Total afucosylated glycoforms of antibodies produced in cell culture

Abstract

The present disclosure provides methods for generating antibody compositions that comprise a desired or predetermined or preselected level of total afucosylated (TAF) glycoform. In an exemplary embodiment, the method comprises maintaining glycosylation-capable cells in a cell culture medium comprising fucose and / or glucose at a specific concentration as described herein, the specific concentration being dependent on the level of the desired TAF glycoform. Related compositions comprising glycosylated proteins and their TAF glycoforms are also provided. Cell culture media are also provided.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 648,308, filed Mar. 26, 2018. The content of each application is incorporated herein by reference.

[0003] Incorporation by reference of electronically - submitted materials

[0004] Incorporated by reference in its entirety is the computer - readable nucleotide / amino acid sequence listing submitted herewith, which is identified as follows: a 28,547 - byte ASCII (text) file named “52249A_Seqlisting.txt”; created on Mar. 26, 2019. Background of the invention

[0005] Glycosylation is one of the most common and important post - translational modifications as it plays a role in multiple cellular functions including, for example, protein folding, quality control, molecular trafficking and sorting, and cell - surface receptor interactions. Glycosylation affects the therapeutic efficacy of recombinant protein drugs as it influences the biological activity, pharmacokinetics, immunogenicity, solubility, and in - vivo clearance of therapeutic glycoproteins. In particular, the Fc glycan profile is an important product quality attribute of recombinant antibodies as they directly affect the clinical efficacy and pharmacokinetics of the antibody.

[0006] It has been found that the high - mannose (HM) glycan 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 glycans not only affect the serum clearance rate of antibodies, but such glycans in addition to the afucosylated (afuco) glycan can also affect antibody effector function or antibody - mediated target cell killing (also known as antibody - dependent cell cytotoxicity (ADCC)).

[0007] Many factors affect the glycan structure and thus the final glycosylation form (glycan type) of a protein (glycoprotein). For example, the cell line expressing the antibody, the cell culture medium, the composition of the feed medium, and the timing of feeding during cell culture can affect the generation of the glycan types of the protein.

[0008] Although research groups have proposed many methods to affect the levels of specific glycan types of antibodies, the biopharmaceutical industry still needs simple and effective methods to manipulate and control the total afucosylated (TAF) glycan levels during the recombinant production of therapeutic antibodies. Summary of the invention

[0009] The following data are described for the first time, which demonstrate that the concentration of fucose and / or glucose in the cell culture medium containing cells that produce recombinant glycosylated proteins (e.g., antibodies or antibody-binding proteins) affects the level of the TAF glycoform of the produced recombinant glycosylated proteins. However, a greater change in the level of the TAF glycoform of a recombinant glycosylated protein (e.g., an antibody or an antibody-binding protein) can be achieved by manipulating the concentration of fucose in the cell culture medium containing cells that produce the recombinant glycosylated protein (e.g., an antibody or an antibody-binding protein). As described herein, a smaller change in the level of the TAF glycoform can be achieved by changing the concentration of glucose in the cell culture medium. In addition, the data indicate that while the glucose concentration of the cell culture medium affects the levels of high-mannose glycans and afucosylated glycans, the fucose concentration of the cell culture medium affects the level of afucosylated glycans and does not affect the level of high-mannose glycans. Each of these sugars (differing only by one oxygen atom in the chemical formula) results in a different effect on the level of the TAF glycoform, and this finding was unexpected. Without being bound by a particular theory, maintaining the cells (which produce recombinant glycosylated proteins (e.g., antibodies or antibody-binding proteins)) in a cell culture medium containing fucose and / or glucose at the concentrations taught herein allows for the production of a recombinant glycosylated protein (e.g., an antibody or an antibody-binding protein) composition having a desired or predetermined or preselected level of the TAF glycoform (e.g., high-mannose glycans and afucosylated glycans). Accordingly, 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) containing a desired or predetermined or preselected level of the TAF glycoform.

[0010] 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 includes maintaining glycosylation-capable cells in a cell culture medium containing fucose and / or glucose at a specific concentration as described herein, the specific concentration depending on the desired level of the TAF glycoform.

[0011] In an exemplary embodiment, the level of the TAF glycoform 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 includes maintaining glycosylation-capable cells in a cell culture medium containing fucose, wherein the fucose is present in the medium at a concentration between about 0.17 g / L and about 1.0 g / L.

[0012] In an exemplary embodiment, the level of the TAF glycoform in a 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 glycosylation-capable cells in a cell culture medium comprising fucose, wherein the fucose is present in the medium at a concentration between about 0.1 g / L and about 1.0 g / L, and wherein the glycosylation-capable cells are not genetically modified to alter the activity of enzymes in the de novo pathway or the salvage pathway.

[0013] 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 glycosylation-capable cells in a cell culture medium comprising fucose and glucose, wherein the fucose is present in the 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 protocol that achieves an average glucose concentration of about 10 g / L or less.

[0014] The present disclosure provides a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) produced by the method of the present disclosure. Additionally, related pharmaceutical compositions and cell culture media are provided. In an exemplary aspect, the cell culture medium comprises an exogenous nucleic acid encoding an antibody (e.g., an IgG antibody) and a 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, the glycosylation-capable cells are not genetically modified to alter the activity of enzymes in the de novo pathway or the salvage pathway. Optionally, the glycosylation-capable cells are not genetically modified to knockout the gene encoding GDP-keto-6-deoxymannose-3,5-epimerase,4-reductase. In some aspects, the medium further comprises glucose at a concentration less than about 10 g / L, optionally less than about 9 g / L or about 6 g / L or less (e.g., about 0.5 g / L to about 4 g / L). In an exemplary case, the pH of the 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 an exemplary aspect, the antibody is specific for a tumor-associated antigen, such as an antigen comprising SEQ ID NO:3.

[0015] The present disclosure further provides methods for altering or modulating the levels of TAF glycans of recombinant glycoprotein compositions (e.g., antibody compositions or antibody-binding protein compositions) produced by glycosylation-capable cells in a cell culture medium. In an exemplary aspect, the method comprises (A) adding fucose to the cell culture medium containing glycosylation-capable cells to achieve a fucose concentration of from about 0.1 g / L to about 1.0 g / L, thereby reducing the level of TAF glycans; (B) adding glucose to the cell culture medium containing glycosylation-capable cells to achieve a glucose concentration of less than about 10 g / L, thereby increasing the TAF level; or (C) a combination of both (A) and (B).

[0016] Also provided are methods for modulating the afucosylated glycan levels of recombinant glycoprotein compositions (e.g., antibody compositions or antibody-binding protein compositions) produced by glycosylation-capable cells. In an exemplary embodiment, the method comprises (A) adding fucose to the cell culture medium containing glycosylation-capable cells to achieve a fucose concentration of from about 0.1 g / L to about 1.0 g / L, thereby reducing the level of afucosylated glycans; (B) adding glucose to the cell culture medium containing glycosylation-capable cells to achieve a glucose concentration of less than or about 10 g / L, thereby increasing the afucosylated glycan level; or (C) a combination of (A) and (B).

[0017] The present disclosure further provides methods for modulating the high-mannose (HM) glycan levels of recombinant glycoprotein compositions (e.g., antibody compositions or antibody-binding protein compositions) produced by glycosylation-capable cells. In an exemplary embodiment, the method comprises adding glucose to the cell culture medium containing glycosylation-capable cells to achieve a glucose concentration of less than about 10 g / L, thereby increasing the level of HM glycans.

[0018] The present disclosure also provides methods for modulating the afucosylated glycan levels of recombinant glycoprotein compositions (e.g., antibody compositions or antibody-binding protein compositions) produced by glycosylation-capable cells, the method comprising reducing the pH of the cell culture medium by about 0.03 to about 1.2, thereby reducing the level of afucosylated 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 afucosylated glycans of the composition by about 0.5% to about 2%.

[0019] The present disclosure provides a method for reducing the afucosylated glycan levels of recombinant glycoprotein compositions (e.g., antibody compositions or antibody-binding protein compositions) produced by glycosylation-capable cells 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.

[0020] In addition, provided is a method of reducing the afucosylated glycan level of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) produced by a cell having glycosylation ability by about 0.5% to about 1.1%, the method comprising reducing the pH of the cell culture medium by about 0.03 - 0.07.

[0021] The present disclosure further provides a method of increasing the afucosylated glycan level of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) produced by a cell having glycosylation ability 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.

[0022] Also provided is a method of increasing the afucosylated glycan level of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) produced by a cell having glycosylation ability 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.

[0023] The present disclosure further provides a method of modulating the TAF glycan level of a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) produced by a cell having glycosylation ability, the method comprising modulating, reducing or increasing the level of afucosylated glycan of the composition according to the methods of modulating, reducing or increasing the afucosylated glycan level currently disclosed.

[0024] The present disclosure provides a method of producing a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) wherein the level of afucosylated glycan in the composition is from about 6.2% to about 8.4%, the method comprising maintaining a cell having glycosylation ability 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 varies by no more than 2°C or the method does not include culturing the cells in a cell culture medium containing manganese or betaine or (D) a combination of two or three of (A), (B) and (C). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1A is an illustration of three types of N-glycans (oligomannose, complex and hybrid) and common symbols for such sugars.

[0026] Figure 1B is a diagrammatic illustration of exemplary glycan structures.

[0027] Figure 2It is a diagram of the salvage and de novo pathways 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. Then, GDP-fucose is transported from the cytoplasm to the Golgi lumen by GDP-Fuc transferase and transferred to acceptor oligosaccharides and proteins. The other reaction product, GDP, is converted to guanosine 5'-monophosphate (GMP) and inorganic phosphate (Pi) by a luminal nucleotide diphosphatase. The former is exported to the cytoplasm (via an antiport system coupled to the transport of GDP-fucose), while the latter is presumably exported from the Golgi lumen via the Golgi anion channel GOLAC. See, e.g., Nordeen et al. 2000; Hirschberg et al. 2001.

[0028] Figure 3 It is a diagram depicting (A) the 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) the fucose concentration (g / L) of a cell culture using the second test medium (line with open squares) during a cell culture run, and (C) the TAF glycan level (%) 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 4 It is a diagram depicting (A) the 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) the high-mannose (HM) glycan level (%) 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.

[0030] Figure 5is a graph depicting (A) the glucose concentration (g / L) of cell cultures using a control medium (line with hollow triangles), a first test medium (line with hollow circles), and a second test medium (line with hollow hexagons) during a cell culture run, and (B) the afucosylated (afuc) glycan level (%) 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.

[0031] Figure 6 is a graph showing the change in TAF glycan level (%) with fucose concentration (g / L) in cell culture media containing 0X glucose, 1X glucose, or 2X glucose.

[0032] Figure 7 is a graph showing the change in ADCC level (expressed as % relative to a control antibody with the same amino acid sequence) with fucose concentration (g / L) in cell culture media containing 0X glucose, 1X glucose, or 2X glucose.

[0033] Figure 8 is a graph of a model illustrating the effect of glucose and fucose on the TAF glycan level (%). The minimum, maximum, and average TAF are shown according to QTPP. The equation in the lower graph shows the mathematical relationship between glucose, fucose, and TAF.

[0034] Figure 9A is a series of graphs showing: (i) the change in TAF glycan level (%) with fucose concentration (g / L) in cell culture media (upper left quadrant), or with glucose concentration (g / L) in cell culture media (upper right quadrant), and (ii) the change in ADCC level (expressed as % relative to a control antibody with the same amino acid sequence) with fucose concentration (g / L) in cell culture media (lower left quadrant) or with glucose concentration (g / L) in cell culture media (lower right quadrant). The TAF glycan level (%) ranges from 3.30684 to 3.73083, and the ADCC level ranges from 78.3092 - 90.4408. At 0.2 g / L fucose and 3.0 g / L glucose, the TAF glycan level (%) is 3.518836, and the ADCC level (%) is 84.37501.

[0035] Figure 9BA series of graphs showing: (i) the variation of TAF glycan levels (%) in cell culture medium with fucose concentration (g / L) (upper left quadrant), or with glucose concentration (g / L) in cell culture medium (upper right quadrant), and (ii) the variation of ADCC levels (expressed as % relative to an innovative antibody or a commercially available antibody) in cell culture medium with fucose concentration (g / L) (lower left quadrant) or with glucose concentration (g / L) in cell culture medium (lower right quadrant). The range of TAF glycan levels (%) is from 3.52301 to 4.0559, and the range of ADCC levels is from 75.6911 - 90.9385. At 0 g / L fucose and 0.554 g / L glucose, the TAF glycan level (%) is 3.789458, and the ADCC level (%) is 83.3148.

[0036] Figure 9C A series of graphs showing: (i) the variation of TAF glycan levels (%) in cell culture medium with fucose concentration (g / L) (upper left quadrant), or with glucose concentration (g / L) in cell culture medium (upper right quadrant), and (ii) the variation of ADCC levels (expressed as % relative to an innovative antibody or a commercially available antibody) in cell culture medium with fucose concentration (g / L) (lower left quadrant) or with glucose concentration (g / L) in cell culture medium (lower right quadrant). The range of TAF glycan levels (%) is from 2.9975 - 3.68468, and the range of ADCC levels is from 79.2215 - 98.8836. At 0.492 g / L fucose and 6.0 g / L glucose, the TAF glycan level (%) is 3.341092, and the ADCC level (%) is 89.05256.

[0037] Figure 10 A graph plotting the osmotic pressure against the fucose concentration.

[0038] Figure 11 A graph plotting the fucose concentration in cell culture against time (duration).

[0039] Figure 12 A graph of % TAF and fucose feed, each plotted against time.

[0040] Figure 13 A pair of graphs demonstrating that controlling glucose within the target range from day 6 or earlier results in equivalent TAF outcomes.

[0041] Figure 14 A graph plotting the % defucosylation against the culture time. Detailed Description

[0042] Many secreted proteins undergo post-translational glycosylation, a process by which a sugar moiety (e.g., glycan, sugar) is covalently attached to specific amino acids of the protein. In eukaryotic cells, two types of glycosylation reactions occur: (1) N-linked glycosylation, in which the glycan is attached to asparagine of the recognition sequence Asn-X-Thr / Ser, where "X" is any amino acid except proline; and (2) O-linked glycosylation, in which the glycan is attached to serine or threonine. Regardless of the type of glycosylation (N-linked or O-linked), there is microheterogeneity of protein glycoforms due to the wide range of glycan structures associated with each site (O or N).

[0043] 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 the following three types: (A) high-mannose (HM) or oligomannose (OM) type, which consists of two N-acetylglucosamine (GalNAc) moieties and a large number (e.g., 5, 6, 7, 8, or 9) of mannose (Man) residues; (B) complex type, which contains more than two GlcNAc moieties and any number of other sugar types; or (C) hybrid type, which contains 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 Edition, Cold Spring Harbor Laboratory Press; 2009) shows the three types of N-glycans.

[0044] N-linked glycans typically contain one or more of the monosaccharides 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-doxynononic acid (Kdn), fucose (Fuc), glucuronic acid (GLcA), iduronic acid (IdoA), galacturonic acid (Gal A), mannuronic acid (Man A). The common symbols for such sugars are shown in Figure 1A and. Exemplary glycans and their identities are shown in Figure 1B and.

[0045] 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).

[0046] 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.

[0047] 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.

[0048] In an exemplary embodiment, the level of TAF glycan is adjusted. As used herein, "total afucosylated glycan" or "TAF glycan" or "total afucosylated glycoform" or "TAF glycoform" refers to the total amount of high-mannose (HM) glycan and afucosylated glycan. In an exemplary embodiment, the level of HM glycan is adjusted. As used herein, the term "high-mannose glycan" or "HM glycan" or "high-mannose glycoform" or "HM glycoform" or "HM" encompasses glycans containing 5, 6, 7, 8, or 9 mannose residues, abbreviated as Man5, Man6, Man7, Man8, and Man9, respectively. In an exemplary embodiment, the level of afucosylated glycan is adjusted. As used herein, the term "afucosylated glycan" or "afuco glycan" or "afucosylated glycoform" or "Afuco" refers to a glycoform lacking a core fucose (e.g., α1,6-linked fucose) on the GlcNAc residue participating in the amide bond with Asn of the N-glycosylation site. Afucosylated glycoforms include, but are not limited to, A1G0, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5. Additional afucosylated glycans include, for example, A1G1a, G0[H3N4], G0[H4N4], G0[H5N4], FO-N[H3N3]. See, e.g., Reusch and Tejada, Glycobiology 25(12):1325-1334 (2015). In an exemplary aspect, the level of TAF and the amounts of HM glycoforms and afucosylated glycoforms are determined by hydrophilic interaction liquid chromatography (HILIC), as further described in Example 1 herein. After enzymatic cleavage of 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 = sample total area / standard total area x 100%. The calculation for determining the % TAF can be performed as follows:

[0049] % afucosylated glycoform = % A1G0 + % A2G0 + % A2G1a + % A2G1b + % A2G2 + % A1G1M5.

[0050] % high-mannose glycoform = % Man5 (if detectable) + % Man6 (if detectable) + % Man7 (if detectable) + % Man8 (if detectable) + % Man9 (if detectable)

[0051] The present disclosure provides methods for generating 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 glycosylation-capable cells in a cell culture medium comprising fucose and / or glucose at a specific concentration as described herein, the specific concentration being dependent on the level of the desired TAF glycoform.

[0052] Fucose

[0053] In embodiments of the methods disclosed herein, fucose is present in the culture medium at a concentration 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 an exemplary case, fucose is present in the culture medium at a concentration below or about 1.2 g / L. In an exemplary case, fucose is present in the culture medium at a concentration from about 0.1 g / L to about 1.0 g / L. In an exemplary case, the culture medium contains fucose at a concentration 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 an exemplary aspect, fucose is present in the culture medium at a concentration from about 0.17 g / L to about 1.2 g / L. In an exemplary case, fucose is present in the culture medium at a concentration from about 0.17 g / L to about 1.0 g / L. In an exemplary case, the culture medium contains fucose at a concentration 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 an exemplary case, fucose is present in the culture medium at a concentration from about 0.2 g / L to about 1.0 g / L. In an exemplary case, fucose is present in the culture medium at a concentration below or about 1.0 g / L. For example, in some cases, the fucose concentration in 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 in 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 an exemplary aspect, 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 an exemplary case, fucose is present in the culture medium at a concentration below or about 0.75 g / L, or from about 0.25 g / L to about 0.75 g / L, such as from about 0.4 g / L to about 0.5 g / L, or about 0.6 g / L. In an exemplary aspect, fucose is present in the culture medium at a concentration below about 0.6 g / L, such as from about 0.2 g / L to about 0.5 g / L.

[0054] In an exemplary aspect, a method of generating a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) includes maintaining glycosylation-capable cells in two different cell culture media. In an exemplary aspect, a method of generating a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) includes maintaining glycosylation-capable cells in a first cell culture medium for an initial period of time and subsequently maintaining these glycosylation-capable cells 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 an exemplary case, the initial period of time begins when the cells are inoculated into a bioreactor containing a cell culture medium (e.g., the cell culture medium). In some aspects, the initial period of time is about 1 day to about 3 days, such as about 24 hours to about 72 hours. In an exemplary aspect, the initial period of time 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 an exemplary aspect, the initial period of time is about 3, about 4, about 5, about 6, about 7, about 8, or about 9 days. In an exemplary aspect, the method includes adding fucose to the culture medium after the initial period of time. In some aspects, fucose is added to the first medium to obtain the second cell culture medium. For example, in various aspects, the method includes adding fucose after about 1 day to about 3 days, after about 3 days but less than about 10 days, or after about 3, about 4, about 5, about 6, about 7, about 8, or about 9 days. In an exemplary aspect, 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 inoculation of the cell culture. In an exemplary aspect, fucose is added to a final 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. In an exemplary aspect, the first cell culture medium does not contain fucose. In an exemplary aspect, the first cell culture medium contains fucose, but the concentration is undetectable or immeasurable, or the concentration is substantially lower than the fucose concentration of the second cell culture medium, e.g., substantially lower than 0.1 g / L, lower than about 0.17 g / L or lower than about 0.2 g / L.

[0055] In an alternative aspect, the method includes maintaining glycosylation-capable 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-capable cells are maintained in the cell culture or for most 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) includes inoculating glycosylation-capable cells into a bioreactor containing a cell culture medium comprising fucose and maintaining the cells in the cell culture medium at a concentration of fucose that remains substantially the same throughout the cell culture.

[0056] In an exemplary embodiment, the concentration of fucose fluctuates very little during the cell culture. In an exemplary embodiment, the fucose concentration fluctuates by about 0.2 g / L or less during the maintenance of glycosylation-capable cells 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 maintenance of glycosylation-capable cells in a cell culture medium comprising fucose. In an exemplary aspect, when fucose is added to the cell culture medium (e.g., after an initial cell culture period), fucose is added to the medium no more than one or two times during the cell culture period.

[0057] Glucose

[0058] In an exemplary embodiment of the method disclosed herein, glucose is present in the culture medium. In an exemplary aspect, 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 an exemplary aspect, glucose is present in the culture medium at a concentration from about 0.5 g / L to about 4.0 g / L. In an exemplary aspect, glucose is present at a concentration from about X g / L to about Y g / L, where 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, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0, provided that X is less than Y.

[0059] In an exemplary aspect, the method includes maintaining the glucose concentration in the cell culture medium for a period of time corresponding to the cell culture period. In an exemplary case, maintaining the glucose concentration in the cell culture medium includes periodically (e.g., once per hour, once every two hours, once every 3, 4, 5, or 6 hours, once a day, twice a day, three times a day, or four times a day, etc.) sampling the cell culture medium, measuring the glucose concentration of the sampled cell culture medium, and if the glucose concentration of the sampled cell culture medium is lower than the desired maintained glucose concentration, adding glucose to the cell culture. In an exemplary aspect, maintaining the glucose concentration in the cell culture medium includes measuring the glucose concentration of the cell culture medium via a glucose sensor. In an exemplary aspect, the glucose concentration is measured at regular intervals (e.g., once per hour, once every two hours, once every 3, 4, 5, or 6 hours, once a day, twice a day, three times a day, or four times a day, etc.) via the glucose sensor, and if it is determined via the glucose sensor that the glucose concentration is lower than the desired maintained glucose concentration, glucose is added to the cell culture. In an alternative aspect, the method includes maintaining glycosylation-capable cells in a cell culture medium containing glucose, but only maintaining the glucose concentration in the cell culture medium after an initial period of time. In an exemplary embodiment, the initial period of time is from about 1 day to about 3 days (about 24 hours to about 72 hours). In some cases, the initial period of time is less than or about 6 days, optionally, wherein the initial period of time is 3 days or 4 days or 5 days after cell culture inoculation. In an exemplary aspect, the method includes maintaining glycosylation-capable cells in a cell culture medium containing glucose and maintaining the glucose concentration in the cell culture medium on the sixth day after inoculation and thereafter. In an exemplary aspect, the glucose concentration is maintained for at least about 4 days or about 5 days after the initial period of time, or optionally at least about 6 days after the initial period of time.

[0060] In an exemplary aspect, for an initial time period, the cell culture medium comprises an initial glucose concentration. For example, in various aspects, the initial glucose concentration is from about 1.0 g / L to about 15 g / L, from about 1.0 to about 12 g / L, or from 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 an exemplary aspect, the initial glucose concentration is the glucose concentration of the cell culture medium used during the initial time period. In an exemplary aspect, the initial glucose concentration is the glucose concentration of the cell culture medium maintained during the initial time period.

[0061] In an exemplary aspect, the initial glucose concentration is the same as the glucose concentration maintained after the initial time period. In an alternative aspect, the initial glucose concentration is different from the glucose concentration maintained after the initial time period. In an exemplary aspect, the method includes adding glucose to the cell culture medium after the initial time period and maintaining the glucose at a different concentration relative to the initial glucose concentration. In an exemplary aspect, the method includes adding glucose to the cell culture medium after the initial time period to maintain the glucose at a different concentration relative to the initial glucose concentration, wherein the step of adding glucose achieves a glucose concentration of about 10 g / L or lower (e.g., about 9 g / L or lower, about 6 g / L or lower, from about 0.5 g / L to about 4 g / L).

[0062] In an exemplary aspect, the method includes adding glucose to the cell culture medium according to a glucose feeding protocol. In some aspects, the glucose feeding protocol begins after an initial period. For example, in some aspects, the initial period is at least 3 or 4 days, and the glucose feeding protocol begins about 4 to about 6 days after cell culture seeding (e.g., about 4 days, about 5 days, about 6 days after cell culture seeding). In an exemplary case, the glucose feeding protocol achieves 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). The term "average glucose concentration" refers to the average concentration of glucose in the cell culture medium determined by a glucose sensor over a period of time (e.g., 1 to 2 days). In an exemplary case, the glucose feeding protocol achieves an average glucose concentration based on the fucose concentration of the cell culture medium. In some aspects, the average glucose concentration is calculated based on Equation I:

[0063] T = 3.354 - 1.388F + 0.111G + [F - 0.4375] x [1.9527(F - 0.4375)]

[0064] (Equation I)

[0065] where T is the target total afucosylated (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 medium (g / L), and G is the average glucose concentration (g / L).

[0066] In an exemplary case, (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.

[0067] TAF, HM, and afucosylated glycan levels

[0068] In an exemplary embodiment, 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 glycan in the composition is less than or about 10%. In an exemplary aspect, the level of TAF glycan 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 an exemplary aspect, the level of TAF glycan in the composition is greater than or about 4%, e.g., between about 4% and 10%. In some aspects, the level of TAF glycan in the composition is about 2% to about 6% or about 2.5% to about 5%. In some aspects, the level of TAF glycan 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 an exemplary aspect, the level of TAF glycan is about 2% to about 5% or about 2% to about 4%.

[0069] In an exemplary aspect of a method for 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 glycan in the composition is less than about 10%.

[0070] In an exemplary embodiment, 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 glycan 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 an exemplary aspect, the level of high-mannose glycan 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%.

[0071] In an exemplary embodiment, the methods disclosed herein produce recombinant glycosylated protein compositions (e.g., antibody compositions or antibody-binding protein compositions), wherein the level of afucosylated glycan 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 an exemplary aspect, the level of afucosylated glycan in the antibody composition is from 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%.

[0072] Methods for glycan measurement

[0073] There are a variety of methods known in the art for assessing the glycan types present in a composition containing a glycoprotein or for determining, detecting, or measuring the glycan profile of a particular sample containing a glycoprotein. Suitable methods include, but are not limited to, cationic MALDI-TOF analysis, anionic 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 [Biochemical Journal] 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 B], 2005, Vol. 825, No. 2, pp. 124-133; Ruhaak L.R., Anal Bioanal Chem [Analytical and Bioanalytical Chemistry], 2010, Vol. 397:3457-3481, and Geoffrey, R.G. et al. Analytical Biochemistry [Biochemical Analysis] 1996, Vol. 240, pp. 210-226. In addition, the examples described herein depict suitable methods for assessing the glycan types present in a composition containing a glycoprotein.

[0074] Regarding the present disclosure, cell cultures can be maintained under any set of conditions suitable for the production of recombinant glycosylated proteins. 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 prior to inoculation is shaken (e.g., at 70 rpm) in a CO2 incubator under standard humidified conditions at 5% CO2. In an exemplary aspect, the cell culture is inoculated at an inoculation density of about 10 6 cells / mL in 1.5 L of medium.

[0075] In an exemplary aspect, the method of the present disclosure includes maintaining the glycosylation-capable cells in a cell culture medium having a pH of from about 6.85 to about 7.05, for example, in various aspects, 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 some aspects, the cell culture medium has a pH of from about 6.9 to about 7.0.

[0076] In an exemplary aspect, the method includes maintaining the cell culture at a temperature between 30°C and 40°C. In an exemplary embodiment, the temperature is between about 32°C and about 38°C or between about 35°C and about 38°C.

[0077] In an exemplary aspect, the method includes maintaining the osmotic pressure between about 200 mOsm / kg and about 500 mOsm / kg. In an exemplary aspect, the method includes maintaining the osmotic pressure between about 225 mOsm / kg and about 400 mOsm / kg or between about 225 mOsm / kg and about 375 mOsm / kg. In an exemplary aspect, the method includes maintaining the osmotic pressure between about 225 mOsm / kg and about 350 mOsm / kg. In various aspects, the 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.

[0078] In an exemplary aspect, the method includes maintaining the dissolved oxygen (DO) level of the cell culture at about 20% to about 60% oxygen saturation during an initial cell culture period. In an exemplary instance, 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 an initial cell culture period. In an exemplary instance, 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 an initial cell culture period. In an exemplary aspect, the DO level is about 35 mm Hg to about 85 mm Hg or about 40 mm Hg to about 80 mm Hg or about 45 mm Hg to about 75 mm Hg.

[0079] The cell culture is maintained in any one or more media. In an exemplary aspect, the cell culture is maintained in a medium suitable for cell growth and / or provided with one or more feed media according to any suitable feeding scheme. In an exemplary aspect, the method includes maintaining the cell culture in a medium containing glucose, lactate, ammonia, glutamine, and / or glutamate. In an exemplary aspect, the method includes maintaining the cell culture in a medium containing less than about 1 μM manganese during an initial cell culture period. In an exemplary aspect, the method includes maintaining the cell culture in a medium containing about 0.25 μM to about 1 μM manganese. In an exemplary aspect, the method includes maintaining the cell culture in a medium containing a negligible amount of manganese. In an exemplary aspect, the method includes maintaining the cell culture in a medium containing less than or about 50 ppb copper during an initial cell culture period. In an exemplary aspect, the method includes maintaining the cell culture in a medium containing less than or about 40 ppb copper during an initial cell culture period. In an exemplary aspect, the method includes maintaining the cell culture in a medium containing less than or about 30 ppb copper during an initial cell culture period. In an exemplary aspect, the method includes maintaining the cell culture in a medium containing less than or about 20 ppb copper during an initial cell culture period. In an exemplary aspect, the medium contains more than or about 5 ppb or more than or about 10 ppb copper. In an exemplary aspect, the cell medium contains mannose. In an exemplary aspect, the cell medium does not contain mannose.

[0080] In an exemplary embodiment, the type of cell culture is fed-batch culture or continuous perfusion culture. However, the methods of the present disclosure are advantageously not limited to any particular type of cell culture.

[0081] Cell

[0082] This disclosure relates to methods of generating recombinant glycosylated protein compositions (e.g., antibody compositions or antibody-binding protein compositions), the methods comprising maintaining glycosylation-capable cells in a cell culture medium. In exemplary aspects, the glycosylation-capable cells are eukaryotic cells, including but not limited to yeast cells, filamentous fungal cells, protozoan cells, algal cells, insect cells, or mammalian cells. Such host cells are described in the art. See, for example, Frenzel et al., Front Immunol [Frontiers in Immunology] 4:217 (2013). In exemplary aspects, the eukaryotic cells are mammalian cells. In exemplary aspects, the eukaryotic cells are non-human mammalian cells. In some aspects, the cells are Chinese hamster ovary (CHO) cells and their derivatives (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 their derivatives (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 fibroblasts L929, mouse neuroblastoma cells N2a, human breast cancer cells MCF-7, retinoblastoma cells Y79, human retinoblastoma cells SO-Rb50, human hepatoma cells Hep G2, mouse B myeloma cells J558L, or baby hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull [Advanced Pharmaceutical Bulletin] 3(2):257-263 (2013)).

[0083] Cells that are not glycosylation-capable can also be converted to glycosylation-capable cells, for example, by transfecting them with genes encoding the relevant enzymes necessary for glycosylation. Exemplary enzymes include but are not limited to oligosaccharyltransferase, glycosidases, glucosidase I, glucosidase II, calnexin / calreticulin, glycosyltransferases, mannosidases, GlcNAc transferases, galactosyltransferases, and sialyltransferases.

[0084] In an exemplary embodiment, the glycosylation-capable cells have not been genetically modified to alter the activity of enzymes in the de novo pathway or salvage pathway. These two fucose metabolic pathways are shown in Figure 2In an exemplary embodiment, the glycosylation-capable cells are not genetically modified to alter the activity of any one or more of the following: fucosyltransferases (FUT, such as FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9), fucose kinase, 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 glycosylation-capable cells are not genetically modified to knockout the gene encoding FX.

[0085] In an exemplary embodiment, the glycosylation-capable cells are not genetically modified to alter the activity of β(1,4)-N-acetylglucosaminyltransferase III (GNTIII) or GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD). In an exemplary aspect, the glycosylation-capable cells are not genetically modified to overexpress GNTIII or RMD.

[0086] Recombinant glycosylated protein

[0087] In an exemplary embodiment, the recombinant glycosylated protein comprises an amino acid sequence containing one or more N-glycosylation consensus sequences of the following formula:

[0088] Asn-Xaa1-Xaa2

[0089] wherein Xaa1 is any amino acid other than Pro, and Xaa2 is Ser or Thr.

[0090] In an exemplary embodiment, the recombinant glycosylated protein comprises a crystallizable fragment (Fc) polypeptide. As used herein, the term "Fc polypeptide" includes polypeptides of native and mutant protein forms derived from the Fc region of an antibody. Also included are truncated forms of such polypeptides containing a hinge region that promotes dimerization. Fusion proteins containing an Fc portion (and oligomers formed therefrom) offer the advantage of facile purification 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.

[0091] 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 form, comprising a heavy chain and a light chain and comprising variable and constant regions. For example, an antibody can be IgG, which is a "Y-shaped" structure of two pairs of identical polypeptide chains, each pair having one "light" chain (usually having a molecular weight of about 25 kDa) and one "heavy" chain (usually having a molecular weight of about 50-70 kDa). Antibodies have variable and constant regions. In the IgG form, the variable region is generally about 100-110 or more amino acids, contains three complementarity determining regions (CDRs), which are mainly responsible for antigen recognition and vary greatly from other antibodies that bind 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).

[0092] Briefly, in the antibody framework, the CDRs are embedded within the framework in the variable regions of the heavy and light chains, where these CDRs constitute the regions mainly responsible for antigen binding and recognition. The variable region contains at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883), within the framework regions (designated framework regions 1-4 by Kabat et al., 1991, i.e., FR1, FR2, FR3, and FR4; see also Chothia and Lesk, 1987, supra).

[0093] Human light chains are divided into kappa and lambda light chains. Heavy chains are divided into mu, delta, gamma, alpha, or epsilon, and the isotypes of antibodies are 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 can be, for example, a kappa or lambda type light chain constant region, such as a human kappa or lambda type light chain constant region. The heavy chain constant region can be, for example, an alpha, delta, epsilon, gamma, or mu type heavy chain constant region, such as a human alpha, delta, epsilon, gamma, or mu type heavy chain constant region. Thus, 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.

[0094] In various aspects, the antibody can be a monoclonal antibody or a polyclonal antibody. In some aspects, the antibody comprises a sequence that is 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 can be considered a mammalian antibody, such as a mouse antibody, rat antibody, rabbit antibody, goat antibody, horse antibody, chicken antibody, hamster antibody, pig antibody, human antibody, etc. In certain aspects, the recombinant glycosylated protein is a human antibody. In certain 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 that contains a constant domain from one species and a variable domain from a second species, or more generally, an antibody that contains amino acid sequence segments from at least two species. The term "humanized," when used in reference to an antibody, refers to an antibody that has at least the CDR regions from a non-human source and has been engineered to have a structure and immunological function that is more similar to a true human antibody. For example, humanization can involve grafting the CDRs from a non-human antibody (such as a mouse antibody) into a human antibody. Humanization can also involve selecting amino acid substitutions to make the non-human sequence look more like a human sequence.

[0095] In various aspects, antibodies are cleaved into fragments by enzymes such as papain and pepsin. Papain cleaves an antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves an antibody to produce an F(ab’)2 fragment and a pFc’ fragment. In an exemplary aspect, the recombinant glycosylated protein is an antibody fragment that retains at least one glycosylation site, such as Fab, Fc, F(ab’)2, or pFc’. With respect to the methods of the present disclosure, the antibody may lack certain portions of the antibody and may be an antibody fragment. In multiple aspects, the antibody fragment contains a glycosylation site. In some aspects, the fragment is a “glycosylated Fc fragment” that includes at least a portion of the Fc region of an antibody that is post-translationally glycosylated in a eukaryotic cell.

[0096] Architectures of antibodies have been developed to generate an increasing number of alternative antibody forms that span a molecular weight range of at least or about 12 - 150 kDa and valencies (n) from monomer (n = 1), dimer (n = 2), and trimer (n = 3) to tetramer (n = 4) and potentially higher; such alternative antibody forms are referred to herein as “antibody protein products” or “antibody binding proteins”.

[0097] Antibody protein products can be antibody fragments that retain full antigen-binding ability, such as antigen-binding forms of scFv, Fab, and VHH / VH. The smallest antigen-binding fragment that retains its complete antigen-binding site is the Fv fragment, which consists entirely of variable (V) regions. A soluble flexible amino acid peptide linker is used to link the V regions to an scFv (single-chain fragment variable) fragment to stabilize the molecule, or a constant (C) domain is added to the V region to produce a Fab fragment [antigen-binding fragment]. Both scFv and Fab are widely used fragments that can be readily produced in a prokaryotic host. Other antibody protein products include disulfide-stabilized scFv (ds-scFv), single-chain Fab (scFab), and dimeric and multimeric antibody forms such as bispecific, trispecific, and tetravalent antibodies, or different forms of miniantibodies (miniAb) that comprise scFv linked to an oligomeric domain. The smallest fragments are the VHH / VH of camelid heavy-chain Abs and single-domain Abs (sdAb). The building block most commonly used to generate novel antibody forms is the single-chain variable (V) domain antibody fragment (scFv), which comprises V domains from the heavy and light chains (VH and VL domains) linked 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 bioactive peptide grafted onto an Fc domain. Peptibodies are well described in the art. See, for example, Shimamoto et al., mAbs 4(5):586 - 591 (2012).

[0098] Other antibody protein products include single-chain antibodies (SCA), bispecific antibodies, trispecific antibodies, tetraspecific antibodies; bispecific or trispecific antibodies, etc. Bispecific antibodies can be divided into five major categories: BsIgG, appended IgG, BsAb fragments, bispecific fusion proteins, and BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology 67(2) Part A:97-106 (2015).

[0099] In an exemplary aspect, the recombinant glycosylated protein comprises any one of these antibody protein products (e.g., scFv, FabVHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g., bispecific antibody, trispecific antibody, tetraspecific antibody), miniAb, peptibody, VHH / VH of camel heavy-chain antibody, sdAb, diabody; trispecific antibody; tetraspecific antibody; bispecific or trispecific antibody, BsIgG, appended 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 an exemplary aspect, the antibody protein product can be a glycosylated Fc fragment conjugated to an antibody-binding fragment (“glycosylated Fc fragment antibody product”).

[0100] 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.

[0101] Advantageously, these methods are not limited to the antigen specificity of antibodies. Thus, antibodies can have any binding specificity for almost any antigen. In exemplary aspects, the antibodies bind to hormones, growth factors, cytokines, cell surface receptors, or any of their ligands. In exemplary aspects, the antibodies bind to proteins expressed on the cell surface of immune cells. In exemplary aspects, the antibodies bind to cluster of differentiation molecules 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, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, 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, CD79α, 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, CD120a, CD120b, CD121a, CDw121b, CD122, CD123, CD124, CD125, CD126, CD127, CDw128, CD129, CD130, CDw131, CD132, CD134, CD135, CDw136CDw137, 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.

[0102] In an exemplary aspect, the antibody is one of those described in: U.S. Patent No. 7,947,809 and U.S. Patent Application Publication No. 2009 / 0041784 (glucagon receptor); U.S. Patent No. 7,939,070, U.S. Patent No. 7,833,527, U.S. Patent No. 7,767,206 and U.S. Patent No. 7,786,284 (IL-17 receptor A); U.S. Patent No. 7,872,106 and U.S. Patent No. 7,592,429 (sclerostin); U.S. Patent No. 7,871,611, U.S. Patent No. 7,815,907, U.S. Patent No. 7,037,498, U.S. Patent No. 7,700,742 and U.S. Patent Application Publication No. 2010 / 0255538 (IGF-1 receptor); U.S. Patent No. 7,868,140 (B7RP1); U.S. Patent No. 7,807,159 and U.S. Patent Application Publication No. 2011 / 0091455 (myostatin); U.S. Patent No. 7,736,644, U.S. Patent No. 7,628,986, U.S. Patent No. 7,524,496 and U.S. Patent Application Publication No. 2010 / 0111979 (deleted mutant of epidermal growth factor receptor); U.S. Patent No. 7,728,110 (SARS coronavirus); U.S. Patent No. 7,718,776 and U.S. Patent Application Publication No. 2010 / 0209435 (OPGL); U.S. Patent No. 7,658,924 and U.S. Patent No. 7,521,053 (angiopoietin-2); U.S. Patent No. 7,601,818, U.S. Patent No. 7,795,413, U.S. Patent Application Publication No. 2009 / 0155274, U.S. Patent Application Publication No. 2011 / 0040076 (NGF); U.S. Patent No. 7,579,186 (TGF-β type II receptor); U.S. Patent No. 7,541,438 (connective tissue growth factor); U.S. Patent No. 7,438,910 (IL1-R1); U.S. Patent No. 7,423,128 (properdin); U.S. Patent No. 7,411,057, U.S. Patent No. 7,824,679, U.S. Patent No. 7,109,003, U.S. Patent No. 6,682,736, U.S. Patent No. 7,132,281 and U.S. Patent No. 7,807,797 (CTLA-4); U.S. Patent No. 7,084,257, U.S. Patent No. 7,790,859, U.S. Patent No. 7,335,743, U.S. Patent No. 7,084,257 and U.S. Patent Application Publication No. 2011 / 0045537 (interferon-γ); U.S. Patent No. 7,932,372 (MAdCAM); U.S. Patent No. 7,906,625, U.S. Patent Application Publication No. 2008 / 0292639 and U.S. Patent Application Publication No. 2011 / 0044986 (amyloid); U.S. Patent No. 7,815,907 and U.S. Patent No. 7,700,742 (insulin-like growth factor I); U.S. Patent No. 7,566,772 and U.S. Patent No. 7,964,193 (interleukin-1β);U.S. Patent No. 7,563,442, U.S. Patent No. 7,288,251, U.S. Patent No. 7,338,660, U.S. Patent No. 7,626,012, U.S. Patent No. 7,618,633, and U.S. Patent Application Publication No. 2010 / 0098694 (CD40); U.S. Patent No. 7,498,420 (c-Met); U.S. Patent No. 7,326,414, U.S. Patent No. 7,592,430, and U.S. Patent No. 7,728,113 (M-CSF); U.S. Patent No. 6,924,360, U.S. Patent No. 7,067,131, and U.S. Patent No. 7,090,844 (MUC18); U.S. Patent No. 6,235,883, U.S. Patent No. 7,807,798, and U.S. Patent Application Publication No. 2010 / 0305307 (epidermal growth factor receptor); U.S. Patent No. 6,716,587, U.S. Patent No. 7,872,113, U.S. Patent No. 7,465,450, U.S. Patent No. 7,186,809, U.S. Patent No. 7,317,090, and U.S. Patent No. 7,638,606 (interleukin-4 receptor); U.S. Patent Application Publication No. 2011 / 0135657 (β-KLOTHO); U.S. Patent No. 7,887,799 and U.S. Patent No. 7,879,323 (fibroblast growth factor-like polypeptide); U.S. Patent No. 7,867,494 (IgE); U.S. Patent Application Publication No. 2010 / 0254975 (α-4β-7); U.S. Patent Application Publication No. 2010 / 0197005 and U.S. Patent No. 7,537,762 (activin receptor-like kinase 1); U.S. Patent No. 7,585,500 and U.S. Patent Application Publication No. 2010 / 0047253 (IL-13); U.S. Patent Application Publication No. 2009 / 0263383 and U.S. Patent No. 7,449,555 (CD148); U.S. Patent Application Publication No. 2009 / 0234106 (activin A); U.S. Patent Application Publication No. 2009 / 0226447 (angiopoietin-1 and angiopoietin-2); U.S. Patent Application Publication No. 2009 / 0191212 (angiopoietin-2); U.S. Patent Application Publication No. 2009 / 0155164 (C-FMS); U.S. Patent No. 7,537,762 (activin receptor-like kinase-1); U.S. Patent No. 7,371,381 (galanin); U.S. Patent Application Publication No. 2007 / 0196376 (insulin-like growth factor); U.S. Patent No. 7,267,960 and U.S. Patent No. 7,741,115 (LDCAM); US7265212 (CD45RB); U.S. Patent No. 7,709,611, U.S. Patent Application Publication No. 2006 / 0127393, and U.S. Patent Application Publication No. 2010 / 0040619 (DKK1); U.S. Patent No. 7,807,795, U.S. Patent Application Publication No. 2003 / 0103978, and U.S. Patent No. 7,923,008 (osteoprotegerin); U.S. Patent Application Publication No. 2009 / 0208489 (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 (hepatic bactericidal 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. 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 protein-1-like polypeptide); U.S. Patent No. 7695948 and U.S. Patent No. 7199224 (TNF-α converting enzyme); U.S. Patent 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. 7081523 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 antigen); 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 (matrix metalloproteinase inhibitor); U.S. Patent No. 6596852 (LERK-5);U.S. Patent No. 6,232,447 (LERK-6); U.S. Patent No. 6,500,429 (Brain-derived neurotrophic factor); U.S. Patent No. 6,184,359 (Epithelial-derived T cell factor); U.S. Patent No. 6,143,874 (Neurotrophic factor NNT-1); U.S. Patent Application Publication No. 2011 / 0027287 (Proprotein convertase subtilisin / kexin type 9 (PCSK9)); U.S. Patent Application Publication No. 2011 / 0014201 (IL-18 receptor); and U.S. Patent Application Publication No. 2009 / 0155164 (C-FMS). For the purposes of their disclosure of 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 related to the corresponding targets of antigen-binding proteins or antibodies containing variable domains, the above patents and published patent applications are incorporated herein by reference in their entirety.;

[0103] In an exemplary embodiment, the antibody is one of the following: muromonab-CD3 (commercially available under the trade name Orthoclone ), abciximab (commercially available under the trade name ), rituximab (commercially available under the trade name ), basiliximab (commercially available under the trade name ), daclizumab (commercially available under the trade name ), palivizumab (commercially available under the trade name ), infliximab (commercially available under the trade name ), trastuzumab (commercially available under the trade name ), alemtuzumab (commercially available under the trade name ), adalimumab (commercially available under the trade name ), tositumomab-I131 (commercially available under the trade name ), efalizumab (commercially available under the trade name ), cetuximab (commercially available under the trade name ), ibritumomab tiuxetan (commercially available under the trade name ), omalizumab (commercially available under the trade name ), bevacizumab (commercially available under the trade name ), natalizumab (commercially available under the trade name ), ranibizumab (commercially available under the trade name ), panitumumab (commercially available under the trade name ), eculizumab (commercially available under the trade name Commercially available products), certolizumab (under the trade name Commercially available products), golimumab (under the trade name Commercially available products), canakinumab (under the trade name Commercially available products), catumaxomab (under the trade name Commercially available products), ustekinumab (under the trade name Commercially available products), tocilizumab (under the trade name Commercially available products), ofatumumab (under the trade name Commercially available products), denosumab (under the trade name Commercially available products), belimumab (under the trade name Commercially available products), raxibacumab, ipilimumab (under the trade name Commercially available products) and pertuzumab (under the trade name Commercially available products). In an exemplary embodiment, the antibody is one of the following: an anti-TNFα antibody, such as adalimumab, infliximab, etanercept, golimumab, and pegsiticizumab; 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, an anti-BAFF antibody, such as belimumab; an anti-CD20 antibody, such as rituximab; an anti-CD22 antibody, such as epratuzumab; an anti-CD25 antibody, such as daclizumab; an anti-CD30 antibody, such as iratumumab; an anti-CD33 antibody, such as gemtuzumab; an anti-CD52 antibody, such as alemtuzumab; an anti-CD152 antibody, such as ipilimumab; an anti-EGFR antibody, such as cetuximab; an anti-HER2 antibody, such as trastuzumab and pertuzumab; an anti-IL6 antibody, such as siltuximab; and an anti-VEGF antibody, such as bevacizumab; an anti-IL6 receptor antibody, 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 sequences of SEQ ID NO:1 and 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 its biosimilar. The term rituximab refers to an IgG1κ chimeric mouse / human monoclonal antibody that binds to the CD20 antigen (see CAS No.: 174722-31-7; DrugBank - DB00073; Kyoto Encyclopedia of Genes and Genomes (KEGG) entry D02994). In an exemplary aspect, the antibody comprises a light chain containing CDR1, CDR2, and CDR3 listed in Table A. In an exemplary aspect, the antibody comprises a heavy chain containing CDR1, CDR2, and CDR3 listed in Table A. In different cases, the antibody comprises VH and VL listed in Table A or comprises VH-IgG1 and VL-IgGκ sequences.

[0104] Table A: Amino Acid Sequence of Rituximab

[0105]

[0106]

[0107] LC, light chain; HC, heavy chain; VL, variable light chain; VH, variable heavy chain.

[0108] In exemplary aspects, the antibody is an anti-EGFR antibody, e.g., 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 that binds to the HER2 / neu antigen (see CAS No.: 180288-69-1; DrugBank - DB00072; Kyoto Encyclopedia of Genes and Genomes (KEGG) entry D03257). 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 various cases, the antibody comprises VH and VL listed in Table B or comprises VH-IgG1 and VL-IgGκ sequences.

[0109] Table B: Amino Acid Sequence of Trastuzumab

[0110]

[0111]

[0112] LC, light chain; HC, heavy chain; VL, variable light chain; VH, variable heavy chain.

[0113] Additional Steps

[0114] In multiple aspects, the methods disclosed herein include additional steps. For example, in some aspects, the method includes one or more upstream or downstream steps related to the production, purification, and formulation of recombinant glycosylated proteins. In an exemplary embodiment, the method includes the step of generating a host cell that expresses 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, 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, e.g., Frenzel et al., Front Immunol [Frontiers in Immunology] 4:217 (2013) and the "Cells" section herein. For example, in some cases, the method includes introducing a vector containing nucleic acid into the host cell, the nucleic acid comprising a nucleotide sequence encoding the recombinant glycosylated protein or a polypeptide chain thereof.

[0115] In an exemplary embodiment, the methods disclosed herein include steps for separating and / or purifying a recombinant glycosylated protein (e.g., a recombinant antibody) from a culture. In an exemplary aspect, the method includes one or more chromatography steps, the one or more chromatography including but not limited to, for example, affinity chromatography (e.g., Protein A affinity chromatography), ion exchange chromatography, and / or hydrophobic interaction chromatography. In an exemplary aspect, the method includes steps for generating crystalline biomolecules from a solution comprising a recombinant glycosylated protein.

[0116] 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.

[0117] Composition

[0118] The present disclosure provides compositions comprising recombinant glycosylated proteins. In an exemplary embodiment, the composition is prepared by the inventive method of producing the recombinant glycosylated protein compositions described herein. In an exemplary aspect, the recombinant glycosylated protein is an antibody. Accordingly, 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 the TAF glycoform, the HM glycoform, and / or the afucosylated glycoform of the antibody. Compositions comprising antibody fragments or antibody protein products are also provided. In various aspects, the antibody fragment, antibody protein product, glycosylated Fc fragment, or glycosylated Fc fragment antibody product comprises a glycosylation site. In an exemplary embodiment, the antibody composition is produced by a method comprising maintaining glycosylation-capable cells in a cell culture medium comprising fucose, wherein fucose is present in the 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 glycosylation-capable cells in a cell culture medium comprising fucose, wherein fucose is present in the medium at a concentration between about 0.1 g / L and about 1.0 g / L, and wherein the glycosylation-capable cells have not been genetically modified to alter the activity of enzymes in the de novo pathway or salvage pathway. In an exemplary embodiment, the antibody composition is produced by a method comprising maintaining glycosylation-capable cells in a cell culture medium comprising fucose and glucose, wherein fucose is present in the medium at a concentration from about 0.1 g / L to about 1.0 g / L, and adding glucose to the cell culture medium according to a glucose feeding protocol to achieve an average glucose concentration of about 10 g / L or less. In an exemplary embodiment, the antibody composition is produced after implementing a method of modulating the level of TAF glycan, afucosylated glycan, or high-mannose glycan of the antibody composition produced by the glycosylation-capable cells. In an exemplary aspect, the antibody composition is produced after implementing a method of modulating the level of TAF glycan, the method comprising (A) adding fucose to a cell culture medium comprising glycosylation-capable cells to achieve a fucose concentration of about 0.1 g / L to about 1.0 g / L, thereby reducing the level of TAF glycan; (B) adding glucose to a cell culture medium comprising glycosylation-capable cells to achieve a glucose concentration below about 10 g / L, thereby increasing the level of TAF glycan; or (C) both (A) and (B).In an exemplary aspect, the antibody composition is produced after implementing a method of modulating the level of afucosylated glycans of an antibody composition produced by a cell having glycosylation ability, the method comprising (A) adding fucose to a cell culture medium containing a cell having glycosylation ability to achieve a fucose concentration of from about 0.1 g / L to about 1.0 g / L, thereby reducing the level of afucosylated glycans; (B) adding glucose to a cell culture medium containing a cell having glycosylation ability to achieve a glucose concentration of less than or about 10 g / L, thereby increasing the level of afucosylated glycans; or (C) both (A) and (B). In an exemplary case, the antibody composition is produced after implementing a method of modulating the level of high-mannose glycans of an antibody composition produced by a cell having glycosylation ability, the method comprising adding glucose to a cell culture medium containing a cell having glycosylation ability to achieve a glucose concentration of less than or about 10 g / L, thereby increasing the level of high-mannose glycans.

[0119] In an exemplary aspect, less than or about 50% (such as 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 the TAF glycoform. In an exemplary aspect, less than about 10% (such as 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 the TAF glycoform. In an exemplary aspect, about 4% to about 10% of the recombinant glycosylated proteins in the composition are the TAF glycoform. In an exemplary aspect, about 2% to about 6% of the recombinant glycosylated proteins in the composition are the TAF glycoform. In an exemplary aspect, about 2.5% to about 5% of the recombinant glycosylated proteins in the composition are the TAF glycoform. In an exemplary aspect, less than or about 4% of the recombinant glycosylated proteins in the composition are the TAF glycoform. In an exemplary aspect, less than or about 4% and greater than or about 2% of the recombinant glycosylated proteins in the composition are the TAF glycoform.

[0120] In exemplary aspects, the compositions of the present disclosure have a glycan profile with 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 glycoform. In exemplary aspects, the compositions of the present disclosure have a glycan profile with 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 glycoform. In exemplary aspects, the compositions of the present disclosure have a glycan profile comprising from about 4% to about 10% TAF glycoform. In exemplary aspects, the compositions of the present disclosure have a glycan profile of from about 2% to about 6% TAF glycoform. In exemplary aspects, the compositions of the present disclosure have a glycan profile of from about 2.5% to about 5% TAF glycoform. In exemplary aspects, the compositions of the present disclosure have a glycan profile with less than or about 4% TAF glycoform. In exemplary aspects, the compositions of the present disclosure have a glycan profile with less than or about 4% and greater than or about 2% TAF glycoform.

[0121] In exemplary aspects, less than or about 5% of the recombinant glycosylated protein (e.g., an antibody or antibody-binding protein) in the composition is a defucosylated glycoform. In exemplary aspects, less than or about 4% of the recombinant glycosylated protein (e.g., an antibody or antibody-binding protein) in the composition is a defucosylated glycoform. In exemplary aspects, less than or about 3.5% of the recombinant glycosylated protein (e.g., an antibody or antibody-binding protein) in the composition is a defucosylated glycoform. In exemplary aspects, from about 0.8% to about 2.8% of the recombinant glycosylated protein in the composition is a defucosylated glycoform. 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%.

[0122] In exemplary aspects, less than or about 5% of the recombinant glycosylated protein (e.g., an antibody or antibody-binding protein) in the composition is of the high-mannose glycoform. In exemplary aspects, less than or about 4% of the recombinant glycosylated protein (e.g., an antibody or antibody-binding protein) in the composition is of the high-mannose glycoform. In exemplary aspects, less than or about 3.5% of the recombinant glycosylated protein (e.g., an antibody or antibody-binding protein) in the composition is of the high-mannose glycoform. In exemplary aspects, about 0.7% to about 3.0% of the recombinant glycosylated protein in the composition is of the high-mannose glycoform. In some aspects, the level of high-mannose glycan 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%.

[0123] In an exemplary embodiment, the composition is combined with a pharmaceutically acceptable carrier, diluent, or excipient. Accordingly, provided herein is a pharmaceutical composition comprising the 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.

[0124] Cell culture medium

[0125] The present disclosure provides a cell culture medium comprising: (a) glycosylation-capable cells 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 glycosylation-capable cells can be any of the cells described herein. In an exemplary case, the glycosylation-capable cells are not genetically modified to alter the activity of enzymes in the de novo or salvage pathways, optionally, wherein the glycosylation-capable cells are not genetically modified to knockout the genes 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 less than about 10 g / L or less than about 9 g / L, such as about 6 g / L or lower or about 0.5 g / L to about 4 g / L. In some aspects, the culture medium comprises fucose. In an exemplary aspect, the pH of the culture medium is about 6.85 to about 7.05, optionally, about 6.90 to about 7.00. In an exemplary case, the glycosylation-capable cells are not genetically modified to alter the activity of enzymes in the de novo or salvage pathways. For example, the glycosylation-capable cells are not genetically modified to knockout the genes encoding GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase. In an exemplary aspect, the antibody is an IgG antibody, optionally, an IgG1 antibody. In an exemplary aspect, the IgG1 antibody is specific for a tumor-associated antigen (e.g., CD20). In an exemplary aspect, the cell culture medium does not comprise mannose.

[0126] Regulation method

[0127] The present disclosure further provides a method of altering or regulating the level of TAF glycan of a recombinant glycosylated protein (e.g., an antibody composition or an antibody-binding protein composition) produced by glycosylation-capable cells in a cell culture medium. In an exemplary aspect, the method comprises (A) adding fucose to a cell culture medium comprising glycosylation-capable cells to achieve a fucose concentration of about 0.1 g / L to about 1.0 g / L, thereby reducing the level of TAF glycan; (B) adding glucose to a cell culture medium comprising glycosylation-capable cells to achieve a glucose concentration less than about 10 g / L, thereby increasing the level of TAF glycan; or (C) both (A) and (B).

[0128] The present disclosure also provides methods of modulating the afucosylated glycan levels of recombinant glycoproteins (e.g., antibody compositions or antibody-binding protein compositions) produced by glycosylation-capable cells. In exemplary aspects, the methods include (A) adding fucose to a cell culture medium comprising glycosylation-capable cells to achieve a fucose concentration of from about 0.1 g / L to about 1.0 g / L, thereby reducing the level of afucosylated glycans; or (B) adding glucose to a cell culture medium comprising glycosylation-capable cells to achieve a glucose concentration of less than about 10 g / L, thereby increasing the level of afucosylated glycans; or (C) both (A) and (B).

[0129] Also provided are methods of modulating the high-mannose glycan levels of recombinant glycoproteins (e.g., antibody compositions or antibody-binding protein compositions) produced by glycosylation-capable cells. In exemplary embodiments, the methods include adding glucose to a cell culture medium comprising glycosylation-capable cells to achieve a glucose concentration of less than about 10 g / L, thereby increasing the level of HM glycans.

[0130] Thus, in some exemplary embodiments, the methods of the present disclosure relate to increasing the levels of TAF glycans, HM glycans, or afucosylated glycans of proteins (e.g., antibodies) produced by cells in a cell culture. In exemplary aspects, the level of the HM glycoform of the recombinant glycoprotein 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 glycoprotein is increased relative to a control cell culture. In exemplary aspects, the level of the afucosylated glycoform of the recombinant glycoprotein 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 glycoprotein 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 glycoprotein 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 control cells in culture, as determined by methods known to those of skill in the art.

[0131] In some aspects, the methods of the present disclosure increase the levels of TAF, HM, or afuco glycoforms to any degree or level relative to a control cell culture. For example, in some aspects, the increase provided by the methods of the present disclosure relative to a control cell culture 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, the increase provided by the methods of the present disclosure relative to a control cell culture is more than 100%, such as 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or even 1000%. In an exemplary embodiment, the level of the TAF, HM, or afuco glycoform of a protein is increased by at least about 1.5-fold relative to a control cell culture. In an exemplary embodiment, the level of the TAF, HM, or afuco glycoform of a protein is increased by at least about 2-fold relative to a control cell culture. In an exemplary embodiment, the level of the TAF, HM, or afuco glycoform of a protein is increased by at least about 3-fold relative to a control cell culture. In an exemplary embodiment, the level of the TAF, HM, or afuco glycoform of a protein is increased by at least about 4-fold or about 5-fold relative to a control cell culture.

[0132] In an exemplary aspect, an increase in the level of the TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable as early as day 1 after a change in fucose and / or glucose concentration. In an exemplary aspect, an increase in the level of the TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable as early as day 2 after the change. In an exemplary aspect, an increase in the level of the TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable as early as day 3 after the change. In an exemplary aspect, an increase in the level of the TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable as early as about day 4 after the change. In an exemplary aspect, an increase in the level of the TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable after about day 5 after the change. In an exemplary aspect, an increase in the level of the TAF glycoform of a protein is observed or observable or detectable or detectable when harvesting the recombinant glycosylated protein from the cell culture.

[0133] In an exemplary aspect, an increase in the level of the TAF glycoform of a recombinant glycosylated protein is observed for more than about day 4, day 5, or day 6 or more days of cell culture. In an exemplary aspect, an increase in the level of the TAF glycoform of a recombinant glycosylated protein is observed for 7, 8, 9, 10, 11, or 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 (after seeding). In an exemplary aspect, an increase in the level of the TAF glycoform of the protein is observed when harvesting the protein from the cell culture.

[0134] In other aspects, the methods of the present disclosure relate to reducing the level of the TAF glycoform of a protein produced by cells in a cell culture. In an exemplary aspect, the level of the HM glycoform of a recombinant glycosylated protein is reduced relative to a control cell culture. In an exemplary aspect, the level of one or more of Man5, Man6, Man7, Man8, and / or Man9 of a recombinant glycosylated protein is reduced relative to a control cell culture. In an exemplary aspect, the level of the defucosylated glycoform of a recombinant glycosylated protein is reduced relative to a control cell culture. In an exemplary aspect, the level of one or more of A1G0, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5 of a recombinant glycosylated protein is reduced relative to a control cell culture. In an exemplary aspect, the level of one or more of A1G1a, G0[H3N4], G0[H4N4], G0[H5N4], and FO-N[H3N3] of a recombinant glycosylated protein is reduced relative to a control cell culture. In an exemplary aspect, the method is a method of reducing the level of the TAF glycoform by about 1% to about 4%, and the method comprises maintaining glycosylation-capable 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 control cells in culture as determined by methods known to those of skill in the art.

[0135] In some aspects, the methods of the present disclosure reduce the level of one or more of TAF, HM, or afuco glycoforms to any degree or level relative to a control cell culture. For example, 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%) relative to the level of a control cell culture. In an exemplary embodiment, the reduction provided by the methods of the present disclosure is more than about 100%, e.g., 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 a control cell culture. In an exemplary embodiment, the level of the TAF, HM, or afuco glycoform of a protein is reduced by at least or about 1.5-fold relative to a control cell culture. In an exemplary embodiment, the level of the TAF, HM, or afuco glycoform of a protein is reduced by at least about 2-fold relative to a control cell culture. In an exemplary embodiment, the level of the TAF, HM, or afuco glycoform of a protein is reduced by at least about 3-fold relative to a control cell culture. In an exemplary embodiment, the level of the TAF, HM, or afuco glycoform of a protein is reduced by at least about 4-fold or at least about 5-fold relative to a control cell culture.

[0136] In an exemplary aspect, a reduction in the level of the TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable as early as about day 1 post-inoculation. In an exemplary aspect, a reduction in the level of the TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable as early as about day 2 post-inoculation. In an exemplary aspect, a reduction in the level of the TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable as early as about day 3 post-inoculation. In an exemplary aspect, a reduction in the level of the TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable as early as about day 4 post-inoculation. In an exemplary aspect, a reduction in the level of the TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable after about day 5 post-inoculation. In an exemplary aspect, a reduction in the level of the TAF glycoform of a recombinant glycosylated protein is observed or observable or detectable or detectable at about the time of harvesting the protein from the cell culture.

[0137] In exemplary aspects, a decrease in the level of the TAF glycoform of a protein is observed 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, a decrease in the level of the TAF glycoform of a protein is observed 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) during cell culture (after seeding). In exemplary aspects, a decrease in the level of the TAF glycoform of a protein is observed approximately when the protein is harvested from the cell culture.

[0138] With respect to the methods of the present disclosure, the modulation, 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 method of the present invention are not performed. In exemplary aspects, the control is the level of the TAF glycoform of the protein when known methods of recombinant production are performed. In exemplary aspects, the control is the level of the TAF glycoform when known glucose or fucose concentrations are maintained during recombinant production. As used herein, the term "control cell culture" means a cell culture maintained in the same manner as the cell culture in which the steps of the method of the present invention (e.g., the cell culture of the disclosed method) are performed, except for the fucose / glucose concentration. In exemplary aspects, the control cell culture is a cell culture maintained under known operating or standard parameters, including a control fucose / glucose concentration. As used herein, the terms "control fucose concentration" or "control glucose concentration" can refer to known operating fucose / glucose concentrations, such as the fucose / glucose concentration of a cell culture maintained at a first time point or at a time point prior to performing the methods 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.

[0139] In exemplary aspects of modulating the methods of the present disclosure, glycosylation-capable cells are not genetically modified to alter the activity of enzymes of the de novo pathway or the salvage pathway. Optionally, glycosylation-capable cells are not genetically modified to knockout the genes encoding GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase.

[0140] In exemplary aspects, after performing the methods of the present disclosure, the level of TAF glycans in the antibody composition is less than about 10%, e.g., about 2% to about 6%, about 2% to about 5%, or about 2% to about 4%.

[0141] In exemplary aspects, after performing the methods of the present 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%.

[0142] In an exemplary aspect, after performing the methods of the present disclosure, the level of afucosylated glycan in the antibody composition is less than about 3.5%, optionally from about 0.8% to about 2.8%.

[0143] Regarding the modulation methods described herein that include adding fucose, the final fucose concentration of the cell culture medium is 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 in various aspects. However, any fucose concentration described herein is contemplated.

[0144] Regarding the modulation methods described herein that include adding glucose to the cell culture medium, in some aspects, glucose is added according to a glucose feeding protocol 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 Equation I:

[0145] T = 3.354 - 1.388F + 0.111G + [F - 0.4375] x [1.9527(F - 0.4375)]

[0146] Equation I

[0147] where T is the target TAF glycan %, and is from 2.5% to about 6%, from about 2.75% to about 5.5%, or from about 3% to about 5%, F is the concentration of fucose in the medium (g / L), and G is the average glucose concentration (g / L).

[0148] The present disclosure further provides methods of modulating (decreasing or increasing) the fucose - depleted glycan levels of recombinant glycoprotein compositions (e.g., antibody compositions or antibody - binding protein compositions) produced by glycosylation - capable cells. In an exemplary embodiment, the method comprises decreasing the pH of the cell culture medium by about 0.03 to about 1.2 (e.g., 0.05 to about 1.0) to decrease the fucose - depleted glycan levels of the recombinant glycoprotein composition (e.g., antibody composition or 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 the cell culture medium by about 0.03 to about 1.2 (e.g., 0.05 to about 1.0) to increase the fucose - depleted glycan levels of the recombinant glycoprotein composition (e.g., antibody composition or 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 an exemplary aspect, the method comprises decreasing 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 decrease the level of fucose - depleted glycan of the recombinant glycoprotein 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%), 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 fucose - depleted glycan of the recombinant glycoprotein 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 includes reducing 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 afucosylated glycans in 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%) 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 afucosylated glycans in 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%).

[0149] Also provided herein are methods for reducing the level of afucosylated glycans in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) produced by a glycosylation-capable cell by about 1% to about 2%. In an exemplary embodiment, the method includes reducing the pH of the cell culture medium by about 0.05 to about 1.2. Optionally, the method includes reducing the pH by about 0.05 to about 0.07 to reduce about 1% of afucosylated glycans; or reducing the pH by about 0.09 to about 1.2 to reduce more than about 1.5% of afucosylated glycans. In various aspects, the method includes culturing the cells at a pH between about 7.10 and about 7.20, optionally between about 7.12 and about 7.19.

[0150] In addition, methods are provided for reducing the level of afucosylated glycans in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) produced by a glycosylation-capable cell 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 to reduce the afucosylated glycans by about 0.8%. In some aspects, the method includes reducing the pH by about 0.05 to about 0.07 to reduce the afucosylated glycans by about 1%. In various cases, the method includes culturing the cells at a pH between about 7.05 and about 7.15, optionally between about 7.07 and about 7.13.

[0151] The present disclosure provides methods for increasing the afucosylated glycan level of recombinant glycosylated protein compositions (e.g., antibody compositions or antibody-binding protein compositions) produced by glycosylation-capable cells by about 1% to about 2%. In an exemplary embodiment, the method includes increasing the pH of the cell culture medium by about 0.05 to about 1.2. Optionally, the method includes increasing the pH by about 0.05 to about 0.07 to reduce the afucosylated glycan by about 1%; or increasing the pH by about 0.09 to about 1.2 to reduce the afucosylated glycan by more than about 1.5%. In some aspects, the method includes culturing the cells at a pH between about 7.10 and about 7.20, optionally between about 7.12 and about 7.19.

[0152] The present disclosure also provides methods for increasing the afucosylated glycan level of recombinant glycosylated protein compositions (e.g., antibody compositions or antibody-binding protein compositions) produced by glycosylation-capable cells 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 the afucosylated glycan by about 0.8%; or increasing the pH by about 0.05 to about 0.07 to reduce the afucosylated glycan by more than about 1%. In various cases, the method includes culturing the cells at a pH between about 7.05 and about 7.15, optionally between about 7.07 and about 7.13.

[0153] In any of the foregoing methods, throughout the culture, the pH of the cell culture medium is greater than 7.0, optionally higher than 7.05 and lower than 7.2. In some aspects, the afucosylated glycan level in the recombinant glycosylated protein composition (e.g., antibody composition or antibody-binding protein composition) is less than about 10%, e.g., about 6.2% to about 8.4%. In any of the foregoing methods, during the culture period, the temperature change is less than 2°C. For example, in some aspects, the temperature change during the culture is 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 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 medium can be according to any of the teachings provided herein. In an exemplary aspect, the glycosylation-capable cells are not genetically modified to alter the activity of enzymes in 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 protocol that achieves an average glucose concentration of about 10 g / L or less. Glucose can be added according to any of the teachings provided herein.

[0154] As discussed above, the TAF glycan is the sum of the HM glycan and the defucosylated glycan. Thus, the methods for modulating defucosylated glycan disclosed herein will modulate the level of TAF glycan in recombinant glycosylated protein compositions (e.g., antibody compositions or antibody-binding protein compositions) in different situations. Accordingly, provided herein are methods for modulating the level of TAF glycan in recombinant glycosylated protein compositions (e.g., antibody compositions or antibody-binding protein compositions) produced by glycosylation-capable cells. In an exemplary aspect, the method includes modulating the level of defucosylated glycan in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) according to the methods for modulating defucosylated glycan level disclosed herein. In an exemplary embodiment, the method for modulating TAF glycan includes reducing the level of defucosylated glycan in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) according to the methods for reducing defucosylated glycan level disclosed herein or increasing the level of defucosylated glycan in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody-binding protein composition) according to the methods for increasing defucosylated glycan disclosed herein.

[0155] In addition, the present disclosure provides a method for producing an antibody composition, wherein the level of defucosylated glycan in the antibody composition is from about 6.2% to about 8.4%. In an exemplary embodiment, the method includes maintaining the glycosylation-capable cells in a cell culture medium having a pH higher than 7.05 and lower than 7.2,

[0156] wherein, optionally:

[0157] (A) during the culturing period, the pH change of the cell culture medium is less than 0.15 (optionally less than 0.10) or

[0158] (B) the temperature change of the cell culture medium is no more than 2 °C or

[0159] (C) the method does not include culturing the cells in a cell culture medium containing manganese or betaine or

[0160] (D) a combination of two or three of (A), (B), and (C).

[0161] In various aspects, during the cultivation period, the pH is maintained at a pH of from about 7.07 to about 7.19 (such as 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 lower than 7.10, or about 7.10 or higher and lower than 1.15, or about 1.15 or higher up to about 7.19. In various aspects, the level of afucosylated glycan in the antibody composition is less than about 10%, optionally, from about 6.2% to about 8.4%. In various cases, the temperature change during the cultivation period is less than 2 °C, optionally, the temperature change during cultivation 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 an exemplary aspect, the cell culture medium contains from about 0.10 g / L to about 1.0 g / L fucose (optionally, from 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 from about 0.2 g / L to about 0.5 g / L. In some aspects, the glycosylation-capable cells are not genetically modified to alter the activity of enzymes in the de novo pathway or salvage pathway, and in some aspects, glucose is added to the cell culture medium according to a glucose feeding protocol that achieves an average glucose concentration of about 10 g / L or less.

[0162] Exemplary embodiment

[0163] The present disclosure provides methods for generating recombinant glycosylated protein compositions (e.g., antibody compositions or antibody-binding protein compositions). In an exemplary embodiment, the method includes maintaining glycosylation-capable cells in a cell culture medium comprising fucose and / or glucose at a specific concentration as described herein, the specific concentration depending on the level of the desired TAF glycoform. In an exemplary embodiment, the level of the TAF glycoform 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 includes maintaining glycosylation-capable cells in a cell culture medium comprising fucose, wherein fucose is present in the medium at a concentration between about 0.17 g / L and about 1.0 g / L. In an exemplary embodiment, the level of the TAF glycoform 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 includes maintaining glycosylation-capable cells in a cell culture medium comprising fucose, wherein fucose is present in the medium at a concentration between about 0.1 g / L and about 1.0 g / L, and wherein the glycosylation-capable cells are not genetically modified to alter the activity of enzymes in the de novo pathway or salvage pathway. The present disclosure also provides a method for generating a recombinant glycosylated protein composition (e.g., antibody composition or antibody-binding protein composition), the method including maintaining glycosylation-capable cells in a cell culture medium comprising fucose and glucose, wherein fucose is present in the 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 protocol to achieve an average glucose concentration of about 10 g / L or less. In an exemplary aspect, fucose is present in the medium at a concentration less than about 0.75 g / L, optionally less than about 0.6 g / L. In an exemplary case, fucose is present in the medium at a concentration of about 0.2 g / L to about 0.5 g / L. In some aspects, fucose is present in the medium for the entire duration that the glycosylation-capable cells are maintained in the cell culture. In an exemplary case, the method for generating a recombinant glycosylated protein composition (e.g., antibody composition or antibody-binding protein composition) includes maintaining glycosylation-capable cells in a first cell culture medium for an initial period of time and subsequently maintaining these glycosylation-capable cells 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 an alternative aspect, 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 inoculation of the cell culture, fucose is added to the first medium to obtain the second cell culture medium.During the maintenance of cells with glycosylation capacity 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 an initial time period, the cell culture medium contains an initial glucose concentration. Optionally, the initial glucose concentration is from about 1 g / L to about 15 g / L, e.g., about 12 g / L ± 1 g / L. In an exemplary aspect, the method further comprises adding glucose to the cell culture medium according to a glucose feeding scheme. In an exemplary aspect, the glucose feeding scheme begins about 4 to about 6 days after inoculation of the cell culture. For example, the glucose feeding scheme can begin about 6 days after inoculation of the cell culture. In an exemplary case, the glucose feeding scheme reaches an average glucose concentration in the cell culture medium of about 10 g / L or less (e.g., about 9 g / L or less, about 6 g / L or less, from about 0.5 g / L to about 4 g / L). In an exemplary case, the glucose feeding scheme reaches an average glucose concentration based on the fucose concentration in the cell culture medium. In an exemplary aspect, the average glucose concentration is calculated based on Equation I:.

[0164] T = 3.354 - 1.388F + 0.111G + [F - 0.4375] x [1.9527(F - 0.4375)]

[0165] (Equation I)

[0166] Where T is the percent of total afucosylated (TAF) glycan in the antibody composition and is from about 2.5% to about 6%, from about 2.75% to about 5.5%, or from about 3% to about 5%, F is the concentration of fucose (g / L) in the medium, and G is the average glucose concentration (g / L) in the medium. In exemplary aspects, (i) the concentration of fucose is about 0.2 ± 0.1 g / L and the average glucose concentration is from 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 from 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 from about 4.5 to about 9 g / L. In exemplary aspects, the pH of the cell culture medium is from about 6.85 to about 7.05 (e.g., from about 6.90 to about 7.00). In some aspects, the glycosylation-capable cells are not genetically modified to alter the activity of enzymes in the de novo or salvage pathways. For example, the glycosylation-capable cells are not genetically modified to knockout the genes encoding GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase. In an exemplary case, the level of total afucosylated (TAF) glycan in the antibody composition is less than about 10% (e.g., from about 2% to about 6%, from about 2% to about 5%, from about 2% to about 4%). In an exemplary case, the level of high-mannose glycan in the antibody composition is less than about 3.5% (e.g., from about 0.7% to about 3.0%). In an exemplary case, the level of afucosylated glycan in the antibody composition is less than about 3.5% (e.g., from about 0.8% to about 2.8%). In some aspects, the glycosylation-capable cells produce IgG antibodies, optionally IgG1 antibodies. In some aspects, the IgG1 antibody is specific for a tumor-associated antigen (e.g., CD20). In an exemplary aspect, the medium does not contain mannose.

[0167] Provided is a cell culture medium comprising: (a) glycosylation-capable cells comprising exogenous nucleic acid encoding an antibody; and (b) a culture medium comprising fucose at a concentration of from about 0.1 g / L to about 1.0 g / L or from about 0.17 g / L to about 1.0 g / L. In an exemplary aspect, the glycosylation-capable cells are not genetically modified to alter the activity of enzymes in the de novo or salvage pathways, optionally, wherein the glycosylation-capable cells are not genetically modified to knockout the 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 from about 0.5 g / L to about 4 g / L. In an exemplary aspect, the pH of the culture medium is from about 6.85 to about 7.05 (such as from about 6.9 to about 7.0). In some aspects, the cell culture medium does not contain mannose. In an exemplary aspect, the antibody is an IgG antibody, for example, an IgG1 antibody. In an exemplary case, the IgG1 antibody is specific for a tumor-associated antigen (such as CD20).

[0168] The present disclosure further provides methods of altering or modulating the level of TAF glycans in recombinant glycosylated protein compositions (e.g., antibody compositions or antibody-binding protein compositions) produced by glycosylation-capable cells in a cell culture medium. In an exemplary aspect, the method comprises (A) adding fucose to a cell culture medium comprising glycosylation-capable cells to achieve a fucose concentration of from 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 glycosylation-capable cells 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 of modulating the level of defucosylated glycans in recombinant glycosylated protein compositions (e.g., antibody compositions or antibody-binding protein compositions) produced by glycosylation-capable cells. In an exemplary embodiment, the method comprises (A) adding fucose to a cell culture medium comprising glycosylation-capable cells to achieve a fucose concentration of from 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 glycosylation-capable cells 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 methods of modulating the level of high-mannose glycans in recombinant glycosylated protein compositions (e.g., antibody compositions or antibody-binding protein compositions) produced by glycosylation-capable cells. In an exemplary embodiment, the method comprises adding glucose to a cell culture medium comprising glycosylation-capable cells 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 modulating method, the glycosylation-capable cells are not genetically modified to alter the activity of enzymes in the de novo or salvage pathways. For example, in some aspects, the glycosylation-capable cells are not genetically modified to knockout 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 an exemplary aspect, the fucose concentration is from about 0.17 g / L to about 1.0 g / L, optionally from about 0.2 g / L to about 0.5 g / L. In some aspects, the method further comprises adding glucose to the cell culture medium according to a glucose feeding protocol that achieves 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 Equation I:

[0169] T = 3.354 - 1.388F + 0.111G + [F - 0.4375] x [1.9527(F - 0.4375)]

[0170] Equation I

[0171] where T is the target total afucosylated (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 medium (g / L), and G is the average glucose concentration (g / L).

[0172] The following examples are given solely to describe the present disclosure and do not limit its scope in any way.

[0173] Examples

[0174] Example 1

[0175] This example describes the methods and materials used in the experiments of Example 2.

[0176] Cell lines, cell culture, and culture media

[0177] All experiments were performed using clones expressing an antibody that contains a light chain containing SEQ ID NO.1 and a heavy chain containing SEQ ID NO.2. All experiments were performed using separately bottled 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 °C).

[0178] Hydrophilic interaction liquid chromatography (HILIC) glycan profiles

[0179] The glycan profile of the enzymatically released N-linked glycans was determined using HILIC. Briefly, the glycans were incubated with a solution containing PNGase F and sodium phosphate buffer (pH 7.5) at about 37 °C for about 2 hours. Then a labeling solution containing 2-aminobenzoic acid (2-AA) and sodium cyanoborohydride was added to the PNGase F-treated glycans, and the mixture was incubated at about 80 °C for about 75 minutes. After incubation, the mixture was centrifuged to obtain a pellet of precipitated protein. The supernatant was collected and placed in a vial.

[0180] Separation of glycans by HILIC coupled with a fluorescence detector: Glycans are injected and bound to the column under high organic conditions (mobile phase A and mobile phase B are ammonium formate and acetonitrile, respectively), and then eluted with an aqueous ammonium formate buffer with an increasing gradient. High resolution is achieved using a 1.7 μm small particle column format and a 150 mm column length. The total run time (including column re - equilibration) is 155 minutes.

[0181] Example 2

[0182] This example demonstrates the effect of increasing the levels of glucose and fucose in the culture medium on the TAF%.

[0183] Cells expressing an antibody with a light chain containing SEQ ID NO:1 and a heavy chain containing SEQ ID NO:2 are added to a bioreactor containing one of the following three culture media: a control medium, a first test medium, and a second test medium. The first test medium is the same as the control medium except that it contains twice the amount of glucose, and the second test medium is the control medium containing 0.5 g / L fucose. Each medium lacks mannose. The cell culture is maintained at a pH of 6.85 to 7.05 for 12 days.

[0184] Culture medium samples are periodically collected from the bioreactor to measure glucose concentration, TAF levels, and ADCC levels. TAF and / or afucosylated (Afuc) glycan levels are determined via the HILIC N - glycan profiling procedure, and the ability to stimulate ADCC is tested using an in vitro assay. The results are shown in Table 1 below.

[0185] Table 1

[0186] <![CDATA Culture media > <![CDATA TAF level (%) > <![CDATA ADCC (%) > Target range 2.0％-4.2％ 69％-97％ Control 4.00％±0.23％ ～100％ First test 5.87％±0.23％ 152％±15％ Second test ～3.4％ ～85％

[0187] The ADCC level is expressed as the age% of the ADCC level achieved relative to a control, which is a commercially available antibody with the same amino acid sequence.

[0188] Glucose concentrations are determined throughout the culture run, and these measurements are plotted over time and related to the TAF levels of the antibodies produced by each cell culture type. The results are shown in Figure 3 . As shown, the antibodies produced by cells cultured in the first test culture show an increase in TAF, which corresponds to an increase in the glucose level in the cell culture. These results (and the results in Table 1) indicate that glucose can affect TAF levels and ADCC.

[0189] The antibodies produced by cells cultured in the second test medium containing 0.5 g / L fucose show an approximately 1% decrease in TAF ( Figure 3 ). As Figure 3As shown, the fucose concentration did not change significantly during the 12-day operation, probably because the uptake of fucose by cells was negligible.

[0190] Further analyze the different glycan species of the antibodies produced in each different medium. Interestingly, as Figure 4 shown, when cells were cultured in the first test medium (which contained twice the amount of glucose compared to the control medium), the high-mannose (HM) glycan % increased. This increase in HM glycan % was not obtained in cells cultured in the second test medium containing fucose. As Figure 4 shown, the HM glycan % of the antibodies produced by cells cultured in the second test medium containing fucose was roughly the same as that of the antibodies produced by cells cultured in the control medium.

[0191] The effect of culturing in a medium containing twice the amount of glucose (the first test medium) or a medium containing fucose (the second test medium) on the afucosylated glycan % was similar to that on the TAF glycan %. As Figure 5 shown, cells cultured in the first test medium containing a higher glucose concentration showed an increase in afucosylated glycans, while cells cultured in the second test medium containing fucose produced antibodies with reduced afucosylated glycans.

[0192] This example shows that glucose and fucose are the levers used to regulate the levels of high-mannose and afucosylated glycans and affect ADCC.

[0193] Example 3

[0194] This example shows that additional studies indicate that glucose and fucose are the levers for regulating TAF levels and ADCC.

[0195] A follow-up multivariable 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 the amounts 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 medium. Glucose was supplied at rates of 0X, 1X (control), and 2X. 0X means no glucose stock solution was added to the culture, and this translated to a residual glucose concentration of approximately 1 g / L after 6 days of cell culture. Glucose was provided only through the medium containing 12 g / L of sugar. At 1X feeding, the glucose was maintained at an average concentration of 3 ± 1 g / L after the start of glucose feeding. In 2X feeding, the average glucose level was maintained at 6 ± 1 g / L. The results showed that the fucose concentration could be changed to affect the TAF level ( Figure 6 ) and the ADCC level ( Figure 7 ).

[0196] 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 the residual glucose in the bioreactor and the glucose obtained through the perfusion medium until the glucose level dropped within the control range. Based on these experiments, according to Figure 8 the model shown, the TAF values were predicted 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 from about 0.1 g / L to about 1.0 g / L fucose and / or from about 0.5 g / L to about 4.0 g / L glucose. Using this model, the TAF values for the following different fucose and glucose concentrations were predicted: 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 indicate that there are several ways to achieve the desired TAF level and ADCC level.

[0197] To confirm Figures 9A - 9C the predictions, additional experiments were conducted. A summary of the experiments is provided in Table 2.

[0198] Table 2

[0199]

[0200] 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.

[0201] This example shows that both the glucose concentration and the fucose concentration are variable and can be manipulated to change the levels of TAF and ADCC.

[0202] Example 4

[0203] This example shows the effect of fucose on the cell culture.

[0204] Additional analyses were performed on the above cell culture. For example, the osmotic pressure of the cell culture was measured and ranged from about 175 mOsm / kg to about 345 mOsm / kg. A lack of correlation was observed between the cell culture osmotic pressure and the fucose concentration. See Figure 10 . As shown, adding fucose to the medium does not seem to affect the osmotic pressure in any particular way. The osmotic pressure varied greatly under control conditions (without fucose), indicating that the components in the medium (except fucose) have a positive effect on the osmotic pressure.

[0205] In one experiment of this fucose study, cells were inoculated into one of five bioreactors, two of which contained media without any fucose (ctrl_a and ctrl_b), and three of which contained media with 0.5 g / L fucose (fuc_a, fuc_b, and fuc_c). During the entire 12-day culture period, media samples were collected from the five cell cultures on day 0, day 3, day 5, day 7, day 9, and day 12. The fucose concentration of the samples was measured. As Figure 11 shown, the concentration of fucose did not change substantially during the entire culture period, indicating that the consumption of fucose was small and / or slow during the culture process.

[0206] In another experiment of this fucose study, cells were maintained in a cell culture in media without fucose for 12 days. In this particular experiment, the pH of the cell culture was perturbed from 7.1, causing the TAF% to increase to approximately 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 feeding rate of 0.9 g / L per day on day 9 of the culture. As Figure 12 shown, after adding fucose to the media, the TAF% decreased from approximately 5.5%. The TAF% continued to decrease to approximately 3.8% on day 12. These data indicate that the addition of fucose may occur late in the culture period and still cause the regulation of TAF%.

[0207] This example demonstrates the effect of fucose concentration in the media on the TAF level.

[0208] Example 5

[0209] This example demonstrates that maintaining glucose within the target range can occur late in the culture period.

[0210] During the 12-day culture period, three experiments were conducted to monitor the time to reach the target glucose range. For each experiment, the initial glucose concentration of each cell culture ranged from approximately 5.0 g / L to approximately 6.0 g / L. The glucose concentration of the cell media was monitored daily. As Figure 13 shown, each cell culture reached the target glucose concentration (0.5 g / L - 4.0 g / L) on different days of the culture period. In one experiment (the line with open squares), the target range was reached on day 2. In the second experiment (the dotted line with open diamonds), the target range was reached on day 4, while in the third experiment (the line with open circles), the target range was reached on day 6. Despite these differences, each cell culture reached the target range of TAF% (approximately 2.0% to approximately 4.3%) (see Figure 13The left figure; the third experiment is represented by hollow circles; the second experiment is represented by hollow diamonds; the first experiment is represented by hollow squares). These data indicate that glucose maintenance can occur in the late stage of the culture period to achieve the same culture TAF level as in the early stage of the culture period.

[0211] These data indicate that control of glucose concentration in the early and late stages results in similar TAF levels.

[0212] Example 6

[0213] This example demonstrates the effect of reducing the pH of a cell culture with and without the addition of fucose on the defucosylation level of an antibody.

[0214] Cell culture samples were removed from a 2000 L bioreactor and used to inoculate parallel 3 L bioreactors. Using a continuous fed-batch process, the 2000 L and 3 L bioreactors were fed continuously for 12 days with Feed A and Feed B. On day 5, two 3 L bioreactors were fed with fucose to a final concentration of 1.0 g / L. The defucosylation level was measured as described in Example 1, and the results are provided in Table 3.

[0215] Table 3

[0216] pH Fucose addition Defucosylation % 7.09 - 6.29 7.09 - 6.317 7.07 - 6.559 7.07 + 4.384 7.12 - 7.13 7.13 - 7.24 7.18 - 8.365 7.19 - 7.917 7.18 + 6.183

[0217] As shown in Table 3, in the absence of added fucose, when the pH was below 7.1, the average defucosylation % was 6.39, while when the pH was higher than 7.10 but lower than 7.15, the average defucosylation % was higher (defucosylation % = 7.185). When the pH was higher than 7.15, the average defucosylation % was even higher (defucosylation % = 8.276). Thus, a lower pH value is associated with a lower defucosylation %.

[0218] When fucose was added to the culture medium (to reach a final concentration of 1.0 g / L fucose), defucosylation % decreased significantly at both a lower pH (7.07) and a higher pH (7.18). When fucose was added, the average decrease in defucosylation % for each of these pH levels was 2.18%.

[0219] These results indicate that reducing the pH of the cell culture medium and / or adding fucose to the cell culture medium results in a decreased defucosylation percentage. A greater degree of decreased defucosylation percentage was observed when the pH was decreased and fucose was added to the cell culture medium.

[0220] Example 7

[0221] This example further demonstrates the effect of reducing the pH of the cell culture with and without the addition of fucose on the level of afucosylation of the antibody.

[0222] Cell culture samples from a 2000 L bioreactor were used to inoculate parallel 3 L bioreactors. Using a continuous fed-batch process, the 2000 L and 3 L bioreactors were fed continuously for 12 days using Feed A and Feed B. On day 5, some bioreactors (i.e., cell cultures) were fed fucose to a final concentration of 0.25, 0.5, or 1 g / L. The level of afucosylation was measured as described in Example 1, and the results are provided in Figure 14 .

[0223] As Figure 14 shown, the control 3 L bioreactor (control pH 7.1) showed a level of afucosylation similar to that of the original 2000 L bioreactor. Both showed an afucosylation % of about 6.5% or higher. The addition of fucose at any of the tested levels resulted in a significant decrease in afucosylation % (5.5% or lower). The lowest afucosylation percentage was observed with the addition of 0.25 g / L fucose.

[0224] In addition, as Figure 14 shown, reducing the pH from 7.1 to 7.0 without the addition of fucose to the culture medium also resulted in a decrease in afucosylation of at least about 1.0%.

[0225] All references cited herein, including publications, patent applications, and patents, are hereby incorporated by reference in their entirety as if each were individually and specifically indicated to be incorporated by reference in its entirety and set forth herein in full.

[0226] Unless otherwise indicated herein or the context clearly dictates otherwise, the terms “a / an” and “the” and similar indicatives used in the context of describing the present disclosure (particularly in the context of the following claims) will be taken to cover both the singular and the plural. Unless otherwise described, the terms “comprising,” “having,” “including,” and “containing” will be taken to be open terms (i.e., meaning “including but not limited to”).

[0227] Unless otherwise indicated herein, the recitation of numerical ranges herein is merely intended to serve as a shorthand method of referring individually to each separate value and endpoint within the range, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[0228] Unless otherwise indicated herein or otherwise 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 merely intended to better describe the disclosure and does not limit the scope of the disclosure. Language in the specification should not be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0229] Preferred embodiments of the disclosure are described herein, including the best mode known to the inventors for practicing the disclosure. Variations of those preferred embodiments will be apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than as specifically described herein. Accordingly, the disclosure includes all modifications and equivalents of the subject matter recited in the appended claims as permitted by applicable law. In addition, unless otherwise indicated herein or otherwise clearly contradicted by the context, the disclosure covers any combination of the above elements in all possible variations.

Claims

1. A method for adjusting the level of TAF glycan in an antibody composition produced by glycosylation-capable cells, wherein the level of TAF glycan in the antibody composition is less than or equal to 10%, the method comprising (A) adding fucose to a cell culture medium containing the glycosylation-capable cells to achieve a fucose concentration of 0.1 g / L to 1.0 g / L, thereby reducing the level of TAF glycan; and (B) adding glucose to the cell culture medium containing the glycosylation-capable cells according to a glucose feeding protocol that achieves an average glucose concentration of 10 g / L or less, thereby increasing the level of TAF glycan, wherein the glucose feeding protocol achieves the average glucose concentration based on the fucose concentration in the cell culture medium, and wherein the average glucose concentration is calculated based on Equation I: T = 3.354 - 1.388F + 0.111G + [F - 0.4375] x [1.9527(F - 0.4375)] (Equation I) wherein T is the target total afucosylated (TAF) glycan % in the antibody composition and is 2.5% to 6%, F is the concentration of fucose in the medium (g / L), and G is the average glucose concentration in the medium (g / L).

2. The method according to claim 1, wherein T is the target total afucosylated (TAF) glycan % in the antibody composition and is 2.75% to 5.5%.

3. The method according to claim 1, wherein T is the target total afucosylated (TAF) glycan % in the antibody composition and is 3% to 5%.

4. A method for adjusting the afucosylated glycan level in an antibody composition produced by glycosylation-capable cells, wherein the level of TAF glycan in the antibody composition is less than or equal to 10%, the method comprising (A) adding fucose to a cell culture medium containing the glycosylation-capable cells to achieve a fucose concentration of 0.1 g / L to 1.0 g / L, thereby reducing the afucosylated glycan level; and (B) adding glucose to the cell culture medium containing the glycosylation-capable cells according to a glucose feeding protocol that achieves an average glucose concentration of 10 g / L or less, thereby increasing the afucosylated glycan level, wherein the glucose feeding protocol achieves the average glucose concentration based on the fucose concentration in the cell culture medium, and wherein the average glucose concentration is calculated based on Equation I: T = 3.354 - 1.388F + 0.111G + [F - 0.4375] x [1.9527(F - 0.4375)] (Equation I) wherein T is the target total afucosylated (TAF) glycan % in the antibody composition and is 2.5% to 6%, F is the concentration of fucose in the medium (g / L), and G is the average glucose concentration in the medium (g / L).

5. The method according to claim 4, wherein T is the target total afucosylated (TAF) glycan % in the antibody composition and is 2.75% to 5.5%.

6. The method according to claim 4, wherein T is the target total afucosylated (TAF) glycan % in the antibody composition, and is 3% to 5%.

7. The method according to any one of claims 1-6, further comprising increasing the high-mannose glycan level of the antibody composition produced by glycosylation-capable cells, including adding glucose to the cell culture medium containing the glycosylation-capable cells to achieve a glucose concentration less than or equal to 10 g / L.

8. The method according to any one of claims 1-6, wherein the glycosylation-capable cells are not genetically modified to alter the activity of enzymes in the de novo pathway or salvage pathway.

9. The method according to claim 8, wherein the glycosylation-capable cells are not genetically modified to knockout the gene encoding GDP-keto-6-deoxymannose-3,5-epimerase,4-reductase.

10. The method according to any one of claims 1-6, wherein (A) the level of TAF glycan in the antibody composition is 2% to 6%, (B) the level of high-mannose glycan in the antibody composition is less than or equal to 3.5%, and / or (C) the level of afucosylated glycan in the antibody composition is less than or equal to 3.5%.

11. The method according to claim 10, wherein (A) the level of TAF glycan in the antibody composition is 2% to 5%, (B) the level of high-mannose glycan in the antibody composition is 0.7% to 3.0%, and / or (C) the level of afucosylated glycan in the antibody composition is 0.8% to 2.8%.

12. The method according to claim 11, wherein the level of TAF glycan in the antibody composition is 2% to 4%.

13. The method according to any one of claims 1-6, wherein fucose is present in the medium at a concentration of 0.17 g / L to 1.0 g / L.

14. The method according to claim 13, wherein fucose is present in the medium at a concentration less than 0.75 g / L.

15. The method according to claim 14, wherein fucose is present in the medium at a concentration less than 0.6 g / L.

16. The method according to claim 15, wherein fucose is present in the medium at a concentration of 0.2 g / L to 0.5 g / L.

17. The method according to any one of claims 1-6, wherein the fucose is present in the medium throughout the entire duration of maintaining the glycosylation-capable cells in cell culture.

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

19. The method according to claim 18, wherein the initial period of time is 24 to 72 hours.

20. The method according to claim 18, wherein the initial time period is equal to or greater than 72 hours but less than or equal to 156 hours.

21. The method according to claim 18, wherein on the 6th day after inoculation of the cell culture, fucose is added to the first culture medium to obtain the second cell culture medium.

22. The method according to any one of claims 1 - 6, wherein during the maintenance of the glycosylation-capable cells in the cell culture medium containing fucose, the fucose concentration fluctuates by 0.2 g / L or less.

23. The method according to claim 22, wherein during the maintenance of the glycosylation-capable cells in the cell culture medium containing fucose, the fucose concentration fluctuates by 0.1 g / L or less.

24. The method according to any one of claims 1 - 6, wherein the glucose feeding protocol achieves an average glucose concentration of 9 g / L or less in the cell culture medium.

25. The method according to claim 24, wherein the glucose feeding protocol achieves an average glucose concentration of 6 g / L or less in the cell culture medium.

26. The method according to claim 25, wherein the glucose feeding protocol achieves an average glucose concentration of 0.5 g / L to 4 g / L in the cell culture medium.

27. The method according to any one of claims 1 - 6, wherein the glucose feeding protocol starts 4 to 6 days after inoculation of the cell culture.

28. The method according to claim 27, wherein the glucose feeding protocol starts 6 days after inoculation of the cell culture.

29. The method according to any one of claims 1 - 6, wherein for the initial time period, the cell culture medium contains an initial glucose concentration.

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