell culture medium contains fucose at a concentration of about 0.1 g / L to about 1.0 g / L. In some cases, the initial period of time is about 24 to about 72 hours. In alternative aspects, the initial period of time is about or greater than about 72 hours, but less than or about 156 hours. In some aspects, on the 6th day after the cell culture is inoculated, fucose is added to the first culture medium to obtain a second cell culture medium.During the period of maintaining cells with glycosylation ability in a cell culture medium containing fucose, the fucose concentration fluctuates by about 0.2 g / L or less (e.g., 0.1 g / L or less). In some aspects, for the initial time period, the cell culture medium comprises an initial glucose concentration. Optionally, the initial glucose concentration is about 1 g / L to about 15 g / L, for example, about 12 g / L ± 1 g / L. In exemplary aspects, the method further includes adding glucose to the cell culture medium according to a glucose feeding scheme. In exemplary aspects, the glucose feeding scheme starts about 4 to about 6 days after the cell culture is inoculated. For example, the glucose feeding scheme can start about 6 days after the cell culture is inoculated. In exemplary cases, the glucose feeding scheme reaches an average glucose concentration of about 10 g / L or less (e.g., about 9 g / L or less, about 6 g / L or less, about 0.5 g / L to about 4 g / L) in the cell culture medium. In exemplary cases, the glucose feeding scheme reaches an average glucose concentration based on the fucose concentration in the cell culture medium. In exemplary aspects, the average glucose concentration is calculated based on Formula I:.

[0162] T=3.354-1.388F+0.111G+[F-0.4375]x[1.9527(F-0.4375)]

[0163] (Formula I)

[0164] Wherein T is the target total defucosylated (TAF) glycan % in the antibody composition and is about 2.5% to about 6%, about 2.75% to about 5.5%, or about 3% to about 5%, F is the concentration of fucose in the culture medium (g / L), and G is the average glucose concentration in the culture medium (g / L). In exemplary aspects, (i) the concentration of fucose is about 0.2±0.1 g / L, and the average glucose concentration is about 2 to about 4 g / L; (ii) the fucose concentration is about 0.5±0.1 g / L, and the average glucose concentration is about 3 to about 6 g / L; or (iii) the fucose concentration is about 0.75±0.1 g / L, and the average glucose concentration is about 4.5 to about 9 g / L. In exemplary aspects, the pH of the cell culture medium is about 6.85 to about 7.05 (e.g., about 6.90 to about 7.00). In some aspects, the cells with glycosylation capability have not been genetically modified to alter the activity of enzymes of the de novo pathway or the salvage pathway. For example, the cells with glycosylation capability have not been genetically modified to knock out the gene encoding GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase. In exemplary cases, the level of total defucosylated (TAF) glycans in the antibody composition is less than about 10% (e.g., about 2% to about 6%, about 2% to about 5%, about 2% to about 4%). In exemplary cases, the level of high mannose glycans in the antibody composition is less than about 3.5% (e.g., about 0.7% to about 3.0%). In exemplary cases, the level of defucosylated glycans in the antibody composition is less than about 3.5% (e.g., about 0.8% to about 2.8%). In some aspects, the cells with glycosylation capability produce IgG antibodies, optionally, IgG1 antibodies. In some aspects, the IgG1 antibody is specific for tumor-associated antigens (e.g., CD20). In exemplary aspects, the culture medium does not comprise mannose.

[0165] A cell culture medium is provided, the cell culture medium comprising: (a) a cell with glycosylation capability, the cell with glycosylation capability comprising an exogenous nucleic acid encoding an antibody; and (b) a culture medium comprising fucose at a concentration of about 0.1 g / L to about 1.0 g / L or about 0.17 g / L to about 1.0 g / L. In an exemplary aspect, the cell with glycosylation capability has not been genetically modified to change the activity of an enzyme of a de novo pathway or a salvage pathway, and optionally, the cell with glycosylation capability has not been genetically modified to knock out a gene encoding GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase. In an exemplary aspect, the culture medium further comprises glucose at a concentration of less than about 10 g / L, such as less than about 9 g / L, less than about 6 g / L, or about 0.5 g / L to about 4 g / L. In an exemplary aspect, the pH of the culture medium is about 6.85 to about 7.05 (e.g., about 6.9 to about 7.0). In some aspects, the cell culture medium does not contain mannose. In exemplary aspects, the antibody is an IgG antibody, e.g., an IgG1 antibody. In exemplary cases, the IgG1 antibody is specific for a tumor-associated antigen, e.g., CD20.

[0166] Further provided herein are methods for altering or regulating the level of TAF glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) produced by a cell culture medium with glycosylation capability. In an exemplary aspect, the method comprises (A) adding fucose to a cell culture medium containing cells with glycosylation capability to achieve a fucose concentration of about 0.1 g / L to about 1.0 g / L, thereby reducing the level of TAF glycans; (B) adding glucose to a cell culture medium containing cells with glycosylation capability to achieve a glucose concentration of less than about 10 g / L, thereby increasing the level of TAF glycans; or (C) both (A) and (B). Also provided are methods for regulating the level of defucosylated glycans of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) produced by a cell with glycosylation capability. In an exemplary embodiment, the method comprises (A) adding fucose to a cell culture medium comprising cells having glycosylation capability to achieve a fucose concentration of about 0.1 g / L to about 1.0 g / L, thereby reducing the level of defucosylated glycans; (B) adding glucose to a cell culture medium comprising cells having glycosylation capability to achieve a glucose concentration of less than or about 10 g / L, thereby increasing the level of defucosylated glycans; or (C) both (A) and (B). The present disclosure further provides a method for regulating the level of high mannose glycans in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by cells having glycosylation capability. In an exemplary embodiment, the method comprises adding glucose to a cell culture medium comprising cells having glycosylation capability to achieve a glucose concentration of less than about 10 g / L, thereby increasing the level of HM glycans. In an exemplary aspect of the regulation method, the cells having glycosylation capability are not genetically modified to alter the activity of an enzyme of a de novo pathway or a salvage pathway. For example, in some aspects, cells with glycosylation capability are not genetically modified to knock out the gene encoding GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase. In some aspects, the level of TAF glycans in the antibody composition is less than or about 10% (e.g., about 2% to about 6%, about 2% to about 5%, about 2% to about 4%). In some aspects, the level of high mannose glycans in the antibody composition is less than or about 3.5% (e.g., about 0.7% to about 3.0%). In some aspects, the level of defucosylated glycans in the antibody composition is less than about 3.5% (e.g., about 0.8% to about 2.8%). In exemplary aspects, the fucose concentration is about 0.17g / L to about 1.0g / L, optionally, about 0.2g / L to about 0.5g / L. In some aspects, the method further comprises adding glucose to the cell culture medium according to a glucose feeding regimen to achieve an average glucose concentration of about 10 g / L (e.g., less than about 9.0 g / L, less than about 6.0 g / L, less than about 4.0 g / L).In some cases, the average glucose concentration is based on the fucose concentration in the cell culture medium. For example, in some aspects, the average glucose concentration is calculated based on Formula I:.

[0167] T=3.354-1.388F+0.111G+[F-0.4375]x[1.9527(F-0.4375)]

[0168] Formula I

[0169] wherein T is the target total defucosylated (TAF) glycan % in the antibody composition and is about 2.5% to about 6%, about 2.75% to about 5.5%, or about 3% to about 5%, F is the concentration of fucose in the culture medium (g / L), and G is the average glucose concentration (g / L).

[0170] The following examples are given merely to illustrate the present disclosure and are not intended to limit its scope in any way.

[0171] Examples

[0172] Example 1

[0173] This example describes the methods performed and materials used in the experiments of Example 2.

[0174] Cell lines, cell culture, and media

[0175] All experiments were performed using clones expressing antibodies comprising a light chain comprising SEQ ID NO.1 and a heavy chain comprising SEQ ID NO.2. All experiments were performed using individual bottles of cultured cells for 25 days. The following parameters were kept constant: duration (12 days), dissolved oxygen (48 mm Hg to 74 mm Hg), pH (6.85 to 7.05), agitation (350 RPM, 20 W / m 3 ), temperature (36.0℃).

[0176] Hydrophilic Interaction Liquid Chromatography (HILIC) Glycan Mapping

[0177] HILIC is used to determine the glycan profile of N-linked glycans released by enzymatic release. In short, the glycans are incubated together with a solution comprising PNGase F and sodium phosphate buffer (pH 7.5) at about 37°C for about 2 hours. The labeling solution comprising 2-aminobenzoic acid (2-AA) and sodium cyanoborohydride is then added to the glycans processed by PNGase F, and the mixture is incubated at about 80°C for about 75 minutes. After incubation, the mixture is centrifuged to obtain a precipitation of precipitated protein. The supernatant is collected and placed in a vial.

[0178] Glycans were separated by HILIC in conjunction with a fluorescence detector: Glycans were injected and bound to the column under high organic conditions (mobile phase A and mobile phase B were ammonium formate and acetonitrile, respectively), followed by elution with an increasing gradient of aqueous ammonium formate buffer. High resolution was achieved using a 1.7 μm small particle column format and a 150 mm column length. The total run time (including column re-equilibration) was 155 minutes.

[0179] Example 2

[0180] This example demonstrates the effect of increasing glucose and fucose levels in the culture medium on TAF%.

[0181] Cells expressing an antibody comprising a light chain of SEQ ID NO: 1 and a heavy chain of SEQ ID NO: 2 were added to a bioreactor containing one of three culture media: a control culture medium, a first test culture medium, and a second test culture medium. The first test culture medium was identical to the control culture medium except that it contained twice the amount of glucose, and the second test culture medium was a control culture medium containing 0.5 g / L fucose. Each culture medium lacked mannose. The cell cultures were maintained at a pH of 6.85 to 7.05 for 12 days.

[0182] Culture medium samples were collected periodically from the bioreactor to measure glucose concentration, TAF level and ADCC level. TAF and / or defucosylated (Afuc) glycan levels were determined via HILIC N-glycan profile program and the ability to stimulate ADCC was tested using in vitro assays. The results are shown in Table 1 below.

[0183] Table 1

[0184] Culture medium TAF level (%) ADCC(%) Target range 2.0％-4.2％ 69％-97％ Comparison 4.00％±0.23％ ～100％ First Test 5.87％±0.23％ 152％±15％ Second test ～3.4％ ～85％

[0185] ADCC levels are expressed as % of age relative to the ADCC level achieved by a control, which is a commercially available antibody having the same amino acid sequence.

[0186] Glucose concentrations were determined throughout the culture run, and these measurements were plotted over time and related to TAF levels of the antibodies produced in each cell culture medium type. Figure 3 As shown in the figure, the antibodies produced by cells cultured in the first test culture showed an increase in TAF, which corresponds to the increase in glucose levels in the cell culture. These results (and the results of Table 1) indicate that glucose can affect TAF levels and ADCC.

[0187] Antibodies produced by cells cultured in the second test medium containing 0.5 g / L fucose showed about 1% reduction in TAF ( Figure 3 ).like Figure 3As shown, the fucose concentration did not change much over the 12-day run, probably because the uptake of fucose by cells was negligible.

[0188] The antibodies produced in each of the different culture media were further analyzed for different glycan species. Interestingly, e.g. Figure 4 As shown, when cells were cultured in the first test medium (which contained 2 times the amount of glucose compared to the control medium), the % of high mannose (HM) glycans increased. This increase in % of HM glycans was not obtained in cells cultured in the second test medium containing fucose. Figure 4 As shown, the HM glycan % of the antibody produced by cells cultured in the second test medium containing fucose was approximately the same as the HM glycan % of the antibody produced by cells cultured in the control medium.

[0189] The effects of culturing in medium containing twice the amount of glucose (first test medium) or in medium containing fucose (second test medium) on the % defucosylated glycans were similar to the effects on the % TAF glycans. Figure 5 As shown, cells cultured in the first test medium comprising a higher glucose concentration showed an increase in defucosylated glycans, whereas cells cultured in the second test medium comprising fucose produced antibodies with reduced defucosylated glycans.

[0190] This example demonstrates that glucose and fucose are levers used to modulate high mannose and defucosylated glycan levels and influence ADCC.

[0191] Example 3

[0192] This example demonstrates additional studies showing that glucose and fucose are levers for regulating TAF levels and ADCC.

[0193] A follow-up multivariate full factorial experiment was designed to (1) elucidate the main effects, interaction effects, and quadratic effects of the fucose and glucose variables and (2) find out the number of these variables that would result in changes in TAF glycan and ADCC levels. Fucose was evaluated at concentrations of 0 g / L, 0.5 g / L, and 1 g / L in the culture medium. Glucose was supplied at rates of 0X, 1X (control), and 2X. 0X meant that no glucose stock solution was added to the culture, and this translated into a residual glucose concentration of approximately 1 g / L after 6 days of cell culture. Glucose was supplied only via culture medium containing 12 g / L of sugar. In the 1X feed, glucose was maintained at an average concentration of 3 ± 1 g / L after the glucose feed began. In the 2X feed, the average glucose level was maintained at 6 ± 1 g / L. The results show that fucose concentration can be varied to affect TAF levels ( Figure 6 ) and ADCC levels ( Figure 7 ).

[0194] In these experiments, after the start of feeding (i.e., after day 6), the glucose concentration was controlled at 3 ± 1 g / L. If the glucose concentration exceeded 4 g / L, no additional glucose was added, and the cells relied on residual glucose in the bioreactor and glucose obtained by perfusing the medium until the glucose level fell within the control range. Based on these experiments, Figure 8 The model shown predicts TAF values ​​for different fucose and glucose concentrations. The model indicates that a quality target product profile (QTTP) can be obtained after culturing cells in a medium containing about 0.1 g / L to about 1.0 g / L fucose and / or about 0.5 g / L to about 4.0 g / L glucose. The model predicts the following TAF values ​​for different fucose and glucose concentrations: 0.2 g / L fucose and 3 g / L glucose, 0 g / L fucose and 0.554 g / L glucose; and 0.492 g / L fucose and 6 g / L glucose. Figures 9A-9C These results suggest that there are several approaches to achieve desired levels of TAF and ADCC.

[0195] To confirm Figures 9A-9C Additional experiments were performed to predict . A summary of the experiments is provided in Table 2.

[0196] Table 2

[0197]

[0198] 0X, the glucose concentration was measured at about 1 g / L after day 6; 1X, the glucose concentration was measured at about 3±1 g / L; 2X, the glucose concentration was measured at about 6±1 g / L.

[0199] This example demonstrates that both glucose concentration and fucose concentration are variable and can be manipulated to alter the levels of TAF and ADCC.

[0200] Example 4

[0201] This example demonstrates the effect of fucose on cell culture.

[0202] Additional analyses were performed on the cell cultures described above. For example, the osmotic pressure of the cell cultures was measured and ranged from about 175 mOsm / kg to about 345 mOsm / kg. A lack of correlation between cell culture osmotic pressure and fucose concentration was observed. See Fig.10 As shown in the figure, adding fucose to the culture medium does not appear to affect osmotic pressure in any particular way. Under control conditions (without fucose), osmotic pressure varies greatly, indicating that the components of the culture medium (other than fucose) have a positive effect on osmotic pressure.

[0203] In one experiment of this fucose study, cells were inoculated into one of five bioreactors, two of which contained medium without any fucose (ctrl_a and ctrl_b), and three of which contained medium with 0.5 g / L fucose (fuc_a, fuc_b, and fuc_c). Media samples were collected from the five cell cultures at day 0, day 3, day 5, day 7, day 9, and day 12 throughout the 12-day culture period. The fucose concentration of the samples was measured. Fig.11 As shown, the concentration of fucose did not change substantially during the entire culture period, suggesting that the consumption of fucose during the culture process was small and / or slow.

[0204] In another experiment in this fucose study, cells were maintained in cell culture in a medium without fucose for 12 days. In this particular experiment, the pH of the cell culture was disturbed from 7.1, causing the TAF% to increase to about 5.5% from day 5 to day 8. TAF% was measured daily starting from day 5. To adjust the TAF level back to the target of 4.0%, fucose was added at a feed rate of 0.9 g / L per day on day 9 of culture. Fig.12 As shown, TAF% decreased from about 5.5% after fucose was added to the culture medium. TAF% continued to decrease to about 3.8% on day 12. These data suggest that fucose addition may occur late in the culture period and still cause regulation of TAF%.

[0205] This example demonstrates the effect of fucose concentration in the culture medium on TAF levels.

[0206] Example 5

[0207] This example demonstrates that maintaining glucose within the target range can occur late in the culture period.

[0208] During the 12-day culture period, three experiments were performed to monitor the time to reach the target glucose range. For each experiment, the initial glucose concentration of each cell culture ranged from about 5.0 g / L to about 6.0 g / L. The glucose concentration of the cell culture medium was monitored daily. Fig.13 As shown, each cell culture reached the target glucose concentration (0.5 g / L-4.0 g / L) on a different day during the culture period. In one experiment (line with open squares), the target range was reached on day 2. In the second experiment (dotted line with open diamonds), the target range was reached on day 4, and in the third experiment (line with open circles), the target range was reached on day 6. Despite these differences, each cell culture reached the target range for TAF% (about 2.0% to about 4.3%) (see Fig.13(left panel; the third experiment is represented by open circles; the second experiment is represented by open diamonds; the first experiment is represented by open squares). These data suggest that glucose maintenance can occur late in the culture period to achieve the same culture TAF levels maintained early in the culture period.

[0209] These data suggest that manipulation of glucose concentrations during early and late phases results in similar TAF levels.

[0210] Example 6

[0211] This example demonstrates the effect of lowering the pH of cell culture on the defucosylation level of an antibody with and without the addition of fucose.

[0212] Cell culture samples were removed from the 2000L bioreactor and used to inoculate a parallel 3L bioreactor. Using a continuous fed-batch process, the 2000L and 3L bioreactors were fed with Feed A and Feed B for 12 days. On day 5, both 3L bioreactors were fed with fucose to a final concentration of 1.0 g / L. Defucosylation levels were measured as described in Example 1, and the results are provided in Table 3.

[0213] Table 3

[0214]

[0215]

[0216] As shown in Table 3, without the addition of fucose, when the pH was below 7.1, the average defucosylation % was 6.39, while when the pH was above 7.10 but below 7.15, the average defucosylation % was higher (defucosylation % = 7.185). When the pH was above 7.15, the average defucosylation % was even higher (defucosylation % = 8.276). Therefore, lower pH values ​​were associated with lower defucosylation %.

[0217] When fucose was added to the culture medium (to reach a final concentration of 1.0 g / L fucose), the % defucosylation decreased significantly at both the lower pH (7.07) and the higher pH (7.18). The average decrease in % defucosylation when fucose was added was 2.18% for each of these pH levels.

[0218] These results indicate that lowering the pH of the cell culture medium and / or adding fucose to the cell culture medium results in a reduced percentage of defucosylation. When the pH is lowered and fucose is added to the cell culture medium, a greater reduction in the percentage of defucosylation is observed.

[0219] Example 7

[0220] This example further demonstrates the effect of lowering the pH of the cell culture on the defucosylation level of the antibody with and without the addition of fucose.

[0221] A parallel 3L bioreactor was inoculated with a cell culture sample from the 2000L bioreactor. Using a continuous fed-batch process, the 2000L and 3L bioreactors were fed with Feed A and Feed B for 12 days. On day 5, some bioreactors (i.e., cell cultures) were fed with fucose to a final concentration of 0.25, 0.5, or 1 g / L. Defucosylation levels were measured as described in Example 1, and Fig.14 The results are provided in.

[0222] like Fig.14 As shown, the control 3L bioreactor (control pH 7.1) showed similar levels of defucosylation as the initial 2000L bioreactor. Both showed about 6.5% or higher defucosylation%. Addition of fucose at any of the levels tested resulted in a significant decrease in defucosylation (5.5% or lower). The defucosylation percentage was lowest with the addition of 0.25g / L fucose.

[0223] In addition, if Fig.14 As shown, lowering the pH from 7.1 to 7.0, without adding fucose to the culture medium, also resulted in a decrease in defucosylation of at least about 1.0%.

[0224] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0225] Unless otherwise indicated herein or clearly contradicted by context, the use of the terms "a," "an," and "the," and similar referents in the context of describing the present disclosure (especially in the context of the following claims) will be construed to cover both the singular and the plural. The terms "comprising," "having," "including," and "containing" will be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise indicated.

[0226] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value and endpoint within the range, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[0227] Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. Unless otherwise claimed, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better describe the disclosure, rather than to impose limitations on the scope of the disclosure. The language in the specification should not be construed as indicating that any unclaimed element is necessary to implement the disclosure.

[0228] Preferred embodiments of the present disclosure are described herein, including the most preferred modes known to the inventors for implementing the present disclosure. After reading the above description, the variations of those preferred embodiments are apparent to those of ordinary skill in the art. The inventors expect that skilled artisans will adopt such variations as appropriate, and the inventors intend to implement the present disclosure in a manner other than that specifically described herein. Therefore, the present disclosure includes all modifications and equivalents allowed by applicable law of the subject matter narrated in the appended claims. In addition, unless otherwise indicated herein or the context is otherwise obviously contradictory, the present disclosure encompasses any combination of the above-mentioned elements in all possible variations.

Claims

1. A method for producing an antibody composition, wherein the level of total defucosylated (TAF) glycans in the antibody composition is less than about 10%, the method comprising maintaining cells with glycosylation capacity in a cell culture medium comprising fucose, wherein the fucose is present in the culture medium at a concentration of about 0.17 g / L to about 1.0 g / L.

2. A method for producing an antibody composition, wherein the level of total defucosylated (TAF) glycans in the antibody composition is less than about 10%, the method comprising maintaining glycosylation-competent cells in a cell culture medium comprising fucose, wherein the fucose is present in the culture medium at a concentration of about 0.1 g / L to about 1.0 g / L, and wherein the glycosylation-competent cells have not been genetically modified to alter the activity of enzymes of the de novo pathway or the salvage pathway.

3. A method for producing an antibody composition, the method comprising maintaining cells having glycosylation capability in a cell culture medium comprising fucose and glucose, wherein the fucose is present in the culture medium at a concentration of about 0.1 g / L to about 1.0 g / L, and adding glucose to the cell culture medium according to a glucose feeding regimen that achieves an average glucose concentration of about 10 g / L or less.

4. The method of any one of claims 1 to 3, wherein fucose is present in the culture medium at a concentration of less than about 0.75 g / L.

5. The method of claim 4, wherein fucose is present in the culture medium at a concentration of less than about 0.6 g / L.

6. The method of claim 5, wherein fucose is present in the culture medium at a concentration of about 0.2 g / L to about 0.5 g / L.

7. The method of any one of claims 1 to 6, wherein the fucose is present in the culture medium throughout the duration that the glycosylation-competent cells are maintained in cell culture.

8. The method of any one of claims 1 to 6, comprising maintaining the glycosylation-competent cells in a first cell culture medium for an initial period of time and subsequently maintaining the glycosylation-competent cells in a second cell culture medium, wherein The first cell culture medium does not comprise fucose at a concentration of about 0.1 g / L to about 1.0 g / L, and the second cell culture medium comprises fucose at a concentration of about 0.1 g / L to about 1.0 g / L.

9. The method of claim 8, wherein the initial period of time is about 24 to about 72 hours.

10. The method of claim 8, wherein the initial period of time is about or greater than about 72 hours, but less than or about 156 hours.

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