EA045782B1 - COMMON AFUCOSYLATED GLYCOFORM OF ANTIBODIES OBTAINED IN CELL CULTURE - Google Patents

Description

Cross reference to related applications

This application claims priority under U.S. Provisional Patent Application No.

62/648308, filed March 26, 2018. The contents of each application are incorporated herein by reference.

Incorporation by reference of material submitted electronically

A machine-readable nucleotide/amino acid sequence listing, incorporated by reference in its entirety, is filed concurrently with this application and is designated as follows: a 28,547-byte ASCII (text) file entitled 52249A_Seqlisting.txt, created March 26, 2019.

BACKGROUND OF THE INVENTION

Glycosylation is one of the most common but important post-translational modifications as it plays a role in many cellular functions including, for example, protein folding, quality control, molecular transport and sorting, and cell surface receptor interactions. Glycosylation influences the therapeutic efficacy of recombinant protein-based drugs because it influences the bioactivity, pharmacokinetics, immunogenicity, solubility, and in vivo clearance of the therapeutic glycoprotein. Fc glycoform profiles, in particular, are important product quality attributes in the case of recombinant antibodies, as they directly influence the clinical efficacy and pharmacokinetics of the antibodies.

The high mannose (HM) glycoform has been found to influence the pharmacokinetic properties of some therapeutic antibodies (Goetze, et al., (2011) Glycobiology 21, 949-59; Yu, et al., (2012) MAbs 4, 475 -87). HM glycoforms not only influence the rate of antibody clearance in serum, but such glycoforms, in addition to afucosylated (afuco-) glycoforms, are capable of influencing antibody effector function or antibody-mediated killing of target cells, also known as antibody-dependent killing. cellular cytotoxicity (ADCC).

Many factors influence the structure of glycans and, therefore, the final glycosylated form (glycoform) of the protein (glycoprotein). For example, the cell line expressing the antibody, the cell culture medium, the composition of the feeding medium, and the feeding schedule during cell culture can influence the production of protein glycoforms.

Although many ways to influence the levels of specific antibody glycoforms have been proposed by research groups, there is still a need in the biopharmaceutical industry for simple and effective ways to regulate and control the levels of total afucosylated (TAF) glycoforms during the production of therapeutic antibodies using recombinant DNA technology.

Short description

For the first time, data are reported demonstrating that the concentration of fucose and/or glucose in a cell culture medium containing cells producing a recombinant glycosylated protein (eg, an antibody or antibody-based binding protein) influences the level of TAF glycoforms of the recombinant glycosylated protein produced. While large changes in the level of TAF glycoforms of a recombinant glycosylated protein (e.g., antibody or antibody-based binding protein) can be achieved by adjusting the concentration of fucose in the cell culture medium containing cells producing the recombinant glycosylated protein (e.g., antibody or antibody-based binding protein) antibody-based protein), smaller changes in the level of TAF glycoforms can be achieved by changing the glucose concentration in the cell culture medium, as described herein. The data also demonstrate that while the concentration of glucose in the cell culture medium influences the levels of high-mannose and afucosylated glycans, the concentration of fucose in the cell culture medium influences the levels of afucosylated glycans but does not affect the levels of high-mannose glycans. high mannose content. The discovery that each of these sugars, which differ in chemical formula by only one oxygen atom, leads to different effects on levels of TAF glycoforms was unexpected. Without being bound by theory, maintaining cells producing a recombinant glycosylated protein (e.g., an antibody or antibody-based binding protein) in a cell culture medium containing fucose and/or glucose at the concentrations specified herein provides a recombinant glycosylated protein composition. protein (eg, antibody or antibody-based binding protein) having a desired or predetermined or preselected level of TAF glycoforms (eg, high-mannose glycans and afucosylated glycans). Accordingly, the present invention relates to methods for preparing a recombinant glycosylated protein composition (eg, an antibody composition or an antibody binding protein composition) containing TAF glycoforms at a desired or predetermined or preselected level.

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The present invention provides methods for preparing a recombinant glycosylated protein composition (eg, an antibody composition or an antibody binding protein composition). In illustrative embodiments, the method includes maintaining glycosylation competent cells in a cell culture medium containing fucose and/or glucose at a specific concentration described herein, depending on the desired level of TAF glycoforms.

In illustrative embodiments, the level of TAF glycoforms in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) is approximately 10% or less, and in illustrative aspects the method includes maintaining glycosylation-competent cells in a cell culture medium containing fucose, wherein the fucose is present in the culture medium at a concentration of from about 0.17 g/L to about 1.0 g/L.

In illustrative embodiments, the level of TAF glycoforms in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) is approximately 10% or less, and in illustrative aspects the method includes maintaining glycosylation-competent cells in a cell culture medium containing fucose, wherein the fucose is present in the culture medium at a concentration of from about 0.1 g/L to about 1.0 g/L, and wherein the glycosylation competent cells are not genetically modified to providing a change in enzyme activity in the de novo or recycling pathway.

The present invention also provides methods for preparing a recombinant glycosylated protein composition (eg, an antibody composition or an antibody binding protein composition), comprising maintaining glycosylation competent cells in a cell culture medium containing fucose and glucose, wherein fucose is present in the culture medium at a concentration of 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 schedule that achieves an average glucose concentration, of approximately 10 g/L or less.

Provided herein are recombinant glycosylated protein compositions (eg, antibody compositions or antibody binding protein compositions) produced by the methods of the present invention. Additionally, suitable pharmaceutical compositions and cell culture media are provided. In illustrative aspects, the cell culture medium contains an exogenous nucleic acid encoding an antibody (e.g., an IgG antibody), and the culture medium contains 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 approximately 1.0 g/l. In some cases, glycosylation-competent cells have not been genetically modified to provide alteration in de novo or salvage pathway enzyme activity. Optionally, glycosylation competent cells are not genetically modified to provide knockout of the gene encoding GDPketo-6-deoxymannose-3,5-epimerase/-4-reductase. The culture medium in some aspects further comprises glucose at a concentration of less than about 10 g/L, optionally less than about 9 g/L or about 6 g/L or less (e.g., from about 0.5 g/L to about 4 g/L ). In illustrative cases, the pH of the culture medium is from about 6.85 to about 7.05, such as from 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 illustrative aspects, the antibody is specific for a tumor-associated antigen, such as the antigen comprising SEQ ID NO: 3.

Further provided herein are methods of altering or modulating the level of TAF glycans in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by glycosylation competent cells in a culture medium cells. In illustrative aspects, the method includes: (A) adding fucose to a cell culture medium containing glycosylation competent cells to achieve a fucose concentration of from about 0.1 g/L to about 1.0 g/L to reduce the level of TAF glycans; (B) adding glucose to a cell culture medium containing glycosylation competent cells to achieve a glucose concentration of less than about 10 g/L to increase TAF levels; or (C) a combination of both (A) and (B).

Also provided are methods for modulating the level of afucosylated glycans in a recombinant glycosylated protein composition (eg, an antibody composition or an antibody binding protein composition) produced by glycosylation competent cells. In illustrative embodiments, the method comprises: (A) adding fucose to a cell culture medium containing competent

- 2 045782 glycosylation of the cell, achieving a fucose concentration of from about 0.1 g/L to about 1.0 g/L, to reduce the level of afucosylated glycans; (B) adding glucose to a cell culture medium containing glycosylation competent cells to achieve a glucose concentration of approximately 10 g/L or less to increase the level of afucosylated glycans; or (C) a combination of both (A) and (B).

The present invention further provides a method for modulating the level of high mannose (HM) glycans in a recombinant glycosylated protein composition (eg, an antibody composition or an antibody binding protein composition) produced by glycosylation competent cells. In illustrative embodiments, the method includes adding glucose to a cell culture medium containing glycosylation competent cells to achieve a glucose concentration of less than about 10 g/L to increase the level of LMW glycans.

The present invention also provides methods for modulating the level of afucosylated glycans in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by glycosylation competent cells, comprising lowering the pH of the medium to cell culture by a value of from about 0.03 to about 1.2, to reduce the level of afucosylated glycans in the composition by a value of from about 0.5% to about 2%, or increasing the pH of the cell culture medium by an amount, from about 0.03 to about 1.2 to increase the level of afucosylated glycans in the composition by about 0.5% to about 2%.

The present invention provides 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 glycosylation competent cells by a value of between about 1 % to about 2%, including reducing the pH of the cell culture medium by a value of from about 0.05 to about 1.2.

Further provided 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 glycosylation competent cells by a value of from about 0.5 % to approximately 1.1%, including a decrease in the pH value of the cell culture medium by approximately 0.03-0.07.

The present invention further provides methods for increasing the level of afucosylated glycans in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by glycosylation competent cells by a value of from about 1% to about 2%, including increasing the pH of the cell culture medium by a value of from about 0.05 to about 1.2.

Methods are also provided for increasing the level of afucosylated glycans in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by glycosylation competent cells by a value of between about 0.5 % to approximately 1.1%, including increasing the pH value of the cell culture medium by approximately 0.03-0.07.

The invention further provides methods for modulating the level of TAF glycans in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by glycosylation competent cells, including modulating, decreasing, or increasing the level of afucosylated glycans in the composition in accordance with a method disclosed herein for modulating, reducing or increasing the level of afucosylated glycans.

The present invention provides a method for preparing a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) wherein the level of afucosylated glycans in the composition is from about 6.2% to about 8.4%, wherein the method comprises maintaining glycosylation competent cells in a cell culture medium at a pH value of greater than 7.05 to less than 7.2, wherein (A) the pH value of the cell culture medium is changed to less than 0.15 (optionally less than 0 ,10) during the culture period, or (B) the temperature of the cell culture medium is changed by no more than 2°C, or the method does not include culturing cells in a cell culture medium containing manganese or betaine, or (D) a combination of two or three from (A), (B) and (C).

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Brief description of graphic materials

Fig. 1A is an illustration of the three types of N-glycans (oligomannose, complex, and hybrid) and the symbols commonly used to designate such saccharides.

Fig. 1B is an illustration of exemplary glycan structures.

Fig. 2 is a diagram of the reutilization pathway and de novo metabolism pathway of fucose. In the salvage pathway, free L-fucose is converted to GDP-fucose, whereas in the de novo pathway, GDP-fucose is synthesized through three reactions catalyzed by GMD and FX. GDP-fucose is then transported from the cytosol into the lumen of the Golgi apparatus by GDP-Fuc transferase and transferred to acceptor oligosaccharides and proteins. Another reaction product, GDP, is converted to guanosine 5-monophosphate (GMP) and inorganic phosphate (Pi) by luminal nucleotide diphosphatase. The former is exported to the cytosol (via an antiport system that is coupled to GDP-fucose transport), while the latter is predicted to leave the Golgi lumen through the Golgi anion channel, GOLAC. See, for example, Nordeen et al. 2000; Hirschberg et al. 2001.

Fig. 3 is a graph showing (A) glucose concentration (g/L) in cell culture using control medium (open triangle line), first test medium (open circle line), and second test medium (open hexagon line) in during the cell culture cycle, (B) fucose concentration (g/L) in the cell culture when the second test medium (open square line) was applied during the cell culture cycle, and (C) TAF glycan levels (%) in the cell culture when applied control medium (dashed line with filled triangles), first test medium (dashed line with filled circles), and second test medium (dashed line with filled hexagons) during the cell culture cycle.

Fig. 4 is a graph showing (A) glucose concentration (g/L) in a cell culture using control medium (open triangle line), first test medium (open circle line), and second test medium (open hexagon line) in during the cell culture cycle and (B) levels of high mannose (HM) glycans (%) in cell culture using control medium (dashed line with filled triangles), first test medium (dashed line with filled circles), and second test medium ( dotted line with filled hexagons) during the cell culture cycle.

Fig. 5 is a graph showing (A) glucose concentration (g/L) in cell culture using control medium (open triangle line), first test medium (open circle line), and second test medium (open hexagon line) in during the cell culture cycle and (B) levels of afucosylated (afuc.) glycans (%) in cell culture using control medium (dashed line with filled triangles), first test medium (dashed line with filled circles), and second test medium (dashed line with filled hexagons) during the cell culture cycle.

Fig. 6 is a graph of TAF glycan levels (%) versus fucose concentration (g/L) in cell culture medium containing 0X glucose, 1X glucose, or 2X glucose.

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

Fig. 8 is a graph illustrating the pattern of effects of glucose and fucose on TAF glycan levels (%). The minimum, maximum and average TAF levels according to the QTPP are shown. The equation below the graph shows the mathematical relationship between glucose, fucose and TAF.

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

Fig. 9B is a series of graphs showing: (i) TAF glycan levels (%) versus fucose concentration (g/L) in cell culture medium (upper left quadrant) or glucose concentration (g/L) in cell culture medium ( upper right quadrant) and (ii) dependence of ADCC levels (expressed as % relative to the ADCC level of the innovative or commercially available antibody) on the concentration of fucose (g/l) in the cell culture medium (lower left quadrant) or on the concentration glucose (g/L) in cell culture medium (lower right quadrant). The range of TAF glycan levels (%) is 3.52301-4.0559, and the range of ADCC levels is 75.6911-90.9385. At 0 g/L fucose and 0.554 g/L glucose, the TAF glycan level (%) was 3.789458 and the ADCC level (%) was 83.3148.

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

Fig. 10 is a graph of osmolality plotted against fucose concentration.

Fig. 11 is a graph of fucose concentration plotted against time (duration) in cell culture.

Fig. 12 is a plot of % TAF and fucose feed, each plotted against time.

Fig. 13 is a pair of graphs demonstrating that controlling glucose levels within the target range from day 6 or earlier results in equivalent TAF results.

Fig. 14 is a graph of % afucosylation plotted against culture time.

Detailed description

Many secreted proteins undergo post-translational glycosylation, a process by which sugar residues (e.g., glycans, saccharides) are covalently attached to specific amino acids of the protein. Two types of glycosylation reactions occur in eukaryotic cells: (1) N-linkage glycosylation, in which glycans are attached to asparagine with the recognition sequence Asn-X-Thr/Ser, where X is any amino acid except proline, and (2) glycosylation with the formation of an O bond, in which glycans are attached to serine or threonine. Regardless of the type of glycosylation (N-linked or O-linked), microheterogeneity of protein glycoforms exists due to the wide range of glycan structures associated with each site (O or N).

All N-glycans are characterized by a common backbone sugar sequence: Mana1-6(Mana13)Mane1-4GlcNAce1-4GlcNAce-Asn-X-Ser/Thr (Man ₃ GlcNAc ₂ Asn) and belong to one of three types: (A) high type content of mannose (NM) or oligomannose (OM), in which the N-glycan consists of two N-acetylglucosamine (GalNAc) residues and a large number (for example, 5, 6, 7, 8 or 9) mannose residues (Man), (B ) complex type, in which the N-glycan contains more than two GlcNAc residues and any number of other types of sugars, or (C) hybrid type, in which the N-glycan contains a Man residue on one side of the branch and a GlcNAc at the base of the complex branch. In fig. Figure 1A (taken from Chapter 8 in Stanley et al.: N-Glycans, Essentials of Glycobiology, 2nd ed., Cold Spring Harbor Laboratory Press; 2009) shows three types of N-glycans.

N-linked glycans typically contain one or more monosaccharides galactose (Gal), N-acetylgalactosamine (GalNAc), galactosamine (GalN), glucose (GLc), N-acetylglucosamine (ClcNAc), glucosamine (GlcN), mannose (Man ), N-acetylmannosamine (ManNAc), mannosamine (ManN), xylose (Xyl), N-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). Commonly used symbols for such saccharides are shown in FIG. 1A. Exemplary glycans and their composition are shown in FIG. 1B.

N-linked glycosylation begins in the endoplasmic reticulum (ER), where a complex set of reactions results in the attachment of a glycan backbone structure formed primarily of two GlcNAc residues and three Man residues. The glycan complex formed in the ER is modified by enzymes in the Golgi apparatus. If a saccharide is relatively inaccessible to enzymes, it usually remains in its original LMW form. If enzymes can access the saccharide, many Man residues are cleaved off and the saccharide is further modified, resulting in the formation of a complex-type N-glycan structure. For example, mannosidase1, localized in the cis-Golgi compartment, is able to cleave or hydrolyze LMglycan, while fucosyltransferase FUT-8, localized in the medial Golgi compartment, fucosylates the glycan (Hanrue Imai-Nishiya (2007), BMC Biotechnology, 7:84) .

Accordingly, the saccharide composition and structural configuration of the glycan structure vary in

- 5 045782 depending on the glycosylation system in the ER and Golgi apparatus, the accessibility of the glycan structure to the enzymes of the apparatus, the order of action of each enzyme and the stage at which the protein is released from the glycosylation system, among other factors.

The present invention provided herein relates to methods for preparing an antibody composition containing TAF glycoforms at a desired or predetermined or preselected level. In illustrative embodiments, the method includes maintaining glycosylation competent cells in a cell culture medium containing fucose and/or glucose at a specific concentration described herein, depending on the desired level of TAF glycoforms. The present invention also provides a method for preparing an antibody composition containing afucosylated glycoforms at a desired or predetermined or preselected level, for example, the level of afucosylated glycans in the antibody composition is from about 6.2% to about 8.4% . In illustrative embodiments, the method includes maintaining glycosylation competent cells in a cell culture medium at a pH of greater than 7.05 to less than 7.2, wherein (A) the pH of the cell culture medium is changed to less than 0.15 (optionally by less than 0.10) during the culture period, or (B) the temperature of the cell culture medium is changed by no more than 2°C, or the method does not include culturing cells in a cell culture medium containing manganese or betaine, or (D) a combination of the two or three of (A), (B) and (C). Without being bound by theory, it is believed that the methods of the present invention provide a means for producing unique compositions containing defined amounts of specific glycoforms of the antibody in question.

In exemplary embodiments, TAF glycan levels are modulated. As used herein, the terms total afucosylated glycans, or TAF glycans, or total afucosylated glycoforms, or TAF glycoforms, refer to the total amount of high mannose (HM) glycans and afucosylated glycans. In illustrative embodiments, the levels of LMW glycans are modulated. As used herein, the terms high-mannose glycans, or HM-glycans, or high-mannose glycoforms, or LM-glycoforms, or HM, cover glycans containing 5, 6, 7, 8, or 9 mannose residues, abbreviated Man5. Man6, Man7, Man8 and Man9 respectively. In illustrative embodiments, the levels of afucosylated glycans are modulated. As used herein, the terms afucosylated glycan, or afucoglycan, or afucosylated glycoform, or afuco. refer to glycoforms in which there is no fucose at the backbone, for example, a1,6-linked fucose at the GlcNAc residue involved in the formation of an amide bond with Asn at the Nglycosylation 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, for example, Reusch and Tejada, Glycobiology 25(12): 1325–1334 (2015). In illustrative aspects, the level of TAF and the amounts of LMW glycoforms and afucosylated glycoforms are determined using hydrophilic interaction liquid chromatography (HILIC), as further described herein in Example 1. After enzymatic digestion of the N-glycans, HILIC is performed to obtain a chromatogram with multiple peaks each the peak of which represents the average distribution (amount) of an individual glycoform. For these purposes, % peak area = peak area/total peak area x 100%, and % total peak area = total sample area/total standard area x 100%. The calculations used for the purpose of determining % TAF can be carried out as follows:

% afucosylated glycoforms = %A1G0 + %A2G0 + %A2G1a + %A2G1b + %A2G2 + %A1G1M5;

% High Mannose Glycoforms = %Man5 (if detectable) + %Man6 (if detectable) + %Man7 (if detectable) + %Man8 (if detectable) + %Man9 (if detectable).

The present invention provides methods for preparing a recombinant glycosylated protein composition. In illustrative embodiments, the recombinant glycosylated protein composition is an antibody composition. In illustrative embodiments, the method includes maintaining glycosylation competent cells in a cell culture medium containing fucose and/or glucose at a specific concentration described herein, depending on the desired level of TAF glycoforms.

Fucose.

In illustrative embodiments of the methods disclosed herein, fucose is present in the culture medium at a concentration of from about 0.1 g/L to about 2.0 g/L, optionally from about 0.1 g/L to about 1 .75 g/L, about 0.1 g/L to about 1.5 g/L, or about 0.1 g/L to about 1.2 g/L. In illustrative cases, fucose is present in the culture medium at a concentration of approximately 1.2 g/L or less. In illustrative cases, fucose is present in the culture medium

- 6 045782 in a concentration of from about 0.1 g/l to about 1.0 g/l. In illustrative cases, the culture medium contains fucose at a concentration of from about 0.17 g/L to about 2.0 g/L, from about 0.17 g/L to about 1.75 g/L, from about 0. 17 g/L to about 1.5 g/L or from about 0.17 g/L to about 1.2 g/L. In illustrative aspects, fucose is present in the culture medium at a concentration of from about 0.17 g/L to about 1.2 g/L. In illustrative cases, fucose is present in the culture medium at a concentration of from about 0.17 g/L to about 1.0 g/L. In illustrative cases, the culture medium contains fucose at a concentration of from about 0.2 g/L to about 2.0 g/L, from about 0.2 g/L to about 1.75 g/L, from about 0. 2 g/L to about 1.5 g/L or about 0.2 g/L to about 1.2 g/L. In illustrative cases, fucose is present in the culture medium at a concentration of from about 0.2 g/L to about 1.0 g/L. In illustrative cases, fucose is present in the culture medium at a concentration of approximately 1.0 g/L or less. For example, in some cases, the concentration of fucose 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 l, approximately 0.15 g/L, approximately 0.16 g/L, approximately 0.17 g/L, approximately 0.18 g/L, approximately 0.19 g/L or approximately 0.20 g/L. In some cases, the concentration of fucose 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, approximately 0.8 g/L or approximately 0.9 g/L. In illustrative aspects, the concentration of fucose is no more than 1.0 g/L or about 1.0 g/L, no more than 0.9 g/L or about 0.9 g/L, no more than 0.8 g/L or about 0.8 g/l or not more than 0.7 g/l or approximately 0.7 g/l. In illustrative cases, fucose is present in the culture medium at a concentration of about 0.75 g/L or less, 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 approximately 0.6 g/l. In illustrative aspects, fucose is present in the culture medium at a concentration of less than about 0.6 g/L, such as from about 0.2 g/L to about 0.5 g/L.

In illustrative aspects, a method for producing a recombinant glycosylated protein composition (eg, an antibody composition or an antibody binding protein composition) involves maintaining glycosylation-competent cells in two different cell culture media. In illustrative aspects, a method of producing a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) includes maintaining glycosylation competent cells in a first cell culture medium for an initial period of time and subsequently maintaining glycosylation competent cells. with respect to glycosylation of cells in the second cell culture medium, optionally, wherein the first cell culture medium does not contain fucose at a concentration of from about 0.1 g/L to about 1.0 g/L, and the second cell culture medium contains fucose at a concentration of from about 0.1 g/L to about 1.0 g/L, and the second cell culture medium contains fucose, for example at one of the above concentrations. In illustrative cases, the initial period of time begins when cells are inoculated into a bioreactor containing cell culture medium, such as cell culture medium. In some aspects, the initial time period is from about 1 day to about 3 days, such as from about 24 hours to about 72 hours. In illustrative aspects, the initial time period is more than about 3 days (about 72 hours) but less than about 10 days (about 72 hours). 240 hours) or less than about 156 hours. In illustrative aspects, the initial period of time is about 3, about 4, about 5, about 6, about 7, about 8, or about 9 days. In illustrative aspects, the method includes adding fucose to the culture medium after an initial period of time. In some aspects, fucose is added to the first culture medium to form a 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 approximately 9 days. In illustrative aspects, the method includes adding fucose to the cell culture medium, such as the first cell culture medium, on day 6, day 7, day 8, or day 9 after inoculation with the cell culture. In illustrative aspects, fucose is added at a final concentration of greater than about 0.1 g/L, greater than about 0.17 g/L, or greater than about 0.2 g/L, and less than about 2.0 g/L. In illustrative aspects, the first cell culture medium does not contain fucose. In illustrative aspects, the first cell culture medium contains fucose, but at a concentration that is undetectable or measurable, or at a concentration that is significantly lower than the concentration of fucose in the second cell culture medium, such as significantly

- 7 045782 below 0.1 g/l, below approximately 0.17 g/l or below approximately 0.2 g/l.

In alternative aspects, the methods include maintaining glycosylation competent cells in a cell culture medium containing fucose (e.g., at a concentration of greater than about 0.1 g/L, greater than about 0.17 g/L, or greater than about 0.2 g/L and less than about 2.0 g/L) for the entire duration of maintenance of glycosylation competent cells in 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) comprises inoculating glycosylation competent cells into a bioreactor containing a cell culture medium containing fucose and maintaining cells in the cell culture medium while maintaining the fucose concentration almost the same throughout the entire cell culture period.

In illustrative embodiments, the fucose concentration fluctuates slightly during cell culture. In illustrative aspects, the concentration of fucose fluctuates by about 0.2 g/L or less during the time of maintaining glycosylation competent cells in a cell culture medium containing fucose. In illustrative aspects, the concentration of fucose fluctuates by about 0.1 g/L or less during the time of maintaining glycosylation competent cells in a cell culture medium containing fucose. In illustrative aspects, when fucose is added to the cell culture medium, for example, after an initial cell culture period, the fucose is added to the medium no more than once or twice during the cell culture period.

Glucose.

In illustrative embodiments of the methods disclosed herein, glucose is present in the culture medium. In illustrative aspects, glucose is present in the culture medium at a concentration of about 10 g/L or less, about 9.0 g/L or less, or about 6.0 g/L or less. In illustrative aspects, glucose is present in the culture medium at a concentration of from about 0.5 g/L to about 4.0 g/L. In illustrative aspects, glucose is present at a concentration of 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 equals approximately 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.

In illustrative aspects, the method includes maintaining a glucose concentration in the cell culture medium for a period of time that is equivalent to the cell culture period. In illustrative cases, maintaining the glucose concentration in the cell culture medium involves sampling the cell culture medium on a regular basis, such as once every hour, once every two hours, once every 3, 4, 5 or 6 hours, once a day , twice a day, three times a day or 4 times a day, etc., measuring the glucose concentration in selected samples of cell culture medium, and adding glucose to the cell culture if the glucose concentration in selected samples of cell culture medium below the required maintenance glucose concentration. In illustrative aspects, maintaining the glucose concentration in the cell culture medium involves measuring the glucose concentration in the cell culture medium using a glucose sensor. In illustrative aspects, the glucose concentration is measured using a glucose sensor at regular intervals, such as once per hour, once every two hours, once every 3, 4, 5 or 6 hours, once per day, twice per day, three times per day or 4 times a day, etc., and glucose is added to the cell culture if the glucose concentration detected by the glucose sensor is lower than the required maintenance glucose concentration. In alternative aspects, the method includes maintaining glycosylation competent cells in a cell culture medium containing glucose, but maintaining the glucose concentration in the cell culture medium only after an initial period of time. In illustrative embodiments, the initial period of time is from about 1 day to about 3 days (from about 24 hours to about 72 hours). In some cases, the initial time period is about 6 days or less, optionally where the initial time period is 3 days, or 4 days, or 5 days after inoculation with the cell culture. In illustrative aspects, the method includes maintaining glycosylation competent cells in a cell culture medium containing glucose, and maintaining a glucose concentration in the cell culture medium on day 6 after inoculation and thereafter. In illustrative aspects, the glucose concentration is maintained for at least about 4 days or about 5 days after the initial time period, or optionally maintained for at least about 6 days after the initial time period.

In illustrative aspects, the cell culture medium contains an initial concentration

- 8 045782 glucose during the initial period of time. 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 g/L 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, approximately 4.0 g/l, approximately 4.5 g/l, approximately 5.0 g/l, approximately 5.5 g/l, approximately 6.0 g/l, approximately 6.5 g /l, approximately 7.0 g/l, approximately 7.5 g/l, approximately 8.0 g/l, approximately 8.5 g/l, approximately 9.0 g/l, approximately 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 illustrative aspects, the initial glucose concentration is the concentration of glucose in the cell culture medium applied during the initial period of time. In illustrative aspects, the initial glucose concentration is the glucose concentration in the cell culture medium maintained for an initial period of time.

In illustrative aspects, the initial glucose concentration is the same as the glucose concentration maintained after the initial period of time. In alternative aspects, the initial glucose concentration is different from the glucose concentration maintained after the initial period of time. In illustrative aspects, the method includes adding glucose to the cell culture medium after an initial period of time and maintaining the glucose at a concentration different from the initial glucose concentration. In illustrative aspects, the method includes adding glucose to the cell culture medium after an initial period of time, maintaining the glucose at a concentration different from the initial glucose concentration, wherein the glucose addition step results in a glucose concentration of about 10 g/L or less (e.g., about 9 g/L or less, about 6 g/L or less, about 0.5 g/L to about 4 g/L).

In illustrative aspects, the method includes adding glucose to the cell culture medium according to a glucose feeding schedule. In some aspects, the glucose feeding regimen is initiated after an initial period of time. For example, in some aspects, the initial time period is at least 3 days or 4 days, and the glucose feeding regimen is initiated after a time period of about 4 to about 6 days after inoculation with the cell culture, e.g., after about 4 days, about 5 days, approximately 6 days after cell culture inoculation. In illustrative cases, the glucose feeding regime 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 approximately 4 g/l). The term average glucose concentration refers to the average glucose concentrations in the cell culture medium determined by a glucose sensor over a period of time (eg, 1-2 days). In illustrative cases, the glucose feeding regime is used to achieve an average glucose concentration based on the fucose concentration in the cell culture medium. In some aspects, the average glucose concentration is calculated using Formula I

T=3.354-l.388F+0.lllG + [F - 0.4375] x [1.9527(F - 0.4375)] (Formula I), where T is the specified % of total afucosylated (TAF) glycans 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 (g/L) of fucose in the medium, and G is is the average glucose concentration (g/l).

In illustrative cases, (i) the fucose concentration is about 0.2 ± 0.1 g/L, and the average glucose concentration is from about 2 g/L 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 g/L 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.

Levels of TAF-, NM- and afucosylated glycans.

In illustrative embodiments, a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) is prepared using the methods disclosed herein, wherein the level of TAF glycans in the composition is approximately 10% or less. In illustrative aspects, the level of TAF glycans in the composition is about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or

- 9 045782 less. In illustrative aspects, the level of TAF glycans in the composition is greater than or about 4%, such as from about 4% to about 10%. In some aspects, the level of TAF glycans in the composition is from about 2% to about 6%, or from about 2.5% to about 5%. In some aspects, the level of TAF glycans is about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5%, about 5 .5% or approximately 6.0%. In illustrative aspects, the level of TAF glycans is from about 2% to about 5%, or from about 2% to about 4%.

In exemplary aspects of methods for preparing 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 of from about 0.1 g/L to about 1 g/L. 0 g/L, or from about 0.17 g/L to about 1.0 g/L, and the level of TAF glycans in the composition is less than about 10%.

In illustrative embodiments, a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) is prepared using the methods disclosed herein, wherein the level of high mannose glycans in the antibody composition is is about 3.5% or less, for example about 3.25% or less, about 3.0% or less, about 2.5% or less, about 2.0% or less. In illustrative aspects, the level of high mannose glycans in the antibody composition is from about 0.7% to about 3.0%, optionally about 0.7%, about 0.8%, about 0.9%, about 1. 0%, approximately 1.1%, approximately 1.2%, approximately 1.3%, approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8% , approximately 1.9%, approximately 2.0%, approximately 2.1%, approximately 2.2%, approximately 2.3%, approximately 2.4%, approximately 2.5%, approximately 2.6%, approximately 2.7%, approximately 2.8%, approximately 2.9%, or approximately 3.0%.

In illustrative embodiments, a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) is prepared using the methods disclosed herein, wherein the level of afucosylated glycans in the antibody composition is approximately 3 .5% or less, for example about 3.25% or less, about 3.0% or less, about 2.5% or less, about 2.0% or less. In illustrative aspects, the level of afucosylated glycans 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%, approximately 1.2%, approximately 1.3%, approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, approximately 2 .0%, approximately 2.1%, approximately 2.2%, approximately 2.3%, approximately 2.4%, approximately 2.5%, approximately 2.6%, approximately 2.7% or approximately 2.8 %.

Measurement methods for assessing glycoforms.

Various methods are known in the art for assessing the glycoforms present in a composition containing glycoproteins, or for determining, detecting or measuring the profile of glycoforms in a particular sample containing glycoproteins. Suitable methods include, but are not limited to, MALDI-TOF analysis in positive ion mode, MALDI-TOF analysis in negative ion mode, weak anion exchange (WAX) chromatography, normal phase chromatography (NP-HPLC), exoglycosidase digestion, Biochromatography -Gel P-4, anion exchange chromatography and one-dimensional NMR spectroscopy and combinations thereof. See, for example, Mattu et al., JBC 273: 2260-2272 (1998); Field et al., Biochem J 299(Pt 1): 261-275 (1994); Yoo et al., MAbs 2(3): 320-334 (2010) Wuhrer M. et al., Journal of Chromatography B, 2005, Vol.825, Issue 2, pages 124-133; Ruhaak L.R., Anal Bioanal Chem, 2010, Vol. 397:3457-3481 and Geoffrey, R.G. et. al. Analytical Biochemistry 1996, Vol. 240, pages 210-226. In addition, the examples provided herein describe a suitable method for assessing the glycoforms present in a composition containing glycoproteins.

With respect to the present invention, the cell culture can be maintained in accordance with any set of conditions suitable for the production of recombinant glycosylated protein. For example, in some aspects, the cell culture is maintained at a particular pH, temperature, cell density, culture volume, dissolved oxygen level, pressure, osmolality, and the like. In illustrative aspects, the cell culture is shaken (eg, 70 rpm) at 5% CO2 under standard humidity conditions in a CO2 incubator prior to inoculation. In illustrative aspects, a 1.5 L volume of cell culture media is inoculated at a seeding density of approximately 10 cells/mL.

In illustrative aspects, the methods of the present invention include maintaining glycosylation competent cells in a cell culture medium at a pH of from about 6.85 to about 7.05, for example, in various aspects, at

- 10 045782 approximately 6.85, approximately 6.86, approximately 6.87, approximately 6.88, approximately 6.89, approximately 6.90, approximately 6.91, approximately 6.92, approximately 6.93, approximately 6 .94, approximately 6.95, approximately 6.96, approximately 6.97, approximately 6.98, approximately 6.99, approximately 7.00, approximately 7.01, approximately 7.02, approximately 7.03, approximately 7 .04 or approximately 7.05. In some aspects, the cell culture medium has a pH value of from about 6.9 to about 7.0.

In illustrative aspects, the methods include maintaining the cell culture at a temperature of 3040°C. In illustrative embodiments, the temperature is from about 32°C to about 38°C, or from about 35°C to about 38°C.

In illustrative aspects, the methods include maintaining osmolality in the range of from about 200 mOsm/kg to about 500 mOsm/kg. In illustrative aspects, the method includes maintaining an osmolality in the range of from about 225 mOsm/kg to about 400 mOsm/kg, or from about 225 mOsm/kg to about 375 mOsm/kg. In illustrative aspects, the method includes maintaining an osmolality in the range of from about 225 mOsm/kg to about 350 mOsm/kg. In various aspects, the osmolality (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.

In illustrative aspects, the methods include maintaining the dissolved oxygen (DO) level in the cell culture at an oxygen saturation level of from about 20% to about 60% during the initial period of cell culture. In illustrative cases, the method includes maintaining the DO level in the cell culture at an oxygen saturation level of from about 30% to about 50% (eg, from about 35% to about 45%) during the initial period of cell culture. In illustrative cases, the method includes maintaining the DO level in the cell culture at an oxygen saturation level of about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%, during the initial cell culture period. In illustrative aspects, the DO level is from about 35 mm Hg. Art. up to approximately 85 mm Hg. Art., or from approximately 40 mm Hg. Art. up to approximately 80 mm Hg. Art., or from approximately 45 mm Hg. Art. up to approximately 75 mm Hg. Art.

The cell culture is maintained in any one or more culture media. In illustrative aspects, the cell culture is maintained in a medium suitable for cell growth and/or provided with one or more feeding media in accordance with any suitable feeding regime. In illustrative aspects, the method includes maintaining a cell culture in a medium containing glucose, lactate, ammonia, glutamine and/or glutamate. In illustrative aspects, the method includes maintaining the cell culture in a medium containing manganese at a concentration of approximately 1 μM or less during the initial cell culture period. In illustrative aspects, the method includes maintaining a cell culture in a medium containing from about 0.25 μM to about 1 μM manganese. In illustrative aspects, the method includes maintaining a cell culture in a medium containing negligible amounts of manganese. In illustrative aspects, the method includes maintaining a cell culture in a medium containing copper at a concentration of about 50 ppb or less during the initial cell culture period. In illustrative aspects, the method includes maintaining a cell culture in a medium containing copper at a concentration of about 40 ppb or less during the initial cell culture period. In illustrative aspects, the method includes maintaining a cell culture in a medium containing copper at a concentration of about 30 ppb or less during the initial cell culture period. In illustrative aspects, the method includes maintaining a cell culture in a medium containing copper at a concentration of about 20 ppb or less during the initial cell culture period. In illustrative aspects, the medium contains copper at a concentration of about 5 ppb or greater or about 10 ppb or greater. In illustrative aspects, the cell culture medium contains mannose. In illustrative aspects, the cell culture medium does not contain mannose.

In illustrative embodiments, the cell culture type is a fed-batch culture or a continuous perfusion culture. However, the methods of the present invention are not primarily limited to any particular type of cell culture.

Cells.

The present invention relates to methods for preparing a recombinant glycosylated protein composition (eg, an antibody composition or an antibody binding protein composition), comprising maintaining glycosylation competent cells in a cell culture medium. In illustrative aspects, glycosylation competent cells are eukaryotic cells, including, without limitation, 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

- 11 045782 measures, Frenzel, et al., Front Immunol 4: 217 (2013). In illustrative aspects, eukaryotic cells are mammalian cells. In illustrative aspects, mammalian cells are non-human mammalian cells. In some aspects, the cells are Chinese hamster ovary (CHO) cells and derivatives thereof (e.g., CHO-Κι, CHO pro3), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2/0), cells engineered to be deficient dihydrofolate reductase (DHFR) activity (e.g. DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or their derivatives (e.g. HEK293T, HEK293EBNA), African green monkey kidney cells (e.g. COS cell line, VERO cell line) , human cervical cancer cells (eg, HeLa), U2-OS human osteosarcoma bone epithelial cells, human A549 adenocarcinoma alveolar epithelial basal cells, HT1080 human fibrosarcoma cells, CAD mouse brain tumor cells, P19 embryonal carcinoma cells, embryonal fibroblast cells NIH 3T3 mouse, L929 mouse fibroblast cells, N2a mouse neuroblastoma cells, MCF-7 human breast cancer cells, Y79 retinoblastoma cells, SO-Rb50 human retinoblastoma cells, Hep G2 human liver cancer cells, J558L mouse B cell myeloma cells or newborn hamster kidney (BHK) (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2): 257-263 (2013)).

Cells that are not glycosylation competent can also be transformed into glycosylation competent cells, for example, by transfecting them with genes encoding the appropriate enzymes required for glycosylation. Exemplary enzymes include, but are not limited to, oligosaccharyltransferases, glycosidases, glucosidase I, glucosidase II, calnexin/calreticulin, glycosyltransferases, mannosidases, GlcNAc transferases, galactosyltransferases, and sialyltransferases.

In illustrative embodiments, the glycosylation competent cells are not genetically modified to provide an alteration in de novo pathway or salvage pathway enzyme activity. These two pathways of fucose metabolism are shown in Fig. 2. In illustrative embodiments, the glycosylation competent cells are not genetically modified to alter the activity of any one or more of the following: fucosyltransferase (FUT, e.g. FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9), fucosokinase, GDP-fucose pyrophosphorylase, GDP-D-mannose-4,6-dehydratase (GMD) and GDPketo-6-deoxymannose-3,5-epimerase-4-reductase (FX). In illustrative embodiments, the glycosylation competent cells are not genetically modified to provide knockout of the gene encoding FX.

In illustrative embodiments, the glycosylation competent cells are not genetically modified to alter e(1,4)-Nacetylglucosaminyltransferase III (GNTIII) or GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD) activity. In illustrative aspects, the glycosylation competent cells are not genetically modified to overexpress GNTIII or RMD.

Recombinant glycosylated proteins.

In illustrative embodiments, the recombinant glycosylated protein comprises an amino acid sequence comprising one or more N-glycosylation consensus sequences of the formula

Asn-Xaai-Haag, where Xaa1 is any amino acid except Pro, and Xaa ₂ is Ser or Thr.

In illustrative embodiments, the recombinant glycosylated protein comprises a crystallizable fragment (Fc) polypeptide. As used herein, the term Fc polypeptide includes native and mutant forms of polypeptides 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 Fc moieties (and oligomers formed therefrom) provide the advantage of easy purification by affinity chromatography on Protein A or Protein G columns. In illustrative embodiments, the recombinant glycosylated protein comprises Fc IgG, such as human IgG . In illustrative aspects, the recombinant glycosylated protein comprises an IgG1 or IgG2 Fc. In illustrative aspects, the recombinant glycosylated protein is an antibody, an antibody-based protein product, a peptibody, or an Fc fusion protein.

In illustrative aspects, the recombinant glycosylated protein is an antibody. As used herein, the term antibody refers to a protein in a conventional immunoglobulin format containing heavy and light chains and containing variable and constant regions. For example, the antibody may be an IgG, which is characterized by a Y-shaped structure of two identical pairs of polypeptide chains, each pair containing one light chain (typically having a molecular weight of approximately 25 kDa) and one heavy chain.

- 12 045782 chain (typically having a molecular weight of approximately 50-70 kDa). An antibody has a variable region and a constant region. In IgG formats, the variable region typically contains approximately 100-110 or more amino acids, contains three complementarity determining regions (CDRs), is primarily responsible for antigen recognition, and is significantly different from other antibodies that bind to different antigens. See, for example, 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).

Briefly, in the antibody backbone, CDRs are integrated into the framework of the variable region of the heavy and light chains, where they constitute the regions primarily 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, N.I.H. Public Health Service, 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 region (designated framework regions 1-4, FR1, FR2, FR3 and FR4, after Kabat et al., 1991 ; see also Chothia and Lesk, 1987, above).

Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon chains, and they define the isotype of the antibody 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 invention include all such classes or isotypes of antibodies. The light chain constant region may be, for example, a kappa or lambda light chain constant region, for example a human kappa or lambda light chain constant region. The heavy chain constant region may be, for example, alpha, delta, epsilon, gamma or mu heavy chain constant regions, for example an alpha, delta, epsilon, gamma or mu heavy chain constant region person. Accordingly, in illustrative embodiments, the antibody is an antibody of the IgA, IgD, IgE, IgG, or IgM isotype, including any of IgG1, IgG2, IgG3, or IgG4.

In various aspects, the antibody may be a monoclonal antibody or a polyclonal antibody. In some aspects, the antibody contains a sequence that is substantially similar to the sequence of a naturally occurring antibody produced by a mammal, such as a mouse, rat, rabbit, goat, horse, chicken, hamster, pig, human, and the like. In this regard, the antibody can be considered as a mammalian antibody, such as a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody, a horse antibody, a chicken antibody, a hamster antibody, a pig antibody, a human antibody, and the like. In certain aspects, the recombinant glycosylated protein is a human monoclonal antibody. In certain aspects, the recombinant glycosylated protein is a chimeric antibody or a humanized antibody. The term chimeric antibody is used herein to mean an antibody comprising constant domains from one species and variable domains from a second, or more generally, comprising stretches of amino acid sequence from at least two species. The term humanized, when applied to antibodies, refers to antibodies containing at least CDR regions from a non-human source engineered to have a structure and immunological function more similar to those of actual human antibodies than to antibodies from the source. source. For example, humanization may involve grafting a CDR from a non-human antibody, such as a mouse antibody, onto a human antibody. Humanization may also include selective amino acid substitutions to make the non-human sequence more similar to the human sequence.

In various aspects, the antibody is cleaved into fragments by the action of enzymes such as, for example, papain and pepsin. Papain cleaves the antibody to form two Fab fragments and one Fc fragment. Pepsin cleaves the antibody to form the F(ab') ₂ fragment and the pFc' fragment. In illustrative aspects, the recombinant glycosylated protein is an antibody fragment, such as a Fab, Fc, F(ab')2, or pFc', in which at least one glycosylation site remains. With respect to the methods of the present invention, the antibody may not contain certain parts of the antibody and may be a fragment of the antibody. In various aspects, the antibody fragment contains a glycosylation site. In some aspects, the fragment is a glycosylated Fc fragment that contains at least a portion of an antibody Fc region that is post-translationally glycosylated in eukaryotic cells.

Antibody architecture has been used to create an increasing range of alternative antibody formats that span a molecular weight range of at least or approximately 12-150 kDa and a range of valencies (n) from monomeric (n=1), dimeric (n=2) and trimeric (n=3) to tetrameric (n=4) and potentially higher; such alternative antibody formats are referred to herein as antibody protein products or antibody binding proteins.

- 13 045782

Antibody protein products may be those whose antigen binding format is based on antibody fragments, such as scFv, Fab and VHH/VH, which retain full antigen binding capacity. 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 attach V regions to the scFv fragment (single chain variable fragment) to stabilize the molecule, or constant (C) domains are added to the V regions to produce a Fab fragment [antigen binding fragment]. Both scFv and Fab are widely used fragments that can be easily produced in prokaryotic hosts. Other antibody-based protein products include disulfide-stabilized scFv (ds-scFv), single-chain Fab (scFab), and di- and multimeric antibody formats such as dia-, tria-, and tetrabodies, or miniantibodies (miniAbs), which include various formats consisting of scFvs associated with oligomerization domains. The smallest fragments are VHH/VH from camelid heavy chain Abs, as well as single domain Abs (sdAbs). The structural element most often used to create new antibody formats is the antibody fragment, which is a single chain variable (V) domain (scFv), which contains heavy and light chain V domains (VH and VL domain) linked via a peptide linker of ~15 amino acid residues. The peptibody or peptide-Fc fusion product is another antibody-based protein product. The peptibody structure consists of a biologically active 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).

Other antibody-based protein products include single chain antibody (SCA); diabody; triatelo; tetrabody; bispecific or trispecific antibodies, etc. Bispecific antibodies can be divided into five main classes: BsIgG, complemented IgG, BsAb fragments, bispecific fusion proteins and BsAb conjugates. See, for example, Spiess et al., Molecular Immunology 67(2) Part A: 97106 (2015).

In illustrative aspects, the recombinant glycosylated protein comprises any of these antibody-based protein products (e.g., scFv, VHH/VH Fab, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g., diabody, tribody, tetrabody), mini-Ab, VHH-/VH camelid heavy chain antibody, sdAb, diabody; tribody; tetrabody; bispecific or trispecific antibody, BsIgG, complemented IgG, BsAb fragment, bispecific fusion protein and BsAb conjugate) and contains one or more Nglycosylation consensus sequences optionally one or more Fc polypeptides. In various aspects, the antibody protein product contains a glycosylation site. In illustrative aspects, the antibody protein product may be a glycosylated Fc moiety conjugated to an antibody binding moiety (glycosylated Fc moiety antibody product).

The recombinant glycosylated protein may be an antibody-based protein product in monomeric form or in polymeric, oligomeric or multimeric form. In certain embodiments in which the antibody contains two or more distinct regions that constitute antigen-binding moieties, the antibody is considered bispecific, trispecific, or multispecific, or bivalent, trivalent, or multivalent, depending on the number of different epitopes that are recognized and bound by the antibody.

Advantageously, the methods are not limited by the specificity of the antibody to the antigen. Accordingly, the antibody has any specificity for binding to virtually any antigen. In illustrative aspects, the antibody binds to a hormone, growth factor, cytokine, cell surface receptor, or any ligand thereof. In illustrative aspects, the antibody binds to a protein expressed on the cell surface of an immune system cell. In illustrative aspects, the antibody binds to a cluster of differentiation molecule selected from the group consisting of the following: CD 1a, 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, CD79a, CD79p, 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, CD1 21a , CDw121b, CD122, CD123, CD124, CD125, CD126, CD127, CDw128, CD129, CD130, CDw131, CD132, CD134, CD135, CDw136, CDw137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143 , CD144, CD145 , CD146, CD147, CD148, CD150, CD151, CD152, CD153, CD154, CD155, CD156, CD157, CD158a, CD158b, CD161, CD162, CD163, CD164, CD165, CD166 and CD182.

- 14 045782

In illustrative aspects, the antibody is one of the antibodies described in US Pat. No. 7,947,809 and US Patent Application Publication No. 20090041784 (glucagon receptor), US Pat. No. 7,939,070, US Pat. No. 7,833,527, US Pat. No. 7,767,206, and US Pat. No. 7,786,284 (receptor). A IL-17), US Patent No. 7872106 and US Patent No. 7592429 (sclerostin), US Patent No. 7871611, US Patent No. 7815907, US Patent No. 7037498, US Patent No. 7700742 and US Patent Application Publication No. 20100255538 (IGF-receptor 1), US Patent No. 7868140 (B7RP1), US Patent No. 7807159 and US Patent Application Publication No. 20110091455 (myostatin), US Patent No. 7736644, US Patent No. 7628986, US Patent No. 7524496 and US Patent Application Publication No. 20100111979 ( epidermal growth factor receptor deletion mutants), US Patent No. 7728110 (SARS coronavirus), US Patent No. 7718776 and US Patent Application Publication No. 20100209435 (OPGL), US Patent No. 7658924 and US Patent No. 7521053 (Angiopoietin-2), Patent US Patent No. 7601818, US Patent No. 7795413, US Patent Application Publication No. 20090155274, US Patent Application Publication No. 20110040076 (NGF), US Patent No. 7579186 (TGF-β Type II Receptor), US Patent No. 7541438 (Connective Tissue Growth Factor ), US Patent No. 7438910 (IL1-R1), US Patent No. 7423128 (Properdin), US Patent No. 7411057, US Patent No. 7824679, US Patent No. 7109003, US Patent No. 6682736, US Patent No. 7132281 and US Patent No. 7807797 ( CTLA-4), US Patent No. 7084257, US Patent No. 7790859, US Patent No. 7335743, US Patent No. 7084257 and US Patent Application Publication No. 20110045537 (Interferon Gamma), US Patent No. 7932372 (MAdCAM), US Patent No. 7906625, US Patent Application Publication No. 20080292639 and US Patent Application Publication No. 20110044986 (amyloid), US Patent No. 7815907 and US Patent No. 7700742 (insulin-like growth factor I), US Patent No. 7566772 and US Patent No. 7964193 (interleukin1β), US Patent No. 7563442, US Patent No. 7288251, US Patent No. 7338660, US Patent No. 7626012, US Patent No. 7618633 and US Patent Application Publication No. 20100098694 (CD40), US Patent No. 7498420 (c-Met), US Patent No. 7326414, Patent US Patent No. 7592430 and US Patent No. 7728113 (M-CSF), US Patent No. 6924360, US Patent No. 7067131 and US Patent No. 7090844 (MUC18), US Patent No. 6235883, US Patent No. 7807798 and US Patent Application Publication No. 20100305307 ( epidermal growth factor receptor), US Patent No. 6716587, US Patent No. 7872113, US Patent No. 7465450, US Patent No. 7186809, US Patent No. 7317090 and US Patent No. 7638606 (interleukin-4 receptor), US Patent Application Publication No. 20110135657 ( BETA-KLOTO), US Patent No. 7887799 and US Patent No. 7879323 (fibroblast growth factor-like polypeptides), US Patent No. 7867494 (IgE), US Patent Application Publication No. 20100254975 (ALPHA-4 BETA-7), US Patent Application Publication No. 20100254975 (ALPHA-4 BETA-7), US Patent Application Publication No. US Patent No. 20100197005 and US Patent No. 7537762 (ACTIVIN RECEPTOR-LIKE KINASE-1), US Patent No. 7585500 and US Patent Application Publication No. 20100047253 (IL-13), US Patent Application Publication No. 20090263383 and US Patent No. 7449555 ( CD148), US Patent Application Publication No. 20090234106 (ACTIVIN A), US Patent Application Publication No. 20090226447 (Angiopoietin-1 and Angiopoietin-2), US Patent Application Publication No. 20090191212 (Angiopoietin-2), US Patent Application Publication No. No. 20090155164 (C-FMS), US Patent No. 7537762 (Activin Receptor-Like Kinase-1), US Patent No. 7371381 (Galanin), US Patent Application Publication No. 20070196376 (INSULIN-LIKE GROWTH FACTORS), US Patent No. 7267960 and US Patent No. 7741115 (LDCAM), US 7265212 (CD45RB), US Patent No. 7709611, US Patent Application Publication No. 20060127393 and US Patent Application Publication No. 20100040619 (DKK1), US Patent No. 7807795, US Patent Application Publication No. 20030103978 and Patent US Patent Application Publication No. 7923008 (osteoprotegerin), US Patent Application Publication No. 20090208489 (OV064), US Patent Application Publication No. 20080286284 (PSMA), US Patent Application Publication No. 7888482, US Patent Application Publication No. 20110165171, and US Patent Application Publication No. 20110059063 ( PAR2), US Patent Application Publication No. 20110150888 (HEPCIDIN), US Patent No. 7939640 (B7L-1), US Patent No. 7915391 (c-Kit), US Patent No. 7807796, US Patent No. 7193058, and US Patent No. 7427669 (ULBP ), US Patent No. 7786271, US Patent No. 7304144 and US Patent Application Publication No. 20090238823 (TSLP), US Patent No. 7767793 (SIGIRR), US Patent No. 7705130 (HER-3), US Patent No. 7704501 (Ataxin-1- similar polypeptide), US Patent No. 7695948 and US Patent No. 7199224 (TNF-a-converting enzyme), US Patent Application Publication No. 20090234106 (ACTIVIN A), US Patent Application Publication No. 20090214559 and US Patent No. 7438910 (IL1-R1 ), US Patent No. 7,579,186 (Type II TGF-β Receptor), US Patent No. 7,569,387 (TNF Receptor-Like Molecules), US Patent No. 7,541,438, (Connective Tissue Growth Factor), US Patent No. 7,521,048 (TRAIL Receptor-2), US Patent No. 6319499, US Patent No. 7081523 and US Patent Application Publication No. 20080182976 (erythropoietin receptor), US Patent Application Publication No. 20080166352 and US Patent No. 7435796 (B7RP1), US Patent No. 7423128 (Properdin), US Patent No. 7422742 and US Patent No. 7141653 (interleukin-5), US Patent No. 6740522 and US Patent No. 7411050 (RANKL), US Patent No. 7378091 (tumor antigen carbonic anhydrase IX (CA IX)), US Patent No. 7318925 and US Patent No. 7288253 (parathyroid hormone), US Patent No. 7285269 (TNF), US Patent No. 6692740 and US Patent No. 7270817 (ACPL), US Patent

- 15 045782 No. 7202343 (monocyte chemoattractant protein-1), US patent No. 7144731 (SCF), US patent No. 6355779 and US patent No. 7138500 (4-1BB), US patent No. 7135174 (PDGFD), US patent No. 6630143 and patent US Patent No. 7,045,128 (Flt-3 ligand), US Patent No. 6,849,450 (metalloproteinase inhibitor), US Patent No. 6,596,852 (LERK-5), US Patent No. 6,232,447 (LERK-6), US Patent No. 6,500,429 (brain-derived neurotrophic factor), U.S. Patent No. 6,184,359 (Epithelium-derived T-cell factor), U.S. Patent No. 6,143,874 (Neurotrophic Factor NNT-1), U.S. Patent Application Publication No. 20110027287 (Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9)), Publication Application No. US Patent No. 20110014201 (IL-18 RECEPTOR) and US Patent Application Publication No. 20090155164 (C-FMS). The foregoing patents and published patent applications are incorporated herein by reference in their entirety for the purpose of disclosing variable domain polypeptides, nucleic acids encoding variable domains, host cells, vectors, methods for producing polypeptides encoding said variable domains, pharmaceutical compositions, and methods of treating diseases associated with a corresponding target of a variable domain-containing antigen-binding protein or antibody.

In illustrative embodiments, the antibody is one of the following: muromonab-CD3 (marketed under the brand name Orthoclone Okt3®), abciximab (marketed under the brand name Reopro®), rituximab (marketed under the brand name Reopro®), rituximab (marketed under the brand name Reopro®), MabThera®, Rituxan®), basiliximab (marketed under the brand name Simulect®), daclizumab (marketed under the brand name Zenapax®), palivizumab (marketed under the brand name Synagis®) , infliximab (marketed under the brand name Remicade®), trastuzumab (marketed under the brand name Herceptin®), alemtuzumab (marketed under the brand name MabCampath®, Campath-1H®), adalimumab ( marketed under the brand name Humira®), tositumomab-I31 (marketed under the brand name Bexxar®), efalizumab (marketed under the brand name Raptiva®), cetuximab (marketed under the brand name Raptiva®), cetuximab (marketed under the brand name Erbitux®), ibritumomab tiuxetan (marketed under the brand name Zevalin®), omalizumab (marketed under the brand name Xolair®), bevacizumab (marketed under the brand name Avastin®), natalizumab (marketed under the brand name Tysabri®), ranibizumab (marketed under the brand name Lucentis®), panitumumab (marketed under the brand name Vectibix®), eculizumab (marketed under the brand name Vectibix®), eculizumab (marketed under the brand name brand Soliris®), certolizumab pegol (marketed under the brand name Cimzia®), golimumab (marketed under the brand name Simponi®), canakinumab (marketed under the brand name Ilaris®), catumaxomab ( marketed under the brand name Removab®), ustekinumab (marketed under the brand name Stelara®), tocilizumab (marketed under the brand name RoActemra®, Actemra®), ofatumumab (marketed under the brand name RoActemra®, Actemra®), ofatumumab (marketed under the brand name RoActemra®, Actemra®), ofatumumab (marketed as under the brand name Arzerra®), denosumab (marketed under the brand name Prolia®), belimumab (marketed under the brand name Benlysta®), raxibacumab, ipilimumab (marketed under the brand name Yervoy®) , and pertuzumab (marketed under the brand name Perjeta®). In illustrative embodiments, the antibody is one of the anti-TNF alpha antibodies, such as adalimumab, infliximab, etanercept, golimumab, and certolizumab pegol; antibodies to J.beta, such as canakinumab; antibodies to IL12/23 (p40), such as ustekinumab and briakinumab; and anti-IL2R antibodies such as daclizumab. In illustrative aspects, the antibody binds to a tumor-associated antigen and is an anticancer antibody. Examples of suitable anti-cancer antibodies include, but are not limited to, anti-BAFF antibodies such as belimumab; anti-CD20 antibodies such as rituximab; anti-CD22 antibodies such as epratuzumab; anti-CD25 antibodies such as daclizumab; anti-CD30 antibodies such as iratumumab, anti-CD33 antibodies such as gemtuzumab, anti-CD52 antibodies such as alemtuzumab; antibodies to CD152, such as ipilimumab; antibodies to EGFR, such as cetuximab; anti-HER2 antibodies such as trastuzumab and pertuzumab; antibodies to IL6, such as siltuximab; and anti-VEGF antibodies such as bevacizumab; IL6 receptor antibodies such as tocilizumab. In illustrative aspects, the tumor-associated antigen is CD20, and the antibody is an anti-CD20 antibody. In illustrative aspects, the tumor-associated antigen comprises SEQ ID NO: 3. In illustrative cases, the antibody comprises the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2. In illustrative aspects, the antibody is an anti-CD20 antibody, such as an anti-CD20 monoclonal antibody . In alternative aspects, the IgG1 antibody is rituximab or a biosimilar thereof. The term rituximab refers to a chimeric mouse/human IgG1 kappa monoclonal antibody that binds CD20 antigen (see CAS number: 174722-31-7; DrugBank - DB00073; Kyoto Encyclopedia of Genes and Genomes (KEGG) entry D02994). In illustrative aspects, the antibody contains a light chain containing CDR1, CDR2 and CDR3, presented in table. A. In illustrative aspects, the antibody contains a heavy chain containing CDR1, CDR2 and CDR3, presented in table.

A. In various cases, the antibody contains VH and VL or contains the sequences VH-IgGl and VLIgG kappa, presented in table. A.

Table A

Amino acid sequences of rituximab

Description Subsequence SEQ GO NO: CDR1LC RASSSVSYIH 4 CDR2LC ATSNLAS 5 CDR3 LC QQWTSNPPT 6 CDR1 NS SYNMH 7 CDR2 NS AIYPGNGDTSYNQKFKG 8 CDR3 NS STYYGGDWYFNV 9 VL QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGS SPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDA ATYYCQQWTSNPPTFGGGTKLEIK 10 VH QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVK QTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSST AYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTT VTVSA eleven VL-IgG kappa QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGS SPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDA ATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGL SSPVTKSFNRGEC 12 VH-IgGl QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVK QTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSST AYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTT VTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSS LGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 13

LC, light chain; HC, heavy chain; VL, light chain variable region; VH, heavy chain variable region.

In illustrative aspects, the antibody is an anti-EGFR antibody, such as an anti-HER2 monoclonal antibody. In illustrative aspects, the antibody is trastuzumab or a biosimilar thereof. The term trastuzumab refers to a humanized IgGl kappa, a monoclonal antibody that binds the HER2/neu antigen (see CAS number: 180288-69-1; DrugBank - DB00072; Kyoto Encyclopedia of Genes and Genomes (KEGG) entry D03257). In illustrative aspects, the antibody contains a light chain containing CDR1, CDR2 and CDR3, presented in table. IN.

In illustrative aspects, the antibody contains a heavy chain containing CDR1, CDR2 and CDR3, presented in table. B. In various cases, the antibody contains VH and VL or contains the VH-IgG1 and VL-IgG kappa sequences presented in table. IN.

Table B

Amino acid sequences of trastuzumab

Description Subsequence SEQID NO: CDR1LC QDVNTA 14 CDR2LC SAS 15 CDR3 LC QQHYTTPPT 16 CDR1 NS GFNIKDTY 17 CDR2 NS IYPTNGYT 18 CDR3 NS SRWGGDGFYAMDY 19 VL DIQMTQSPSSLSSASVGDRVTITCRASQDVNTAVAWYQQKP GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDF ATYYCQQHYTTPPTFGQGTKVEIK 20 VH EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQA PGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAY LQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVT VSS 21 VL-IgG kappa DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDF ATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSP VTKSFNRGEC 22 VH-IgGl EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQA PGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAY LQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPP 23 VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG

LC, light chain; HC, heavy chain; VL, light chain variable region; VH, heavy chain variable region.

Additional stages.

The methods disclosed herein include additional steps in various aspects. For example, in some aspects, the methods include one or more upstream or downstream steps used to produce, purify, and formulate a recombinant glycosylated protein. In illustrative embodiments, the method includes the steps of obtaining host cells that express a recombinant glycosylated protein (eg, an antibody or antibody-based binding protein). The host cells are, in some aspects, prokaryotic host cells, such as E. coli or Bacillus subtilis, or the host cells, in some aspects, are eukaryotic host cells, such as yeast cells, mycea cells

- 18 045782 lial fungi, protozoan cells, insect cells or mammalian cells (for example, CHO cells). Such host cells are described in the art. See, for example, Frenzel, et al., Front Immunol 4: 217 (2013) and herein under Cells. For example, in some cases, the methods include introducing into host cells a vector containing a nucleic acid containing a nucleotide sequence encoding a recombinant glycosylated protein or a polypeptide chain thereof.

In illustrative embodiments, the methods disclosed herein include the steps of isolating and/or purifying a recombinant glycosylated protein (eg, a recombinant antibody) from a culture. In illustrative aspects, the method includes one or more chromatography steps, including, without limitation, for example, affinity chromatography (eg, protein A affinity chromatography), ion exchange chromatography, and/or hydrophobic interaction chromatography. In illustrative aspects, the method includes the steps of obtaining crystalline biomolecules from a solution containing recombinant glycosylated proteins.

The methods of the present invention in various aspects include one or more steps of preparing a composition, including in some aspects a pharmaceutical composition comprising a purified recombinant glycosylated protein. Such compositions are discussed below.

Compositions.

Provided herein are compositions containing recombinant glycosylated proteins. In illustrative embodiments, the compositions are prepared using the methods for producing the recombinant glycosylated protein composition of the present invention described herein. In illustrative aspects, the recombinant glycosylated protein is an antibody. Accordingly, antibody compositions are provided herein. In illustrative embodiments, the antibody composition contains various glycoforms of the antibody. In illustrative embodiments, the antibody composition contains TAF glycoforms, LMW glycoforms, and/or afucosylated glycoforms of the antibody. Compositions containing antibody fragments or antibody-based protein products are also provided. In various aspects, antibody fragments, antibody protein products, glycosylated Fc fragments, or antibody products with a glycosylated Fc fragment contain a glycosylation site. In illustrative embodiments, the antibody composition is prepared by a method comprising maintaining glycosylation competent cells in a cell culture medium containing fucose, wherein the fucose is present in the culture medium at a concentration of from about 0.17 g/L to about 1.0 g/l. In illustrative embodiments, the antibody composition is prepared by a method comprising maintaining glycosylation competent cells in a cell culture medium containing fucose, wherein the fucose is present in the culture medium at a concentration of from about 0.1 g/L to about 1.0 g/L, and where the glycosylation competent cells are not genetically modified to provide alteration of de novo or recycle pathway enzyme activity. In illustrative embodiments, the antibody composition is prepared by a method comprising maintaining glycosylation competent cells in a cell culture medium containing fucose and glucose, wherein the fucose is present in the culture medium at a concentration of between about 0.1 g/L to about 1.0 g/L, and adding glucose to the cell culture medium according to a glucose feeding schedule that achieves an average glucose concentration of about 10 g/L or less. In illustrative embodiments, an antibody composition is prepared by practicing a method of modulating the level of TAF glycans, afucosylated glycans, or high mannose glycans in an antibody composition produced by glycosylation competent cells. In illustrative aspects, an antibody composition is prepared by employing a method of modulating TAF glycan levels comprising (A) adding fucose to a cell culture medium containing glycosylation competent cells to achieve a fucose concentration of about 0.1 g/ l to approximately 1.0 g/l, to reduce the level of TAF glycans; (B) adding glucose to a cell culture medium containing glycosylation competent cells to achieve a glucose concentration of less than about 10 g/L to increase the level of TAF glycans; or (C) both (A) and (B). In illustrative aspects, an antibody composition is prepared by practicing a method of modulating the level of afucosylated glycans in an antibody composition produced by glycosylation competent cells, comprising (A) adding fucose to a cell culture medium containing glycosylation competent cells, achieving a fucose concentration of from about 0.1 g/L to about 1.0 g/L to reduce the level of afucosylated glycans; (B) adding glucose to a cell culture medium containing glycosylation competent cells to achieve a glucose concentration of approximately 10 g/L or less to increase the level

- 19 045782 afucosylated glycans; or (C) both (A) and (B). In illustrative cases, the antibody composition is prepared by practicing a method of modulating the level of high mannose glycans in an antibody composition produced by glycosylation competent cells, comprising adding glucose to a cell culture medium containing glycosylation competent cells, achieving a glucose concentration of approximately 10 g/L or less to increase the level of high-mannose glycans.

In illustrative aspects, about 50% or less (e.g., about 40% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less) of the recombinant glycosylated protein in the composition is TAF glycoforms . In illustrative aspects, less than about 10% (e.g., about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3%, or less, approximately 2% or less) of the recombinant glycosylated protein in the composition are TAF glycoforms. In illustrative aspects, from about 4% to about 10% of the recombinant glycosylated protein in the composition is TAF glycoforms. In illustrative aspects, from about 2% to about 6% of the recombinant glycosylated protein in the composition is TAF glycoforms. In illustrative aspects, from about 2.5% to about 5% of the recombinant glycosylated protein in the composition is TAF glycoforms. In illustrative aspects, approximately 4% or less of the recombinant glycosylated protein in the composition is TAF glycoforms. In further exemplary aspects, from about 4% or less to about 2% or more of the recombinant glycosylated protein in the composition is TAF glycoforms.

In illustrative aspects, the compositions of the present invention are characterized by a glycoform profile in which about 50% or less (e.g., about 40% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less ) constitute TAF glycoforms. In illustrative aspects, the compositions of the present invention are characterized by a glycoform profile in which about 10% or less (e.g., about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less , about 4% or less, about 3% or less, about 2% or less) are TAF glycoforms. In illustrative aspects, the compositions of the present invention are characterized by a glycoform profile in which from about 4% to about 10% are TAF glycoforms. In illustrative aspects, the compositions of the present invention are characterized by a glycoform profile in which from about 2% to about 6% are TAF glycoforms. In illustrative aspects, the compositions of the present invention are characterized by a glycoform profile in which from about 2.5% to about 5% are TAF glycoforms. In illustrative aspects, the compositions of the present invention are characterized by a glycoform profile in which approximately 4% or less are TAF glycoforms. In illustrative aspects, the compositions of the present invention are characterized by a glycoform profile that is about 4% or less and about 2% or more TAF glycoforms.

In illustrative aspects, about 5% or less of the recombinant glycosylated protein (eg, antibody or antibody-based binding protein) in the composition is afucosylated glycoforms. In illustrative aspects, approximately 4% or less of the recombinant glycosylated protein (eg, antibody or antibody-based binding protein) in the composition is afucosylated glycoforms. In illustrative aspects, about 3.5% or less of the recombinant glycosylated protein (eg, antibody or antibody-based binding protein) in the composition is afucosylated glycoforms. In illustrative aspects, from about 0.8% to about 2.8% of the recombinant glycosylated protein in the composition is afucosylated glycoforms. In some aspects, the level of afucosylated 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%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, approximately 2.0%, approximately 2.1%, approximately 2.2 %, approximately 2.3%, approximately 2.4%, approximately 2.5%, approximately 2.6%, approximately 2.7% or approximately 2.8%.

In illustrative aspects, about 5% or less of the recombinant glycosylated protein (eg, antibody or antibody-based binding protein) in the composition is high-mannose glycoforms. In illustrative aspects, about 4% or less of the recombinant glycosylated protein (eg, antibody or antibody-based binding protein) in the composition is high-mannose glycoforms. In illustrative aspects, about 3.5% or less of the recombinant glycosylated protein (e.g., an

- 20 045782 antibody or antibody-based binding protein) in the composition are high-mannose glycoforms. In illustrative aspects, from about 0.7% to about 3.0% of the recombinant glycosylated protein in the composition is high mannose glycoforms. In some aspects, the level of high mannose glycans in the antibody composition is about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2% , approximately 1.3%, approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, approximately 2.0%, approximately 2.1%, approximately 2.2%, approximately 2.3%, approximately 2.4%, approximately 2.5%, approximately 2.6%, approximately 2.7%, approximately 2.8%, approximately 2, 9% or approximately 3.0%.

In illustrative embodiments, the composition is combined with a pharmaceutically acceptable carrier, diluent, or excipient. Accordingly, provided herein are pharmaceutical compositions comprising a recombinant glycosylated protein composition (eg, 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 of the standard pharmaceutical carriers such as phosphate-buffered saline, water, emulsions such as oil-in-water or water-in-oil, and various types of wetting agents.

Cell culture medium.

Provided herein is a cell culture medium containing: (a) glycosylation competent cells containing an exogenous nucleotide sequence encoding an antibody; and (b) a culture medium containing 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. Glycosylation competent cells can be any cell described herein. In illustrative cases, the glycosylation competent cells are not genetically modified to provide a change in the activity of a de novo pathway enzyme or a salvage pathway, optionally where the glycosylation competent cells are not genetically modified to provide a knockout of the gene encoding GDP-keto-6-deoxymannose- 3,5-epimerase/-4-reductase. In illustrative embodiments, the culture medium further contains glucose. In some aspects, the culture medium contains glucose at a concentration of less than about 10 g/L or less than about 9 g/L, such as about 6 g/L or less, or about 0.5 g/L to about 4 g/L. In some aspects, the culture medium contains fucose. In illustrative aspects, the pH of the culture medium is from about 6.85 to about 7.05, optionally from about 6.90 to about 7.00. In illustrative cases, the glycosylation competent cells are not genetically modified to alter the activity of a de novo or salvage pathway enzyme. For example, glycosylation competent cells are not genetically modified to knock out the gene encoding GDP-keto-6-deoxymannose-3,5-epimerase-4reductase. In illustrative aspects, the antibody is an IgG antibody, optionally an IgG1 antibody. The IgG1 antibody, in illustrative aspects, is specific for a tumor-associated antigen, such as CD20. In illustrative aspects, the cell culture medium does not contain mannose.

Modulation methods.

Further provided herein are methods of altering or modulating the levels of TAF glycans in a recombinant glycosylated protein (e.g., an antibody composition or an antibody binding protein composition) produced by glycosylation competent cells in a cell culture medium. In illustrative aspects, the method includes: (A) adding fucose to a cell culture medium containing glycosylation competent cells to achieve a fucose concentration of from about 0.1 g/L to about 1.0 g/L to reduce the level of TAF glycans; (B) adding glucose to a cell culture medium containing glycosylation competent cells to achieve a glucose concentration of less than about 10 g/L to increase the level of TAF glycans; or (C) both (A) and (B).

The present invention also provides methods for modulating the level of afucosylated glycans in a recombinant glycosylated protein (eg, an antibody composition or an antibody binding protein composition) produced by glycosylation competent cells. In illustrative aspects, the methods comprise (A) adding fucose to a cell culture medium containing glycosylation competent cells to achieve a fucose concentration of from about 0.1 g/L to about 1.0 g/L to reduce the level of afucosylated glycans; (B) adding glucose to the medium

- 21 045782 for culturing cells containing glycosylation competent cells to achieve a glucose concentration of less than about 10 g/l to increase the level of afucosylated glycans; or (C) both (A) and (B).

Also provided are methods for modulating the level of high mannose glycans in a recombinant glycosylated protein (eg, an antibody composition or an antibody binding protein composition) produced by glycosylation competent cells. In illustrative embodiments, the methods include adding glucose to a cell culture medium containing glycosylation competent cells to achieve a glucose concentration of less than about 10 g/L to increase the level of LMW glycans.

Accordingly, in some illustrative embodiments, the methods of the present invention relate to increasing the levels of TAF-, HM-, or afucosylated glycans in a protein, such as an antibody, produced by cells in cell culture. In illustrative aspects, levels of the LMW glycoforms of the recombinant glycosylated protein are increased relative to those in control cell culture. In illustrative aspects, the levels of one or more of Man5, Man6, Man7, Man8 and/or Man9 in the recombinant glycosylated protein are increased relative to a cell culture control. In illustrative aspects, the levels of afucosylated glycoforms of the recombinant glycosylated protein are increased relative to those in control cell culture. In illustrative aspects, the levels of one or more of A1G0, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5 in the recombinant glycosylated protein are increased relative to those in the control cell culture. In illustrative aspects, the levels of one or more of A1G1a, G0[H3N4], G0[H4N4], G0[H5N4], and FO-N[H3N3] in the recombinant glycosylated protein are increased relative to those in the control cell culture. In some aspects, the increase is an increase relative to a control cell culture as determined by hydrophilic interaction liquid chromatography (HILIC). In some aspects, the increase is an increase relative to a control cell culture, determined using methods known to one of ordinary skill in the art.

In some aspects, the methods of the present invention increase the levels of the TAF, NM, or afuco-glycoform to any extent or level relative to those in a control cell culture. For example, in some aspects, the increase provided by the methods of the present invention is an increase of at least or about 1% to about 10% (e.g., an increase of at least or about 1%, an increase of at least or about 2%, an increase of at least or about 3%, an increase of at least or about 4%, an increase of at least or about 5%, an increase of at least or about 6%, an increase of at least or about 7%, an increase of at least or about 8%, an increase of at least or about 9%, an increase of at least or about 9.5%, an increase of at least or about 9.8%, an increase at least or approximately 10%) relative to the control cell culture. In illustrative embodiments, the increase provided by the methods of the present invention is an increase of greater than 100%, such as 200, 300, 400, 500, 600, 700, 800, 900, or even 1000%, relative to a cell culture control. In illustrative embodiments, the level of TAF-, NM-, or afucosylated glycoforms of the protein is increased by at least about 1.5-fold relative to that in the control cell culture. In illustrative embodiments, the level of TAF-, NM-, or afucosylated glycoforms of the protein is increased by at least about 2-fold relative to that in the control cell culture. In illustrative embodiments, the level of TAF-, NM-, or afucosylated glycoforms of the protein is increased by at least about 3-fold relative to that in the control cell culture. In illustrative embodiments, the level of TAF-, NM-, or afucosylated glycoforms of the protein is increased by at least about 4-fold or about 5-fold relative to that in the control cell culture.

In illustrative aspects, increased levels of TAF glycoforms of the recombinant glycosylated protein are or can be observed or are detected or can be detected as early as day 1 after changing the concentration of fucose and/or glucose. In illustrative aspects, increased levels of TAF glycoforms of the recombinant glycosylated protein are or may be observed or are detected or may be detected as early as day 2 after the change. In illustrative aspects, increased levels of TAF glycoforms of the recombinant glycosylated protein are observed or may be observed or are detected or may be detected as early as day 3 after the change. In illustrative aspects, increased levels of TAF glycoforms of the recombinant glycosylated protein are or may be observed or are detected or may be detected as early as approximately 4 days after the change. In illustrative aspects, increased levels of TAF glycoforms of the recombinant glycosylated protein are observed or may be observed or are detected or may be detected after approximately day 5

- 22 045782 after change. In illustrative aspects, increased levels of TAF glycoforms of the protein are or may be observed or are detected or may be detected during collection of the recombinant glycosylated protein from the cell culture.

In illustrative aspects, increased levels of TAF glycoforms of the recombinant glycosylated protein are observed until about day 4, 5, or 6 of cell culture or longer. In illustrative aspects, increased levels of TAF glycoforms of the recombinant glycosylated protein are observed for 7, 8, 9, 10, 11, or 12 days of cell culture (post inoculation) or longer (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, approximately 2 months, approximately 3 months, approximately 6 months or approximately 1 year). In illustrative aspects, increased levels of TAF glycoforms of the protein are observed during collection of the protein from a cell culture.

In other aspects, the methods of the present invention relate to reducing the levels of TAF glycoforms of the protein produced by cells in cell culture. In illustrative aspects, the levels of the LMW glycoforms of the recombinant glycosylated protein are reduced relative to those in the control cell culture. In illustrative aspects, the levels of one or more of Man5, Man6, Man7, Man8 and/or Man9 in the recombinant glycosylated protein are reduced relative to those in control cell culture. In illustrative aspects, the levels of afucosylated glycoforms of the recombinant glycosylated protein are reduced relative to those in control cell culture. In illustrative aspects, the levels of one or more of A1G0, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5 in the recombinant glycosylated protein are reduced relative to those in control cell culture. In illustrative aspects, the levels of one or more of A1G1a, G0[H3N4], G0[H4N4], G0[H5N4], and FO-N[H3N3] in the recombinant glycosylated protein are reduced relative to those in control cell culture. In illustrative aspects, the method is a method of reducing the level of TAF glycoforms by an amount of from about 1% to about 4%, and the method includes maintaining glycosylation competent cells in the first cell culture medium and increasing the concentration of fucose to a value of from about 0.1 g /l to approximately 1.0 g/l. In some aspects, the reduction is a reduction relative to a control cell culture as determined by HILIC. In some aspects, the reduction is a reduction relative to a control cell culture, determined using methods known to one skilled in the art.

In some aspects, the methods of the present invention reduce the level(s) of TAF-, NM-, or afucosylated glycoform to any extent or level relative to those in a control cell culture. For example, the reduction achieved by the methods of the present invention 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 decrease of at least or about 0.3%, a decrease of at least or about 0.4%, a decrease of at least or about 0.5%, a decrease of at least or about of 0.6%, a decrease of at least or about 0.7%, a decrease of at least or about 0.8%, a decrease of at least or about 0.9%, a decrease of at least or about 0 .95%, a decrease of at least or about 0.98%, a decrease of at least or about 1.0%) relative to the level in the control cell culture. In illustrative embodiments, the reduction achieved by the methods of the present invention is a reduction of greater than about 100%, such as about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800 %, approximately 900% or even approximately 1000% relative to the level in the control cell culture. In illustrative embodiments, the level of TAF-, NM-, or afucosylated glycoforms of the protein is reduced by at least or about 1.5-fold relative to that in the control cell culture. In illustrative embodiments, the level of TAF-, NM-, or afucosylated glycoforms of the protein is reduced by at least about 2-fold relative to that in the control cell culture. In illustrative embodiments, the level of TAF-, NM-, or afucosylated glycoforms of the protein is reduced by at least about 3-fold relative to that in the control cell culture. In illustrative embodiments, the level of TAF-, NM-, or afucosylated glycoforms of the protein is reduced by at least about 4-fold or at least about 5-fold relative to that in the control cell culture.

In illustrative aspects, reduced levels of TAF glycoforms of the recombinant glycosylated protein are or may be observed or are detected or may be detected as early as approximately 1 day after inoculation. In illustrative aspects, reduced levels of TAF glycoforms of the recombinant glycosylated protein are or may be observed or are detected or may be detected as early as approximately day 2 postinoculation. In illustrative aspects, reduced

- 23 045782 the level of TAF glycoforms of the recombinant glycosylated protein is observed or can be observed or is detected or can be detected as early as approximately the 3rd day after inoculation. In illustrative aspects, reduced levels of TAF glycoforms of the recombinant glycosylated protein are or may be observed or detected or may be detected as early as approximately day 4 postinoculation. In illustrative aspects, a reduced level of TAF glycoforms of the recombinant glycosylated protein is or may be observed or detected or may be detected after approximately day 5 post-inoculation. In illustrative aspects, a reduced level of TAF glycoforms of the recombinant glycosylated protein is or may be observed, or is or may be detected around the time of collection of the protein from the cell culture.

In illustrative aspects, reduced levels of TAF glycoforms of the protein are observed longer than before about 4, about 5, or about 6 days of cell culture or after the initial cell culture period. In illustrative aspects, reduced levels of TAF protein glycoforms are observed for about 7, about 8, about 9, about 10, about 11, or about 12 days of cell culture (after inoculation) or longer (e.g., about 2 weeks, about 3 weeks, about 4 weeks, approximately 1 month, approximately 2 months, approximately 3 months, approximately 6 months or approximately 1 year). In illustrative aspects, reduced levels of TAF glycoforms of the protein are observed around the time of protein collection from the cell culture.

With respect to the methods of the present invention, the modulation, increase or decrease affected by such methods is relative to the control or control cell culture. In illustrative aspects, the reference level is the level of TAF glycoforms of the protein observed when the method steps of the present invention are not performed. In illustrative aspects, the reference level is the level of TAF glycoforms of the protein observed when a known production method is carried out using recombinant DNA technology. In illustrative aspects, the control is the level of TAF glycoforms observed when a known concentration of glucose or fucose is maintained during production by recombinant DNA technology. As used herein, the term control cell culture means a cell culture maintained in the same manner as a cell culture with respect to in which the steps of the method of the present invention are carried out (for example, cell culture according to the disclosed methods), with the exception of the concentration of fucose/glucose. In illustrative aspects, a control cell culture is a cell culture maintained at known operating or standard parameters, including a control fucose/glucose concentration. As used herein, the term fucose control concentration or glucose control concentration may refer to a known operating concentration of fucose/glucose, for example, the concentration of fucose/glucose in a cell culture maintained at the first time point or at a time point prior to the implementation of the methods of the present invention. In illustrative aspects, the reference fucose/glucose concentration is the concentration of fucose/glucose in a cell culture for which TAF levels are known or determined.

In exemplary aspects of the modulation methods of the present invention, the glycosylation competent cells are not genetically modified to provide an alteration in de novo pathway or salvage pathway enzyme activity. Optionally, glycosylation competent cells are not genetically modified to provide knockout of the gene encoding GDP-keto-6-deoxymannose-3,5-epimerase-4-reductase.

In illustrative aspects, after implementing the methods of the present invention, the level of TAF glycans in the antibody composition is less than about 10%, such as from about 2% to about 6%, from about 2% to about 5%, or from about 2% to about 4 %.

In illustrative aspects, after implementing the methods of the present invention, the level of high mannose glycans in the antibody composition is less than about 3.5%, optionally from about 0.7% to about 3.0%.

In illustrative aspects, after implementing the methods of the present invention, the level of afucosylated glycans in the antibody composition is less than about 3.5%, optionally from about 0.8% to about 2.8%.

With respect to the modulation methods described herein involving the addition of fucose, the final concentration of fucose in the cell culture medium is in various aspects from about 0.17 g/L to about 1.0 g/L or from about 0.2 g/L l to approximately 0.5 g/l. However, any of the fucose concentrations described in the document are considered.

With respect to the modulation methods described herein involving the addition of glucose to the cell culture medium, in some aspects, the glucose is added according to a glucose feeding schedule 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

- 24 045782 approximately 4.0 g/l. In some cases, the average glucose concentration is based on the fucose concentration in the cell culture medium. For example, the average glucose concentration can be calculated based on formula I

T=3.354-1.388F+O.111G + [F - 0.4375] x [1.9527(F - 0.4375)] (formula I), where T is a given % of TAF glycans and ranges from 2, 5% to about 6%, from about 2.75% to about 5.5%, or from about 3% to about 5%; F represents the concentration (g/L) of fucose in the medium; and G represents the average glucose concentration (g/L).

The present invention further provides methods for modulating (reducing or increasing) the level of afucosylated glycans in a recombinant glycosylated protein composition (eg, an antibody composition or an antibody binding protein composition) produced by glycosylation competent cells. In illustrative embodiments, the method includes lowering the pH of the cell culture medium by a value of from about 0.03 to about 1.2 (for example, from 0.05 to about 1.0) to reduce the level of afucosylated glycans in the composition based on recombinant glycosylated protein (e.g., an antibody composition or an antibody-based binding protein composition) to a value of from about 0.5% to about 2% (for example, from 0.5% to about 1.5%, from 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 to a value of between about 0. 03 to about 1.2 (e.g., 0.05 to about 1.0), 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 a value of from about 0.5% to about 2% (for example, from 0.5% to about 1.5%, from 0.5% to about 1.0%, from 1.0% to about 2%, from 1.5% to approximately 2.0%). In illustrative aspects, the method includes lowering the pH of the cell culture medium by a value of from about 0.05 to about 1.2 (e.g., 0.06, 0.07, 0.08, 0.09, 1.1, 1 ,11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.20), to reduce the level of afucosylated glycans in a composition based on recombinant glycosylated protein (e.g., an antibody composition or an antibody-based binding protein composition) by a value of 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 a value of from about 0.05 to about 1.2 (for example, 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 afucosylated glycans in a composition based on recombinant glycosylated protein (e.g., antibody composition or antibody binding protein composition) to a value of from about 1% to about 2% (e.g., from 1.0 to 1.5%, from 1.5 to 2.0%). In various cases, the method includes lowering the pH value of the cell culture medium by a value of from about 0.03 to about 0.07 (for example, 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 a value of from 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 a value of from about 0.03 to about 0.07 (for example, 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 a value of from about 0.5% to approximately 1.1% (for example, 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%).

Also provided herein are methods of 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 glycosylation competent cells by a value of from about 1% to approximately 2%. In illustrative embodiments, the method includes lowering the pH value of the cell culture medium by a value of from about 0.05 to about 1.2. Optionally, the method includes lowering the pH value by a value of from about 0.05 to about 0.07 to reduce the level of afucosylated glycans by about 1%, or decreasing the pH value by a value of from about 0.09 to about 1.2 to reducing the level of afucosylated glycans by more than approximately 1.5%. In various aspects, the method includes culturing cells at a pH value between

- 25 045782 approximately 7.10 to approximately 7.20, optionally from approximately 7.12 to approximately

7.19.

Methods are also 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 glycosylation competent cells to a value of from about 0 .5% to approximately 1.1%. In illustrative embodiments, the method includes lowering the pH value of the cell culture medium by approximately 0.03 to 0.07. In various aspects, the method includes lowering the pH by an amount of from about 0.03 to about 0.06 to reduce the level of afucosylated glycans by about 0.8%. In some aspects, the method includes lowering the pH by an amount of from about 0.05 to about 0.07 to reduce the level of afucosylated glycans by about 1%. In various cases, the method includes culturing the cells at a pH value of from about 7.05 to about 7.15, optionally from about 7.07 to about 7.13.

The present invention provides methods for increasing the level of afucosylated glycans in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by glycosylation competent cells by a value of between about 1 % to approximately 2%. In illustrative embodiments, the method includes increasing the pH of the cell culture medium by a value of from about 0.05 to about 1.2. Optionally, the method includes increasing the pH value by an amount of from about 0.05 to about 0.07 to reduce the level of afucosylated glycans by about 1% or increasing the pH value by an amount of from about 0.09 to about 1.2 to reducing the level of afucosylated glycans by more than approximately 1.5%. In some aspects, the method includes culturing cells at a pH value of from about 7.10 to about 7.20, optionally from about 7.12 to about 7.19.

The present invention also provides methods for increasing the level of afucosylated glycans in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by glycosylation competent cells by a value of from about 0.5% to approximately 1.1%. In illustrative embodiments, the method includes increasing the pH of the cell culture medium by about 0.03 to about 0.07, or increasing the pH by an amount of about 0.03 to about 0.06, to reduce the level of afucosylated glycans by about 0 .8%, or increasing the pH by about 0.05 to about 0.07 to reduce the level of afucosylated glycans by about 1%. In various cases, the method includes culturing the cells at a pH value of from about 7.05 to about 7.15, optionally from about 7.07 to about 7.13.

In any of the above methods, the pH of the cell culture medium throughout the culture is greater than 7.0, optionally greater than 7.05 to less than 7.2. In some aspects, the level of afucosylated glycans in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) is less than about 10%, such as about 6.2% to about 8.4%. In any of the above methods, the temperature changes by less than 2°C during the culture period. For example, in some aspects, the temperature of the crop changes by no more than 1.5 or 1.0°C. In any of the above methods, the cell culture medium does not contain any detectable amounts of manganese or betaine. The cell culture medium in some aspects 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. The addition or presence of fucose in the culture medium may follow any of the principles provided herein. In illustrative aspects, the glycosylation competent cells are not genetically modified to provide an alteration in the activity of a de novo pathway enzyme or a salvage pathway enzyme. In any of the above methods, glucose is added to the cell culture medium according to a glucose feeding schedule that achieves an average glucose concentration of about 10 g/L or less. Glucose can be added in accordance with any of the principles provided herein.

As discussed above, TAF glycans represent the sum of LM glycans and afucosylated glycans. Accordingly, methods for modulating afucosylated glycans disclosed herein will in various cases provide modulation of the level of TAF-26045782 glycans in a recombinant glycosylated protein composition (eg, an antibody composition or an antibody binding protein composition). Accordingly, provided herein are methods for modulating the level of TAF glycans in a recombinant glycosylated protein composition (eg, an antibody composition or an antibody binding protein composition) produced by glycosylation competent cells. In illustrative aspects, the method includes modulating the level of afucosylated glycans in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) in accordance with a method for modulating the level of afucosylated glycans disclosed herein. In illustrative embodiments, a method for modulating TAF glycans includes reducing the level of afucosylated glycans in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) in accordance with a method of reducing the level of afucosylated glycans disclosed herein or increasing the level of afucosylated glycans in a recombinant glycosylated protein composition (eg, an antibody composition or an antibody binding protein composition) in accordance with a method of increasing the level of afucosylated glycans disclosed herein.

In addition, the present invention provides a method for preparing an antibody composition, wherein the level of afucosylated glycans in the antibody composition is from about 6.2% to about 8.4%. In illustrative embodiments, the method includes maintaining glycosylation competent cells in a cell culture medium at a pH value of greater than 7.05 to less than 7.2, where optionally:

(A) the pH of the cell culture medium is changed to less than 0.15 (optionally less than 0.10) during the culture period, or (B) the temperature of the cell culture medium is changed to no more than 2°C, or (C) the method does not include culturing cells in a cell culture medium containing manganese or betaine, or (D) a combination of two or three of (A), (B) and (C).

In various aspects, the pH is maintained at a pH level of from about 7.07 to about 7.19 (e.g., 7.08, 7.09, 7.10, 7.11, 7.12, 7.13, 7.14, 7.15, 7.16, 7.17, 7.18, or 7.19) during the culture period, optionally, wherein the pH is maintained at or above about 7.07 to less than 7.10, or about 7 .10 or more to less than 7.15, or from about 7.15 or more to about 7.19. In various aspects, the level of afucosylated glycans in the antibody composition is less than about 10%, optionally from about 6.2% to about 8.4%. In various cases, the temperature changes by less than 2°C during the culture period, optionally the culture temperature changes by no more than 1.5 or 1.0°C. In various aspects, the cell culture medium does not contain any detectable amounts of manganese or betaine. In illustrative aspects, 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 fucose is present in the culture medium in concentrations 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 competent cells are not genetically modified to alter de novo or salvage pathway enzyme activity, and in some aspects, glucose is added to the cell culture medium according to a glucose feeding regimen that achieves an average glucose concentration, of approximately 10 g/L or less.

Exemplary Embodiments.

The present invention provides methods for preparing a recombinant glycosylated protein composition (eg, an antibody composition or an antibody binding protein composition). In illustrative embodiments, the method includes maintaining glycosylation competent cells in a cell culture medium containing fucose and/or glucose at a specific concentration described herein, depending on the desired level of TAF glycoforms. In illustrative embodiments, the level of TAF glycoforms in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) is approximately 10% or less, and in illustrative aspects the method includes maintaining glycosylation-competent cells in a cell culture medium containing fucose, wherein the fucose is present in the culture medium at a concentration of from about 0.17 g/L to about 1.0 g/L. In illustrative embodiments, the level of TAF glycoforms in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) is at

- 27 045782 about 10% or less, and in illustrative aspects the method includes maintaining glycosylation competent cells in a cell culture medium containing fucose, wherein the fucose is present in the culture medium at a concentration of from about 0.1 g/L to approximately 1.0 g/L, and where the glycosylation competent cells are not genetically modified to provide alteration in de novo or recycle pathway enzyme activity. The present invention also provides methods for producing a recombinant glycosylated protein composition (eg, an antibody composition or an antibody binding protein composition), comprising maintaining glycosylation competent cells in a cell culture medium containing fucose and glucose, wherein fucose is present in the culture medium at a concentration of 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 schedule that achieves an average glucose concentration, of approximately 10 g/L or less. In illustrative aspects, fucose is present in the culture medium at a concentration of less than about 0.75 g/L, optionally less than about 0.6 g/L. In illustrative cases, fucose is present in the culture medium at a concentration of from about 0.2 g/L to about 0.5 g/L. In some aspects, fucose is present in the culture medium for as long as the glycosylation competent cells are maintained in the cell culture. In illustrative cases, a method of producing a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) comprises maintaining glycosylation competent cells in a first cell culture medium for an initial period of time and then maintaining glycosylation competent cells. with respect to glycosylation of cells in a second cell culture medium, wherein the first cell culture medium does not contain fucose at a concentration of from about 0.1 g/L to about 1.0 g/L, and the second cell culture medium contains fucose at concentration ranging from about 0.1 g/L to about 1.0 g/L. In some cases, the initial time period is from about 24 hours to about 72 hours. In alternative aspects, the initial time period is about 72 hours or more than about 72 hours but less than 156 hours or about 156 hours. In some aspects, fucose is added to the first medium to cultivation on the 6th day after inoculation with the cell culture to obtain a second cell culture medium. The concentration of fucose fluctuates by about 0.2 g/L or less (eg, 0.1 g/L or less) during the time the glycosylation competent cells are maintained in cell culture medium containing fucose. In some aspects, the cell culture medium contains an initial concentration of glucose for an initial period of time. Optionally, the initial glucose concentration is from about 1 g/L to about 15 g/L, for example about 12 g/L ± 1 g/L. In illustrative aspects, the method further includes adding glucose to the cell culture medium in accordance with a glucose feeding regimen. In illustrative aspects, the glucose feeding regimen is initiated after a period of time ranging from about 4 days to about 6 days after inoculation with the cell culture. For example, a glucose feeding regimen can be initiated approximately 6 days after cell culture inoculation. In illustrative cases, the glucose feeding regime 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 in cell culture medium, from about 0. 5 g/l to approximately 4 g/l). In illustrative cases, the glucose feeding regime is used to achieve an average glucose concentration based on the fucose concentration in the cell culture medium. In illustrative aspects, the average glucose concentration is calculated using Formula I

T=3.354-l.388F+0.lllG + [F - 0.4375] x [1.9527(F - 0.4375)] (Formula I), where T is the specified % of total afucosylated (TAF) glycans in 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 (g/L) of fucose in medium, and G represents the average glucose concentration (g/L) in the medium. In illustrative aspects, (i) the fucose concentration is about 0.2 ± 0.1 g/L, and the average glucose concentration is from about 2 g/L 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 g/L 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 illustrative aspects, the pH of the cell culture medium is from about 6.85 to about 7.05 (for example, from about 6.90 to about 7.00). In some aspects, glycosylation competent cells are not genetically modified

- 28 045782 baths to ensure a change in enzyme activity of the de novo pathway or the reutilization pathway. For example, glycosylation competent cells are not genetically modified to knock out the gene encoding GDP-keto-6-deoxymannose-3,5-epimerase 4-reductase. In illustrative cases, the level of total afucosylated (TAF) glycans in the antibody composition is less than about 10% (e.g., about 2% to about 6%, about 2% to about 5%, about 2% to about 4%) . In illustrative cases, the level of high-mannose glycans in the antibody composition is less than about 3.5% (eg, from about 0.7% to about 3.0%). In illustrative cases, the level of afucosylated glycans in the antibody composition is less than about 3.5% (eg, from about 0.8% to about 2.8%). In some aspects, glycosylation competent cells produce IgG antibodies, optionally IgG1 antibodies. In some aspects, IgG1 antibodies are specific for a tumor-associated antigen (eg, CD20). In illustrative aspects, the culture medium does not contain mannose.

A cell culture medium containing: (a) glycosylation competent cells containing an exogenous nucleic acid encoding an antibody; and (b) a culture medium containing 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 illustrative aspects, the glycosylation competent cells are not genetically modified to provide a change in the activity of a de novo pathway enzyme or a salvage pathway, optionally wherein the glycosylation competent cells are not genetically modified to provide a knockout of the gene encoding GDP-keto-6-deoxymannose- 3,5-epimerase, 4-reductase. In illustrative aspects, the culture medium further contains 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 illustrative aspects, 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 illustrative aspects, the antibody is an IgG antibody, such as an IgG1 antibody. In illustrative cases, the IgG1 antibody is specific for a tumor-associated antigen, such as CD20.

Further provided herein are methods of altering or modulating the level of TAF glycans in a recombinant glycosylated protein composition (e.g., an antibody composition or an antibody binding protein composition) produced by glycosylation competent cells in a culture medium cells. In illustrative aspects, the method includes: (A) adding fucose to a cell culture medium containing glycosylation competent cells to achieve a fucose concentration of from about 0.1 g/L to about 1.0 g/L to reduce TAF levels -glycans; (B) adding glucose to a cell culture medium containing glycosylation competent cells to achieve a glucose concentration of less than about 10 g/L to increase the level of TAF glycans; or (C) both (A) and (B). Also provided are methods for modulating the level of afucosylated glycans in a recombinant glycosylated protein composition (eg, an antibody composition or an antibody binding protein composition) produced by glycosylation competent cells. In illustrative embodiments, the method comprises: (A) adding fucose to a cell culture medium containing glycosylation competent cells to achieve a fucose concentration of from about 0.1 g/L to about 1.0 g/L to reduce level of afucosylated glycans; (B) adding glucose to a cell culture medium containing glycosylation competent cells to achieve a glucose concentration of approximately 10 g/L or less to increase the level of afucosylated glycans; or (C) both (A) and (B). The present invention further provides a method for modulating the level of high mannose glycans in a recombinant glycosylated protein composition (eg, an antibody composition or an antibody binding protein composition) produced by glycosylation competent cells. In illustrative embodiments, the method includes adding glucose to a cell culture medium containing glycosylation competent cells to achieve a glucose concentration of less than about 10 g/L to increase the level of LMW glycans. In illustrative aspects of the modulation methods, the glycosylation competent cells are not genetically modified to provide a change in the activity of a de novo pathway enzyme or a salvage pathway enzyme. For example, glycosylation competent cells in some aspects are not genetically modified to provide knockout of 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 about 10% or less (e.g.,

- 29 045782 approximately 2% to approximately 6%, from approximately 2% to approximately 5%, from approximately 2% to approximately 4%). In some aspects, the level of high mannose glycans in the antibody composition is about 3.5% or less (eg, about 0.7% to about 3.0%). In some aspects, the level of afucosylated glycans in the antibody composition is about 3.5% or less (eg, about 0.8% to about 2.8%). In illustrative aspects, the concentration of fucose 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 includes adding glucose to the cell culture medium in accordance with a glucose feeding schedule 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). .0 g/l, less than approximately 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 using Formula I:

T=3.354-l.388F+0.lllG + [F - 0.4375] x [1.9527(F - 0.4375)] (Formula I), where T is the specified % of total afucosylated (TAF) glycans in 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 (g/L) of fucose in medium, and G represents the average glucose concentration (g/L).

The following examples are given only to illustrate the present invention and do not limit its scope in any way.

Examples

Example 1.

This example describes the methods performed and the materials used in the experiments in Example 2.

Cell lines, cell culture and media.

All experiments were performed using a clone expressing an antibody containing a light chain containing SEQ ID NO: 1 and a heavy chain containing SEQ ID NO: 2. All experiments were performed using a separate vial of cells cultured for 25 days. The following parameters were kept constant: duration (12 days), dissolved oxygen (48 to 74 mm Hg), pH value (6.85 to 7.05), stirring (350 rpm, 20 W/m ³ ), temperature (36.0°C).

Glycan map generated by hydrophilic interaction liquid chromatography (HILIC).

Glycan maps, which are enzymatically released N-linked glycans, were generated using HILIC. Briefly, glycans were incubated with a solution containing PNGase F and sodium phosphate buffer (pH 7.5) for ∼2 h at ∼37°C. The labeling solution containing 2-aminobenzoic acid (2-AA) and sodium cyanoborohydride was then added to the PNGase F-treated glycans and the mixture was incubated at ~80°C for approximately 75 min. After incubation, the mixtures were centrifuged to obtain a precipitated protein pellet. Supernatant samples were collected and placed in vials.

Glycans were separated using HILIC with an integrated fluorescence detector: glycans were loaded onto the column and bound to the column under high organic conditions (mobile phase A and mobile phase B were ammonium formate and acetonitrile, respectively) and then eluted using an increasing gradient of buffer based on an aqueous solution of ammonium formate. High resolution was achieved using a small particle column format of 1.7 μm diameter and a column length of 150 mm. The total run time, including column re-equilibration, was 155 min.

Example 2.

This example demonstrates the effects of increasing the levels of glucose and fucose in the culture medium in relation to % TAF.

Cells expressing an antibody containing the light chain of SEQ ID NO: 1 and the heavy chain of SEQ ID NO: 2 were added to a bioreactor containing one of three culture media: a control culture medium, a first test culture medium, and a second test medium. for cultivation. The first test culture medium was identical to the control culture medium except that it contained twice the amount of glucose, and the second test culture medium was the control culture medium with 0.5 g/L fucose. Each of the culture media did not contain mannose. The cell culture was maintained for 12 days at a pH value in the range of 6.85-7.05.

Media samples were periodically taken from the bioreactors to measure glucose concentrations.

- 30 045782 TAF levels and ADCC levels. Levels of TAF- and/or afucosylated (afuc-) glycans were analyzed using a HILIC N-glycan mapping procedure, and ADCC stimulating ability was tested using an in vitro assay. The results are shown in table. 1 below.

__________________________________________________________________Table 1

Culture medium TAF level (%) ADCC(%) Specified 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%

ADCC levels were expressed as % relative to the ADCC level achieved with a control, which is a commercially available antibody having the same amino acid sequence.

Glucose concentrations were determined throughout the culture cycle and the results of these measurements were plotted as a function of time and against TAF levels in antibodies produced using each type of cell culture medium. The results are shown in Fig. 3. As shown in this figure, antibodies produced by cells cultured in the first test culture showed an increase in TAF levels, and this increase corresponded to an increase in glucose levels in the cell culture. These results (and those from Table 1) indicate that glucose may influence TAF and ADCC levels.

Antibodies produced by cells cultured in a second test culture medium containing 0.5 g/L fucose showed an ~1% reduction in TAF levels (Figure 3). As shown in FIG. 3, the concentration of fucose did not change significantly during the 12-day run, probably because the uptake of fucose into cells was negligible.

Additional analysis of individual glycan species in antibodies obtained using each of the different culture media was performed. Interestingly, as shown in FIG. 4.% of high mannose (HM) glycans increased if cells were cultured in the first test medium, which contained 2 times the amount of glucose compared to the control medium. This increase in % LMW glycans was not achieved by cells cultured in the second test medium containing fucose. As shown in FIG. 4.% LMW glycans in antibodies produced by cells cultured in the second test medium containing fucose was approximately the same as % LMW glycans in antibodies produced by cells cultured in control medium.

The effects of culture in media containing twice the amount of glucose (first test medium) or containing fucose (second test medium) on % afucosylated glycans were similar to those on % TAF glycans. As shown in FIG. 5, cells cultured in the first test medium containing a higher concentration of glucose showed increased levels of afucosylated glycans, whereas cells cultured in a second test medium containing fucose produced antibodies with reduced levels of afucosylated glycans.

This example demonstrates that glucose and fucose are levers that can be used to modulate the levels of high-mannose and afucosylated glycans, as well as influence ADCC.

Example 3.

This example demonstrates additional research demonstrating that glucose and fucose are levers for modulating TAF and ADCC levels.

A prospective multivariate full factorial experiment was designed to (1) elucidate the main effects, interaction effects, and quadratic effects for the fucose and glucose variables and (2) determine the amounts of these variables that would result in changes in TAF glycan and ADCC levels. Fucose was evaluated in the culture medium at concentrations of 0, 0.5 and 1 g/L. Glucose was fed at rates of 0X, 1X (control) and 2X. 0X meant that no initial glucose solution was added to the cultures, resulting in a residual glucose concentration after day 6 of cell culture of ~1 g/L. Glucose was supplied only when using media that contained 12 g/l sugar. During 1X feeding, glucose was maintained at an average concentration of 3 ± 1 g/L after the start of glucose feeding. During 2X feeding, mean glucose levels were maintained at 6 ± 1 g/L. The results indicate that fucose concentrations can be varied to provide an effect on TAF levels (Figure 6) and ADCC levels (Figure 7).

In these experiments, glucose concentration was controlled at 3 ± 1 g/L after feeding began (i.e., after day 6). If glucose concentrations exceeded 4 g/L, no additional glucose was given

- 31 045782 was added and the cells were required to use the residual glucose in the bioreactor and the glucose supplied through the perfusion medium until the glucose level fell within the control range. Based on these experiments, TAF values were predicted for different concentrations of fucose and glucose according to the model shown in FIG. 8. The model demonstrates that the target quality product profile (QTTP) can be achieved by culturing cells in culture media containing from about 0.1 g/L to about 1.0 g/L fucose and/or from about 0.5 g/L to approximately 4.0 g/L glucose. Using this model, TAF values were predicted for the following different concentrations of fucose and glucose: 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. Fig. 9A-9C. These results indicate that there are several ways to achieve the required TAF levels and ADCC levels.

To confirm the predictions from FIG. 9A-9C conducted additional experiments. A brief description of the experiments is presented in Table. 2.

table 2

Conditions Fucose (g/l) Glucose (g/l) TAG (95% CI) Actual TAG level ADCC (95% CI) Actual ADCC level 1 0 0X 3,504.06 3.50 75.7-90.9 80.13 2 (control) 0 IX 3.68- 4.22 3.84 84.5-101.1 100.3 3 0.2 IX 3.30- 3.73 3.35 78.3-90.4 85.9 4 0.5 IX 2.67- 3.30 2.94 66.8-83.14 76.2

0X - measured glucose concentration was ~1 g/L after day 6; IX - the measured glucose concentration was ~ 3±1 g/l; 2X - the measured glucose concentration was ~ 6±1 g/l.

This example demonstrates that both glucose concentration and fucose concentration are variables that can be manipulated to alter TAF and ADCC levels.

Example 4.

This example demonstrates the effect of fucose on cell cultures.

Additional analyzes were performed on the cell cultures described above. For example, osmolality was measured for cell cultures and ranged from approximately 175 mOsm/kg to approximately 345 mOsm/kg. There was no correlation between the osmolality of cell cultures and the concentration of fucose. See fig. 10. As shown in this figure, the addition of fucose to the culture medium does not appear to affect osmolality in any particular way. The fact that osmolality varied greatly under control conditions (no fucose) indicates that components in the culture medium (other than fucose) actively influence osmolality.

In one experiment of this fucose study, cells were inoculated into one of five bioreactors, two of which contained culture medium without fucose (ctrl_a and ctrl_b), and three of which contained culture medium with 0.5 g/L fucose (fuc_a, fuc_b and fuc_c). Media samples from five cell cultures were collected throughout the 12-day culture period: day 0, day 3, day 5, day 7, day 9, and day 12. The concentration of fucose in the samples was measured. As shown in FIG. 11, the concentration of fucose remained virtually unchanged throughout the cultivation, indicating a small and/or slow consumption of fucose during this cultivation process.

In another experiment in this study regarding fucose, cells were maintained in cell culture for 12 days in a culture medium containing no fucose. In this particular experiment, cell culture pH was allowed to deviate from 7.1, which caused %TAF to increase to approximately 5.5% from day 5 to ~day 8. %TAF was measured daily starting on day 5. In an attempt to modulate TAF levels back to a target value of 4.0% fucose was added on day 9 of cultivation at a feeding rate of 0.9 g/L per day. As shown in FIG. 12.% TAF was reduced from approximately 5.5% when fucose was added to the culture medium. %TAF continued to decline to ~3.8% on day 12. These data indicate that addition of fucose may occur late in the culture period and still cause modulation of %TAF.

This example demonstrates the effect of fucose concentration in the culture medium.

-32045782 nia to TAR levels.

Example 5.

This example demonstrates that maintaining glucose levels within a given range can occur at the end of the culture period.

Three experiments were performed to track the time to reach a given glucose range over a 12-day culture period. For each experiment, the initial glucose concentration in each cell culture ranged from approximately 5.0 g/L to approximately 6.0 g/L. Glucose concentrations in the cell culture medium were monitored daily. As shown in FIG. 13, each of the cell cultures reached a given glucose concentration (0.5-4.0 g/l) on different days of the culture period. In one experiment (line with open squares), the target range was reached on day 2. In the second experiment (dashed line with open diamonds), the target range was reached on day 4, whereas in the third experiment (dashed line with open circles), the target range was reached on day 6. Despite these differences, each cell culture achieved the target %TAF range (~2.0 to ~4.3%) (see left graph in Fig. 13; third experiment is represented by open circles; second experiment is represented by open diamonds; the first experiment is represented by open squares). These data indicate that glucose can be supplied late in the culture period to achieve the same TAF levels as in cultures supplied earlier in the culture period.

These data demonstrate that early and late control of glucose concentrations result in similar levels of TAF.

Example 6.

This example demonstrates the effects of lowering the pH value in cell culture on the level of afucosylation of an antibody with and without the addition of fucose.

A cell culture sample was collected from a 2000 L bioreactor and used to inoculate parallel 3 L bioreactors. Bioreactors of 2000 L and 3 L were fed using a continuous fed-batch culture method using feed A and feed B for 12 days. Two of the 3 L bioreactors were fed with fucose to achieve a final concentration of 1.0 g/L on day 5. Afucosylation levels were measured as described in Example 1 and the results are presented in Table 1. 3.

Table 3

pH value Adding fucose % afucosylation 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

As shown in table. 3, in the absence of fucose addition, if the pH value was below 7.1, the average % afucosylation was 6.39, while if the pH value was above 7.10 but below 7.15, the average % afucosylation was higher (% afucosylation=7.185). If the pH value was higher than 7.15, the average % afucosylation was even higher (% afucosylation = 8.276). Thus, lower pH was correlated with lower % afucosylation.

When fucose was added to the culture medium (to reach a final concentration of 1.0 g/L fucose), % afucosylation was significantly reduced at both lower pH (7.07) and higher pH (7.18). ). For each of these pH levels, if fucose was added, the average reduction in % afucosylation was 2.18%.

These results indicate that lowering the pH of the cell culture medium and/or adding fucose to the cell culture medium results in a decrease in the percentage of afucosylation. When the pH value was simultaneously decreased and fucose was added to the cell culture medium, a greater decrease in the percentage of afucosylation was observed.

Example 7.

This example further demonstrates the effects of lowering pH in cell culture on the level of antibody afucosylation with and without the addition of fucose.

-

Claims (33)

A cell culture sample from a 2000 L bioreactor was used to inoculate parallel 3 L bioreactors. Bioreactors with a volume of 2000 L and 3 L were fed using a continuous fed-batch culture method using feed A and feed B for 12 days. Some of the bioreactors (ie cell culture) were fed with fucose to a final concentration of 0.25, 0.5 or 1 g/L on day 5. Afucosylation levels were measured as described in Example 1 and the results are presented in FIG. 14. As shown in FIG. 14, the control 3 L bioreactor (control pH 7.1) exhibited levels of afucosylation similar to that of the original 2000 L bioreactor. Both reactors exhibited % afucosylation of approximately 6.5% or greater. Addition of fucose at any of the levels tested resulted in a significant reduction in % afucosylation (5.5% or lower). The percentage of afucosylation was lowest with the addition of 0.25 g/L fucose. Also, as shown in FIG. 14, decreasing the pH from 7.1 to 7.0 without adding fucose to the culture medium also resulted in a reduction in afucosylation of at least about 1.0%. All references, including publications, patent applications and patents cited herein, are hereby incorporated by reference to the same extent as if each document had been separately and specifically identified as being incorporated by reference and had been presented in its entirety in this document. The use of the singular, plural and similar terms in the context of the description of the present invention (especially in the context of the following claims) should be construed to include both the singular and the plural, unless otherwise indicated herein or clearly inconsistent with the context. . The terms including, having, including and containing are to be construed as open terms (i.e., including without limitation) unless otherwise noted. It is intended that the presentation of ranges of values in this document serve solely as a means of abbreviating the individual indication of each individual value within a given range and each endpoint, unless otherwise specified herein, and each individual value and endpoint is included herein. , as if they were individually mentioned herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or otherwise clearly inconsistent with the context. The use of any kind of examples or illustrative language (eg, such as) provided herein is intended solely to better illuminate the present invention and does not limit the scope of the present invention unless otherwise stated. Nothing in this specification should be construed as indicating any unstated element as essential to the practice of the present invention. This document describes preferred embodiments of the present invention, including the best method known to the inventors of the present invention for carrying out the present invention. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The present inventors expect that those skilled in the art will use such variations accordingly, and the present inventors expect that the present invention will be practiced differently than specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter mentioned in the claims appended hereto, to the extent permitted by applicable law. Moreover, any combination of the above-described elements, in all possible variations thereof, is encompassed by the present invention unless otherwise indicated herein or otherwise clearly contradicted by the context. CLAIM 1. A method of producing an antibody composition, wherein the level of total afucosylated (TAF) glycans in the antibody composition is less than 10%, comprising maintaining glycosylation competent cells in a cell culture medium containing from 0.1 to 1.0 g /l fucose and from 0.5 to 10 g/l glucose. 2. The method according to claim 1, where the cultivation medium contains from 0.17 to 1.0 g/l fucose. 3. The method according to claim 2, where the cultivation medium contains from 0.2 to 1.0 g/l or from 0.2 to 0.5 g/l fucose. 4. Method according to any one of claims 1 to 3, wherein fucose is present in the culture medium during the entire period of maintaining glycosylation competent cells in cell culture. 5. The method according to any one of claims 1-3, including maintaining glycosylation-competent - 34 045782 culturing cells in a first cell culture medium for an initial period of time and subsequently maintaining glycosylation competent cells in a second cell culture medium, wherein the first cell culture medium does not contain fucose at a concentration of from 0.1 to 1 .0 g/L, and the second cell culture medium contains fucose at a concentration of 0.1 to 1.0 g/L. 6. The method according to claim 5, where the initial period of time is from 24 to 72 hours, or 72 hours, or more than 72 hours, but less than 156 hours, or 156 hours. 7. The method according to any one of claims 5 or 6, wherein fucose is added to the first culture medium on day 6 after inoculation with the cell culture to obtain a second cell culture medium. 8. The method according to any one of claims 1 to 7, wherein the concentration of fucose fluctuates by 0.2 g/L or less during the time of maintaining glycosylation competent cells in a cell culture medium containing fucose, optionally, wherein the concentration of fucose fluctuates by 0.1 g/L or less during the time of maintaining glycosylation competent cells in a cell culture medium containing fucose. 9. Method according to any one of claims 1 to 8, wherein the culture medium contains 9 g/l, optionally 6 g/l glucose. 10. Method according to any one of claims 1 to 8, where the culture medium contains from 0.5 to 4 g/l glucose. 11. Method according to any one of claims 1 to 8, wherein the culture medium contains glucose in a concentration based on the concentration of fucose, optionally, wherein the glucose concentration is calculated using formula I T=3.354-l.388F+0.lllG + [F - 0.4375] x [1.9527(F - 0.4375)] (Formula I), where T is the specified % of total afucosylated (TAF) glycans in composition based on the antibody and is from 2.5 to 6%, from 2.75 to 5.5% or from 3 to 5%, F is the concentration (g/l) of fucose in the medium and G is the average concentration of glucose ( g/l) in the medium. 12. The method according to claim 11, where (i) the concentration of fucose is 0.2 ± 0.1 g/L and the concentration of glucose is from 2 to 4 g/L; (ii) the fucose concentration is 0.5 ± 0.1 g/L and the glucose concentration is between 3 and 6 g/L; or (iii) the fucose concentration is 0.75 ± 0.1 g/L and the glucose concentration is between 4.5 and 9 g/L. 13. Method according to any one of claims 1 to 12, wherein the pH value of the cell culture medium is from 6.85 to 7.05, optionally from 6.90 to 7.00. 14. The method according to any one of claims 1 to 13, wherein the glycosylation competent cells are not genetically modified to provide a change in the activity of the de novo pathway enzyme or the recycling pathway. 15. The method of claim 14, wherein the glycosylation competent cells are not genetically modified to provide knockout of the gene encoding GDP-keto-b-deoxymannose 3,5 epimerase/-4 reductase. 16. The method according to any one of claims 1 to 15, wherein the level of TAF glycans is from 2 to 6%, optionally from 2 to 5% or from 2 to 4%. 17. The method according to any one of claims 1 to 16, wherein the level of high mannose glycans and/or the level of afucosylated glycans in the antibody composition is less than 3.5%, optionally, wherein the level of high mannose glycans in the antibody composition the antibody is from 0.7 to 3.0% and/or the level of afucosylated glycans in the antibody composition is from 0.8 to 2.8%. 18. The method according to any one of claims 1 to 17, wherein the glycosylation competent cells produce IgG antibodies, optionally IgG1 antibodies. 19. The method of claim 18, wherein the IgG1 antibodies are specific for a tumor-associated antigen, optionally wherein the tumor-associated antigen comprises SEQ ID NO: 3. 20. Cell culture containing: a. glycosylation competent cells containing exogenous nucleic acid encoding an antibody; And b. a culture medium containing fucose at a concentration of 0.1 to 1.0 g/l or 0.17 to 1.0 g/l, and glucose at a concentration of less than 10 g/l. 21. Cell culture according to claim 20, where the glucose concentration in the culture medium is from 0.5 to 4 g/l. 22. Cell culture according to claim 20 or 21, wherein the pH value of the culture medium is from 6.85 to 7.05, optionally from 6.90 to 7.00. 23. Cell culture according to any one of claims 20-22, where the antibody is an IgG antibody, optionally an IgG1 antibody. 24. The cell culture of claim 23, wherein the IgG1 antibody is specific for a tumor-associated antigen, optionally comprising SEQ ID NO: 3. - 35 045782 25. A method of modulating the level of TAF glycans in an antibody composition produced using glycosylation competent cells, comprising: (A) adding fucose to a cell culture medium containing glycosylation competent cells, achieving a fucose concentration of 0.1 to 1.0 g/l, to reduce the level of TAF glycans; and (B) adding glucose to a cell culture medium containing glycosylation competent cells to achieve a glucose concentration of 0.5 to 10 g/L to increase the level of TAF glycans. 26. A method of modulating the level of afucosylated glycans in an antibody composition produced by glycosylation-competent cells, comprising: (A) adding fucose to a cell culture medium containing glycosylation-competent cells to achieve a fucose concentration of 0 ,1 to 1.0 g/l, to reduce the level of afucosylated glycans; and (B) adding glucose to a cell culture medium containing glycosylation competent cells to achieve a glucose concentration of 0.5 to 10 g/L to increase the level of afucosylated glycans. 27. The method of claim 25 or 26, wherein the glycosylation competent cells are not genetically modified to provide a change in the activity of a de novo pathway enzyme or a salvage pathway, optionally wherein the glycosylation competent cells are not genetically modified to provide a knockout of the gene encoding GDP-keto-6deoxymannose-3,5-epimerase/-4-reductase. 28. The method according to any one of claims 25 to 27, wherein the level of TAF glycans in the antibody composition is 10% or less, optionally wherein the level of TAF glycans is 2 to 6%, 2 to 5%, or 2 up to 4%. 29. The method of any one of claims 25 to 28, wherein the level of high mannose glycans in the antibody composition is 3.5% or less, optionally 0.7 to 3.0%. 30. The method according to any one of claims 25 to 29, wherein the level of afucosylated glycans in the antibody composition is 3.5% or less, optionally wherein the level of afucosylated glycans in the antibody composition is from 0.8 to 2.8% . 31. The method according to any one of claims 25 to 30, where the concentration of fucose is from 0.17 to 1.0 g/l, optionally where the concentration of fucose is from 0.2 to 0.5 g/l. 32. The method according to any one of claims 25 to 31, further comprising adding glucose to the cell culture medium in accordance with a glucose feeding regime, which achieves an average glucose concentration of 10 g/l, optionally less, optionally where the average the glucose concentration is less than 6.0 g/L, optionally less than 4.0 g/L. 33. The method of claim 32, wherein the average glucose concentration is obtained from the concentration of fucose in the cell culture medium, optionally wherein the average glucose concentration is calculated using Formula I T=3.354-l.388F+0.lllG + [F - 0.4375] x [1.9527(F - 0.4375)] (formula I), , where T is the predetermined % of total afucosylated (TAF) glycans in the antibody composition and ranges from 3 to 5%, F is the concentration (g/L) of fucose in the medium, and G is the average concentration of glucose (g/L). -