Modulation of total nonfucosylated glycoforms of antibodies produced in cell culture

Abstract

Provided herein are methods for adjusting the level of total nonfucosylated (TAF) glycoforms of a recombinant glycosylated protein produced by glycosylation-competent cells in cell culture. In an exemplary embodiment, the method comprises maintaining the cell culture at a preset pH value for an initial cell culture period. Also provided herein are related compositions comprising the glycosylated protein and its TAF glycoforms.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 471,347, filed March 14, 2017, the contents of which are incorporated herein by reference. [Background technology]

[0002] Glycosylation is one of the most common and important post-translational modifications since it is involved in multiple cellular functions including, for example, protein folding, quality control, molecular trafficking and localization, and cell surface receptor interactions. Glycosylation affects the therapeutic efficacy of recombinant protein drugs by influencing the bioactivity, pharmacokinetics, immunogenicity, solubility, and in vivo clearance of therapeutic glycoproteins. The Fc glycoform profile is an important product quality attribute, especially for recombinant antibodies, since it directly affects the clinical efficacy and pharmacokinetics of the antibody.

[0003] High mannose (HM) glycoform content has been found to affect the pharmacokinetic properties of certain therapeutic antibodies (Non-Patent Document 1) (Non-Patent Document 2). Not only do HM glycoforms affect the serum clearance rate of antibodies, but such glycoforms, in addition to non-fucosylated (non-fuco) glycoforms, may also affect antibody effector functions or antibody-mediated target cell killing, also known as antibody-dependent cellular cytotoxicity (ADCC).

[0004] Many factors influence the glycan structure and therefore the final glycosylation form (glycoform) of a protein. For example, the cell line expressing the antibody, the cell culture medium, the feed medium composition and the timing of addition during cell culture can affect the production of glycoforms of a protein.

[0005] While many methods have been suggested by research groups to influence the levels of specific glycoforms of antibodies, there remains a need in the biopharmaceutical industry for simple and efficient methods to manipulate and regulate the levels of total nonfucosylated (TAF) glycoforms during recombinant production of therapeutic antibodies. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Goetze,et al.,(2011)Glycobiology 21,949-59 [Non-Patent Document 2] Yu, et al., (2012) MAbs 4, 475-87 Summary of the Invention [Means for solving the problem]

[0007] This is the first time that data have been described that reveals that maintaining a desired initial pH (e.g., initial setting pH value) during the initial cell culture period (e.g., the first 2, 3, 4, 5 or 6 days after inoculation) is important for adjusting the level of TAF glycoforms of recombinantly produced glycosylated proteins, while cell culture pH after the initial cell culture period does not significantly affect TAF glycoform levels. The discovery that initial pH, but not later stage pH, affects TAF levels was unexpected. Without being bound to a particular theory, adjusting the initial pH (e.g., initial setting pH value) during the initial cell culture period (e.g., the first 2, 3, 4, 5 or 6 days after inoculation) allows recombinant production of glycosylated proteins to have a desired, or predetermined, or preselected TAF glycoform level. Thus, the present invention relates to a method for producing recombinant glycosylated proteins (glycoproteins) with a desired, or predetermined, or preselected TAF glycoform level.

[0008] The present invention provides a method for modulating TAF glycoform levels of a recombinant glycosylated protein produced by a glycosylation-competent cell in a cell culture. In an exemplary embodiment, the method comprises maintaining a cell culture at an initial pH, e.g., an initial pH value, for an initial cell culture period.

[0009] In a representative embodiment, the initial cell culture period is about 4 days to about 6 days after inoculation, for example, about 4 days, about 5 days, or about 6 days, or about 2 days to about 6 days after inoculation, for example, about 2 days, about 3 days, or about 48 hours to about 144 hours after inoculation, or 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, 78 hours, 79 hours, 80 hours, 81 hours, 82 hours, 83 hours, 84 hours, 85 hours, 86 hours, 87 hours, 88 hours, 89 hours, 90 hours, 91 hours, 92 hours, 9 3 hours, 94 hours, 95 hours, 96 hours, 97 hours, 98 hours, 99 hours, 100 hours, 101 hours, 102 hours, 103 hours, 104 hours, 105 hours, 106 hours, 107 hours, 108 hours, 109 hours, 110 hours, 111 hours, 112 hours, 113 hours, 114 hours, 115 hours, 116 hours, 117 hours, 118 hours, 119 hours, 120 hours, 121 hours, 122 hours, 123 hours, 124 hours, 125 hours, 126 hours, 127 hours, 128 hours, 129 hours, 130 hours, 131 hours, 132 hours, 133 hours, 134 hours, 135 hours, 136 hours, 137 hours, 138 hours, 139 hours, 140 hours, 141 hours, 142 hours, 143 hours, 144 hours, 145 hours, 146 hours, 147 hours, 148 hours, 150 hours, 151 hours, 152 hours, 153 hours, 154 hours, 155 hours, 156 hours, 157 hours, 158 hours, 159 hours, 160 hours, 161 hours, 162 hours, 163 hours, 164 hours, 165 hours, 166 hours, 166 hours, or an increment thereof.

[0010] In a typical embodiment, the initial cell culture period depends on the particular viable cell density (VCD) of the cell culture. In a typical embodiment, the initial cell culture period depends on the VCD of the cell culture being about 6.5x10 6 In an exemplary embodiment, the method comprises the step of: (a) culturing a cell culture having a VCD of about 6.5×10 cells / mL or less; 6 and maintaining the cell culture at the preset pH value until the VCD of the cell culture reaches about 6.9x10 cells / mL. 6 ~about 8.2x10 6 In an exemplary embodiment, the method comprises maintaining the cell culture at the preset pH value until the VCD of the cell culture reaches about 8.2×10 6 ~Approx. 1.94x10 7 In an exemplary embodiment, the method comprises maintaining the cell culture at the preset pH value until the VCD of the cell culture reaches about 1.21×10 7 ~Approx. 3.46x10 7 The method includes maintaining the cell culture at the initial pH value until the initial pH value is reached.

[0011] In an exemplary embodiment, the initial setting pH value is selected from a pH that is greater than about 6.5 and less than about 7.5.

[0012] The present invention also relates to compositions comprising glycosylated proteins and TAF glycoforms thereof. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is an illustration of the three types of N-glycans and commonly used symbols for such sugars. [Diagram 2] FIG. 2 is a table of representative glycan structures. [Diagram 3] FIG. 3 is a set of graphs correlating TAF(%), HM(%) or AF(%) as a function of initial pH or time. [Figure 4] FIG. 4 is a graph of the % TAF present in the cell culture as a function of time for various pH set points. [Diagram 5] 5 is a set of graphs showing the %TAF present in a cell culture (over the indicated times) as a function of the average initial pH of the cell culture. (A) is a graph of %TAF; (B) is a graph of %TAF from days 0 to 6; (C) is a graph of %TAF from days 6 to 9, and (D) is a graph of %TAF from days 9 to 12. [Figure 6] FIG. 6 is a graph of % TAF as a function of pH for cell cultures from days 6 to 12. [Figure 7] FIG. 7 is a pair of graphs of pH or % TAF as a function of time. [Figure 8] FIG. 8 is a set of graphs correlating HM, non-fuco or TAF (%) versus pH or post-shift temperature. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Many secreted proteins are post-translationally glycosylated, a process by which sugar moieties (e.g., glycans, sugars) are covalently attached to specific amino acids of proteins. In eukaryotic cells, two types of glycosylation occur: (1) N-linked glycosylation, in which the glycan is linked to an asparagine at the recognition sequence Asn-X-Thr / Ser (where "X" is any amino acid except proline), and (2) O-linked glycosylation, in which the glycan is linked to a serine or threonine. Regardless of the type of glycosylation (N-linked or O-linked), there is a wide range of glycan structures attached to each site, so there is microheterogeneity in protein glycoforms (O or N).

[0015] All N-glycans share the same core sugar sequence: Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser / Thr(Man 3 GlcNAc 2Asn) and are classified into one of three types: (A) high mannose (HM) or oligomannose (OM) type consisting of two N-acetylglucosamine (GalNAc) moieties and multiple (e.g., 5, 6, 7, 8, or 9) mannose (Man) residues, (B) complex type containing more than two GlcNAc moieties and any number of other sugar types, or (C) hybrid type containing a Man residue at one of the branches and a GlcNAc at the base of the complex branch. Figure 1 (Stanley et al., Chapter 8: N-Glycans, Essentials of Glycobiology, 2 nd ed., Cold Spring Harbor Laboratory Press; 2009) shows three types of N-glycans.

[0016] N-linked glycans generally contain one or more of the following monosaccharides: galactose (Gal), N-acetylgalactosamine (GalNAc), galactosamine (GalN), glucose (GLc), N-acetylglucosamine (ClcNAc), glucosamine (GlcN), mannose (Man), N-acetylmannosamine (ManNAc), mannosamine (ManN), xylose (Xyl), N0-acetylneuraminic acid (Neu5Ac), N-glycosylneuraminic acid (Neu5Gc), 2-keto-3-doxynononic acid (Kdn), fucose (Fuc), glucuronic acid (GLcA), iduronic acid (IdoA), galacturonic acid (GalA), and manuronic acid (ManA). Commonly used symbols for such saccharides are shown in Figure 1. Representative glycans and their individual properties are shown in FIG.

[0017] N-linked glycosylation is initiated in the endoplasmic reticulum (ER) and a complex series of reactions results in the attachment of a core glycan structure, essentially made up of two GlcNAc and three Man residues. The glycan complex formed in the ER is modified by the action of enzymes in the Golgi apparatus. If the sugar is relatively inaccessible to the enzymes, it generally remains in its original HM form. If the sugar is accessible to the enzymes, many of the Man residues are cleaved off and the sugar is further modified, resulting in complex N-glycan structures. For example, mannosidase-1 located in the cis-Golgi can cleave or hydrolyze HM glycans, while fucosyltransferase FUT-8 located in the medial-Golgi fucosylates the glycans (Hanrue Imai-Nishiya (2007), BMC Biotechnology, 7:84).

[0018] Thus, the sugar composition and structural configuration of a glycan structure varies depending, inter alia, on the glycosylation machinery in the ER and Golgi apparatus, the accessibility of the glycan structure to the enzymes of that machinery, the order of action of each enzyme, and the stage at which the protein is released from the glycosylation machinery.

[0019] The invention provided herein relates to methods for modulating the levels of various glycosylation forms (glycoforms) of a protein during recombinant production by glycosylation-competent cells. Without being bound to a particular theory, it is believed that the methods of the present invention provide a means for tailoring compositions containing specific amounts of specific glycoforms of a given recombinant protein.

[0020] In representative embodiments, the level of total nonfucosylated (TAF) glycoforms is adjusted. As used herein, "total nonfucosylated glycoforms" or "TAF glycoforms" or "TAF" or "TAF final" refers to the combined amount of high mannose glycoforms and nonfucosylated glycoforms. As used herein, the term "high mannose" or "HM" or "HM final" encompasses glycoforms containing 5, 6, 7, 8, or 9 mannose residues, abbreviated as Man5, Man6, Man7, Man8, and Man9, respectively. As used herein, the term "nonfucosylated glycoforms" or "nonfuco glycoforms" or "nonfucosylated glycans" or "nonfuco" or "AF" or "nonfucosylated" refers to glycoforms that lack core fucose, e.g., α1,6-linked fucose on GlcNAc residues involved in an amide bond with the Asn of the N-glycosylation site. Nonfucosylated glycoforms include, but are not limited to, A1G0, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5. Additional nonfucosylated 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 an exemplary embodiment, the levels of TAF, the amount of HM glycoforms, and nonfucosylated glycoforms are determined via HILIC, as further described in Example 1 herein. After enzymatic cleavage of N-glycans, HILIC is performed to obtain a chromatogram with several peaks, each peak representing the average distribution (amount) of the various glycoforms. For these purposes, % Peak Area = Peak Area / Total Peak Area x 100% and % Total Peak Area = Total Area of ​​Sample / Total Area of ​​Standard x 100%. The calculations used for purposes of determining %TAF may be performed as follows: %nonfucosylated 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).

[0021] The present invention provides a method for adjusting the level of TAF glycoforms of a recombinant glycosylated protein. In an exemplary embodiment, the recombinant glycosylated protein is produced by glycosylation-competent cells in cell culture. In an exemplary embodiment, the method includes maintaining the cell culture at a preset pH value for an initial cell culture period. As used herein, the term "maintain" means setting a set pH value at a preset pH value and not changing the set pH value during a specified time. As used herein, the term "set pH value" refers to a desired or target pH value as set by a user on a pH control system or device. As used herein, the term "set pH value" refers to a set pH value set by a user during or immediately after inoculation of the cell culture. As one of skill in the art will recognize, the set pH value may differ from the actual pH of the cell culture depending on the calibration limits of a particular pH control system. Typically, the actual pH of the cell culture is ±0.05 of the set pH value, and in some embodiments, the actual pH of the cell culture will be ±0.03 or ±0.02 of the set pH value. In an exemplary embodiment, maintaining the cell culture at an initial pH value for the initial cell culture period means that the pH of the cell culture does not shift by more than 0.05 from the initial pH value during the initial cell culture period. In an exemplary embodiment, the method includes maintaining the cell culture at an initial pH value for the initial cell culture period, the initial pH value being greater than about 6.5 and less than about 7.5. For example, the initial set pH value is 6.50, 6.52, 6.54, 6.56, 6.58, 6.60, 6.62, 6.64, 6.66, 6.68, 6.70, 6.72, 6.74, 6.76, 6.78, 6.80, 6.82, 6.84, 6.86, 6.88, 6.90, 7.10, 7.12, 7.14, 7.16, 7.18, 7.20, 7.22, 7.24, 7.26, 7.28, 7.30, 7.32, 7.34, 7.36, 7.38, 7.40, 7.42, 7.44, 7.46, 7.48, 7.50 or more.For example, the default pH values ​​are 6.50, 6.55, 6.60, 6.65, 6.70, 6.75, 6.80, 6.85, 6.90, 6.95, 7.0, 7.05, 7.10, 7.15, 7.20, 7.25, 7.30, 7.35, 7.40, 7.45, 7.50, and even lower default values ​​of 7.5. For example, the default pH values ​​are 7.48, 7.46, 7.44, 7.42, 7.40, 7.38, 7.36, 7.34, 7.32, 7.30, 7.28, 7.26, 7.24, 7.22, 7.20, 7.18, 7.16, 7.14, 7.12, 7.10, 7.08, 7.06, 7.04, 7.02, 7. In an exemplary embodiment, the initial setting pH value is greater than about 6.55 and less than about 7.5. In an exemplary embodiment, the initial setting pH value is greater than about 6.60 and less than about 7.5. In an exemplary embodiment, the initial setting pH value is greater than about 6.65 and less than about 7.5. In an exemplary embodiment, the initial setting pH value is greater than about 6.7 and less than about 7.5. In an exemplary embodiment, the initial setting pH value is greater than about 6.7 and less than about 7.5. In an exemplary embodiment, the initial setting pH value is greater than about 6.75 and less than about 7.5. In representative embodiments, the initial setting pH value is greater than about 6.80 and less than about 7.5. In representative embodiments, the initial setting pH value is greater than about 6.5 and less than about 7.45. In representative embodiments, the initial setting pH value is greater than about 6.5 and less than about 7.4. In representative embodiments, the initial setting pH value is greater than about 6.5 and less than about 7.35. In representative embodiments, the initial setting pH value is greater than about 6.5 and less than about 7.3. In representative embodiments, the initial setting pH value is greater than about 6.5 and less than about 7.25. In representative embodiments, the initial setting pH value is greater than about 6.5 and less than about 7.2. In representative embodiments, the initial setting pH value is greater than about 6.5 and less than about 7.15. In representative embodiments, the initial setting pH value is greater than about 6.5 and less than about 7.1. In representative embodiments, the initial setting pH value is greater than about 6.85 and less than 7.2.In a representative embodiment, the initial pH value is about 7.0 to 7.1. In a representative embodiment, the initial pH value is about 6.85 or more and about 6.95 or less. In a representative embodiment, the initial pH value is about 6.95 to 7.15.

[0022] In a representative embodiment, the method includes maintaining the cell culture at a preset pH value for an initial cell culture period. As used herein, the phrase "initial cell culture period" refers to the time post-inoculation or thereafter when glycosylation-competent cells are added to a cell culture medium for the purpose of culturing the cells for recombinant protein production. In a representative embodiment, the initial cell culture period is about 4 days to about 6 days (e.g., about 4 days, 5 days, or about 6 days), or about 96 hours to about 144 hours, or increments therein. In a representative embodiment, the initial cell culture period is about 4 days to about 5 days. In a representative embodiment, the initial cell culture period is about 4 days. In a representative embodiment, the initial cell culture period is determined for a particular viable cell density (VCD) of the cell culture. In a representative embodiment, the initial cell culture period is determined for a cell culture having a VCD of about 6.5x10 6 In an exemplary embodiment, the method comprises the step of culturing a cell culture at a concentration of about 6.5×10 cells / mL or less after inoculation. 6 and maintaining the cell culture at the preset pH value until the cell culture achieves a VCD of about 8.2×10 cells / mL. 6 ~Approx. 1.94x10 7 In an exemplary embodiment, the method comprises maintaining the cell culture at the initial set pH value until the cell culture reaches a VCD of about 1.21×10 7 ~Approx. 3.46x10 7 The method includes maintaining the cell culture at the initial pH value until the VCD of

[0023] In a representative embodiment, the initial pH is higher than the control pH of the control cell culture. Without being bound by any particular theory, maintaining a higher pH (relative to the control pH) leads to an increase in TAF glycoforms compared to the control cell culture. Thus, in a representative embodiment, the method of the invention relates to increasing the level of TAF glycoforms of a protein produced by cells in a cell culture. In a representative embodiment, the level of HM glycoforms of a recombinant glycosylated protein is increased compared to the control cell culture. In a representative embodiment, the level of one or more of Man5, Man6, Man7, Man8 and / or Man9 of a recombinant glycosylated protein is increased compared to the control cell culture. In a representative embodiment, the level of non-fucosylated glycoforms of a recombinant glycosylated protein is increased compared to the control cell culture. In a representative embodiment, the level of one or more of A1G0, A2G0, A2G1a, A2G1b, A2G2 and A1G1M5 of a recombinant glycosylated protein is increased compared to the control cell culture. In representative embodiments, the levels of one or more of the recombinant glycosylated proteins A1G1a, G0[H3N4], G0[H4N4], G0[H5N4] and FO-N[H3N3] are increased compared to a control cell culture.

[0024] As used herein, the term "elevate" and words derived therefrom may not be a 100% or complete increase. Rather, there are various degrees of increase that one skilled in the art would recognize as having potential benefits. In this regard, the method of the present invention may increase TAF, HM or non-fucoglycoform levels to any degree or level compared to a control cell culture. In representative embodiments, the increase provided by the method of the present invention is at least or about 10% increase (e.g., at least or about 20% increase, at least or about 30% increase, at least or about 40% increase, at least or about 50% increase, at least or about 60% increase, at least or about 70% increase, at least or about 80% increase, at least or about 90% increase, at least or about 95% increase, at least or about 98% increase) compared to a control cell culture. In representative embodiments, the increase provided by the methods of the invention is greater than 100%, e.g., 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or even 1000% compared to a control cell culture. In representative embodiments, the level of a TAF, HM or non-fucoglycoform of a protein is increased at least about 1.5-fold compared to a control cell culture. In representative embodiments, the level of a TAF, HM or non-fucoglycoform of a protein is increased at least about 2-fold compared to a control cell culture. In representative embodiments, the level of a TAF, HM or non-fucoglycoform of a protein is increased at least about 3-fold compared to a control cell culture. In representative embodiments, the level of a TAF, HM or non-fucoglycoform of a protein is increased at least about 4-fold or 5-fold compared to a control cell culture.

[0025] In representative embodiments, elevated levels of the TAF glycoforms of the protein are observed or observable or detectable or detectable as early as the first day after inoculation. In representative embodiments, elevated levels of the TAF glycoforms of the protein are observed or observable or detectable or detectable as early as the second day after inoculation. In representative embodiments, elevated levels of the TAF glycoforms of the protein are observed or observable or detectable or detectable as early as the third day after inoculation. In representative embodiments, elevated levels of the TAF glycoforms of the protein are observed or observable or detectable or detectable as early as the fourth day after inoculation. In representative embodiments, elevated levels of the TAF glycoforms of the protein are observed or observable or detectable or detectable as early as the fifth day after inoculation. In representative embodiments, elevated levels of the TAF glycoforms of the protein are observed or observable or detectable or detectable when the protein is harvested from the cell culture.

[0026] In representative embodiments, elevated levels of the TAF glycoforms of the protein are observed for greater than the 4th, 5th, or 6th day of cell culture, or over the initial cell culture period. In representative embodiments, elevated levels of the TAF glycoforms of the protein are observed for 7, 8, 9, 10, 11, or 12 days (post-inoculation) of cell culture, or for longer (e.g., 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, 1 year). In representative embodiments, elevated levels of the TAF glycoforms of the protein are observed when the protein is harvested from the cell culture.

[0027] In a representative embodiment, the initial pH is lower than the control pH in the control cell culture. Without being bound to a particular theory, maintaining a lower pH (relative to the control pH) leads to a reduction in TAF glycoforms compared to the control cell culture. Thus, in a representative embodiment, the method of the invention relates to reducing the level of TAF glycoforms of a protein produced by cells in a cell culture. In a representative embodiment, the level of HM glycoforms of the recombinant glycosylated protein is reduced compared to the control cell culture. In a representative embodiment, the level of one or more of Man5, Man6, Man7, Man8, and / or Man9 of the recombinant glycosylated protein is reduced compared to the control cell culture. In a representative embodiment, the level of non-fucosylated glycoforms of the recombinant glycosylated protein is reduced compared to the control cell culture. In a representative embodiment, the level of one or more of A1G0, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5 of the recombinant glycosylated protein is reduced compared to the control cell culture. In representative embodiments, the levels of one or more of the recombinant glycosylated proteins A1G1a, G0[H3N4], G0[H4N4], G0[H5N4] and FO-N[H3N3] are reduced compared to a control cell culture.

[0028] As used herein, the term "reduced" and words derived therefrom may not be a 100% or complete reduction. Rather, there are various degrees of reduction that one skilled in the art would recognize as having potential benefits. In this regard, the method of the present invention may reduce TAF, HM or non-fucoglycoform levels to any degree or level compared to a control cell culture. In representative embodiments, the reduction provided by the method of the present invention is at least or about 10% reduction (e.g., at least or about 20% reduction, at least or about 30% reduction, at least or about 40% reduction, at least or about 50% reduction, at least or about 60% reduction, at least or about 70% reduction, at least or about 80% reduction, at least or about 90% reduction, at least or about 95% reduction, at least or about 98% reduction) compared to a control cell culture. In representative embodiments, the reduction provided by the methods of the invention is greater than 100%, e.g., 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or even 1000% compared to a control cell culture. In representative embodiments, the levels of TAF, HM or non-fucoglycoforms of the protein are reduced at least about 1.5-fold compared to a control cell culture. In representative embodiments, the levels of TAF, HM or non-fucoglycoforms of the protein are reduced at least about 2-fold compared to a control cell culture. In representative embodiments, the levels of TAF, HM or non-fucoglycoforms of the protein are reduced at least about 3-fold compared to a control cell culture. In representative embodiments, the levels of TAF, HM or non-fucoglycoforms of the protein are reduced at least about 4-fold or 5-fold compared to a control cell culture.

[0029] In representative embodiments, a reduction in the level of the TAF glycoform of the protein is observed or observable or detectable or is detectable as early as the first day after inoculation. In representative embodiments, a reduction in the level of the TAF glycoform of the protein is observed or observable or detectable or is detectable as early as the second day after inoculation. In representative embodiments, a reduction in the level of the TAF glycoform of the protein is observed or observable or detectable or is detectable as early as the third day after inoculation. In representative embodiments, a reduction in the level of the TAF glycoform of the protein is observed or observable or detectable or is detectable as early as the fourth day after inoculation. In representative embodiments, a reduction in the level of the TAF glycoform of the protein is observed or observable or detectable or is detectable after about the fifth day after inoculation. In representative embodiments, a reduction in the level of the TAF glycoform of the protein is observed or observable or detectable or is detectable at the time the protein is harvested from the cell culture.

[0030] In representative embodiments, reduced levels of TAF glycoforms of the protein are observed for greater than the 4th, 5th, or 6th day of cell culture, or over the initial cell culture period. In representative embodiments, reduced levels of TAF glycoforms of the protein are observed for greater than 7, 8, 9, 10, 11, or 12 days of cell culture (post-inoculation), or for greater than (e.g., 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, 1 year). In representative embodiments, reduced levels of TAF glycoforms of the protein are observed when the protein is harvested from the cell culture.

[0031] With respect to the methods of the invention, the adjustment, increase or decrease affected by such methods is relative to a "control" or "control cell culture." These terms are used interchangeably herein. In an exemplary embodiment, the control is the level of a TAF glycoform of a protein when a step of a method of the invention is not performed. In an exemplary embodiment, the control is the level of a TAF glycoform of a protein when a known method of recombinant production is performed. In an exemplary embodiment, the control is the level of a TAF glycoform when a known operational pH is maintained during recombinant production. As used herein, the term "control cell culture" refers to a cell culture that is maintained identically to the cell culture in which a step of a method of the invention is performed, except for pH during the initial cell culture period (e.g., a cell culture of a method of the invention). In an exemplary embodiment, a control cell culture is a cell culture that is maintained at known operational or standard parameters, including a control pH. As used herein, the term "control pH" can refer to a known operational pH, e.g., the pH of a cell culture that is maintained at a first time point or at a time point prior to performing a method of the invention. In an exemplary embodiment, the control pH is the pH of a cell culture in which the TAF level is known or determined.

[0032] A variety of methods are known in the art for assessing the glycoforms present in a glycoprotein containing composition or for determining the glycoform profile of a particular sample containing a glycoprotein. Suitable methods include positive ion MALDI-TOF analysis, negative ion MALDI-TOF analysis, weak anion exchange (WAX) chromatography, normal phase chromatography (NP-HPLC), exoglycosidase digestion, Bio-Gel P-4 chromatography, anion exchange chromatography, and one-dimensional nmr spectroscopy and combinations thereof. See, e.g., 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, pp. 124-133; Ruhaak LR, Anal Bioanal Chem, 2010, Vol. 397:3457-3481, and Geoffrey, RGet. al. Analytical Biochemistry 1996, Vol. 240, pp. 210-226. The examples provided herein also describe suitable methods for assessing the glycoforms present in a glycoprotein-containing composition.

[0033] Temperature and other cell culture parameters In an exemplary embodiment, the method further comprises maintaining the cell culture at an initial temperature during the initial cell culture period, and optionally during a second cell culture period following the initial cell culture period, the initial temperature being between 30° C. and 40° C. In an exemplary embodiment, the initial temperature is between about 32° C. and about 38° C. or between about 35° C. and about 38° C. In an exemplary embodiment, maintaining the cell culture at the initial temperature refers to maintaining the cell culture within ±1° C. of the initial temperature during the initial cell culture period. In an exemplary embodiment, maintaining the cell culture at the initial temperature throughout the initial cell culture period means that the temperature of the cell culture does not shift by more than 1° C. from the initial temperature during the initial cell culture period.

[0034] In the context of the present invention, the cell culture can be maintained according to any set of conditions suitable for recombinant protein production. For example, the cell culture can be maintained at a particular cell density, culture volume, dissolved oxygen level, pressure, osmolarity, etc. In an exemplary embodiment, the cell culture can be maintained at a particular cell density, culture volume, dissolved oxygen level, pressure, osmolarity, etc. 2 Incubated in a standard humidified incubator with 5% CO 2 Shake the pre-inoculated cell culture at 400 rpm (e.g., 70 rpm). In a representative embodiment, 10 6 The cell culture is inoculated at a seeding density of 100 cells / mL. In a representative embodiment, the method includes maintaining an osmolarity of about 200 mOsm / kg to about 500 mOsm / kg. In a representative embodiment, the method includes maintaining an osmolarity of about 225 mOsm / kg to about 400 mOsm / kg or about 225 mOsm / kg to about 375 mOsm / kg. In a representative embodiment, the method includes maintaining an osmolarity of about 225 mOsm / kg to about 350 mOsm / kg. In a representative embodiment, the method includes maintaining a dissolved oxygen (DO) level of the cell culture at about 20% to about 60% oxygen saturation during the initial cell culture period. In a representative example, the method includes maintaining a DO level of the cell culture at about 30% to about 50% (e.g., about 35% to about 45%) oxygen saturation during the initial cell culture period. In representative examples, the methods include maintaining the DO level of the cell culture at about 20%, about 30%, about 40%, about 50%, or about 60% oxygen saturation during the initial cell culture period.

[0035] The cell culture may be maintained in any one or more culture media. In representative embodiments, the cell culture may be maintained in a medium suitable for cell growth and / or may be provided in one or more feeding media according to any suitable feeding schedule. In representative embodiments, the method includes maintaining the cell culture in a medium comprising glucose, lactate, ammonia, glutamine and / or glutamate. In representative embodiments, the method includes maintaining the cell culture in a medium comprising manganese at a concentration of less than about 1 μM during the initial cell culture period. In representative embodiments, the method includes maintaining the cell culture in a medium comprising about 0.25 μM to about 1 μM manganese. In representative embodiments, the method includes maintaining the cell culture in a medium comprising negligible amounts of manganese. In representative embodiments, the method includes maintaining the cell culture in a medium comprising copper at a concentration of about 50 ppb or less during the initial cell culture period. In representative embodiments, the method includes maintaining the cell culture in a medium comprising copper at a concentration of about 40 ppb or less during the initial cell culture period. In representative embodiments, the method comprises maintaining the cell culture in a medium containing copper at a concentration of about 30 ppb or less during the initial cell culture period. In representative embodiments, the method comprises maintaining the cell culture in a medium containing copper at a concentration of about 20 ppb or less during the initial cell culture period. In representative embodiments, the medium contains copper at a concentration of about 5 ppb or more or about 10 ppb or more.

[0036] In representative embodiments, the type of cell culture is fed-batch or continuous perfusion, however, the methods of the present invention are advantageously not limited to any particular type of cell culture.

[0037] After the initial cell culture period In an exemplary embodiment, the method of the present invention for modulating TAF glycoform levels of a recombinant glycosylated protein comprises maintaining the cell culture at an initial pH for an initial cell culture period. In an exemplary embodiment, the method further comprises ceasing to maintain the cell culture at the initial pH value after the initial cell culture period. For purposes herein, the concept of "ceasing to maintain the cell culture at the initial pH value" refers to ceasing to take actions required to regulate the cell culture at the initial pH value. For example, one or more settings on a pH control system may be altered to effectively cease maintaining the initial pH value. In an exemplary embodiment, "ceasing to maintain the cell culture at the initial pH value" may refer to tolerating a pH shift or change. For example, "ceasing to maintain the cell culture at the initial pH value" may refer to an active step of shifting the pH, for example, by altering the pH set point in a pH regulation system, or may refer to a non-active step of allowing the pH to shift or ceasing to regulate or maintain a particular pH or pH range. In an exemplary embodiment, maintaining or regulating the cell culture pH is ceased after the first 4-6 days of cell culture. In a representative embodiment, the method of adjusting the TAF glycoform level of a recombinant glycosylated protein produced by a glycosylation-competent cell comprises maintaining the cell culture at an initial pH for an initial cell culture period, ceasing to maintain the cell culture at the initial pH after the initial cell culture period, and allowing the pH of the cell culture to shift, e.g., beyond 0.05. For purposes herein, the concept of "allowing the pH shift" may refer to an active step of shifting the pH, e.g., by changing the set pH value in a pH regulation system, or may refer to an inactive step of allowing the pH shift or ceasing to regulate or maintain a particular pH or pH range. In a representative embodiment, the pH of the cell culture is allowed to shift by about 0.05 to about 2.0 after the initial cell culture period. For example, the pH is allowed to shift by about 0.1 to about 1.5 or about 0.5 to about 1.0 after the initial cell culture period. In certain representative embodiments, the pH is not allowed to fall outside of a pH range that is suitable for antibody production by the cells of the cell culture.For example, the pH cannot be allowed to exceed 9 or fall below 4.

[0038] In a representative embodiment, the method includes a pH shift after an initial cell culture period. In a representative embodiment, the method includes changing the set pH value in a pH control system from an initial pH value to a different set pH value. In a representative embodiment, the method includes shifting the pH (e.g., set pH value) by more than about 0.05 (relative to the initial pH value) after the initial cell culture period (optionally for a second cell culture period). In a representative embodiment, the method includes shifting the pH (e.g., set pH value) by about 0.05 to about 2.0 (relative to the initial pH value) after the initial cell culture period. In a representative embodiment, the pH shift is an increase in pH (e.g., relative to the initial pH value). In certain embodiments, the method includes increasing the pH (e.g., set pH value) by about 0.1 to about 1.5 or about 0.15 to about 1.0. In a representative embodiment, the shift is a decrease in pH (e.g., relative to the initial pH value). In certain embodiments, the methods include lowering the pH (eg, the set pH value) by about 0.1 to about 1.5, or by about 0.15 to about 1.0.

[0039] Methods for adjusting or maintaining the pH of a cell culture, as well as doing so using fully instrumented, high throughput bioreactors that include pH monitoring systems, are known in the art. For example, see Seung Joon Lee's paper "Dissolved Oxygen and pH Monitoring within Cell Culture Media using a Hydrogel Microarray Sensor", Texas A&M University, December 2006; Adami et al., "Development of a pH Sensor with Integrated Reference Electrode for Cell Culture Monitoring", Sensors, Vol 162 Lecture Notes in Electrical Engineering, Chapter 86, pp. 481-485 (2013); U.S. Patent No. 7,429,491; Ge et al., J Biotechnology 122:293-306 (2006); Weuster-Botz et al., Bioprocess. Biosyst. Eng. 28(2):109-119 (2005); Maharbiz et al., Biotechnol. Bioeng. 85(4):376-381 (2004); Zanzotto et al. See, e.g., Hermann et al., Biotechnol. Bioeng. 87(2):243-254 (2005); Hermann et al., Biotechnol. Bioeng. 81:178-186 (2002); EP 3128319; U.S. Patent Publication No. 2015 / 0376647. pH monitors are commercially available, including, for example, Easyferm Plus ARC 225 (Hamilton, Reno, NV). Methods for maintaining cell culture pH are also described in the Examples herein.

[0040] In a representative embodiment, the method of the invention for modulating TAF glycoform levels of a recombinant glycosylated protein comprises maintaining the cell culture at an initial pH value for an initial cell culture period, and further comprises maintaining the cell culture at an initial temperature during the initial cell culture period and optionally during a second cell culture period after the initial cell culture period, the initial temperature being between 30° C. and 40° C. In a representative embodiment, the method further comprises maintaining the initial cell temperature during the initial cell culture period and for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more after the initial cell culture period. In a representative embodiment, the method further comprises ceasing to maintain the initial temperature after the initial cell culture period. In a representative embodiment, the method comprises ceasing to maintain the cell culture at the initial temperature and allowing a temperature shift of about 2° C. or more after the initial cell culture period. In a representative embodiment, a temperature shift of more than about 2° C. is allowed after the initial cell culture period. In a representative embodiment, the method comprises a temperature shift of more than about 2° C. after the initial cell culture period. In a representative embodiment, the method comprises a temperature shift of more than about 2° C. after the initial cell culture period. In any of the embodiments herein, the second cell culture period can be an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more after the initial cell culture period.

[0041] In representative embodiments, it is acceptable to shift the cell culture temperature after the initial cell culture period. In representative embodiments, after the initial cell culture period, the temperature is no longer maintained at ±1°C of the selected initial temperature. In representative embodiments, it is acceptable to shift the temperature by about 1°C to about 6°C after the initial cell culture period. For example, it is acceptable to shift the temperature by about 1°C to about 5°C, or about 1°C to about 4°C, or about 2°C, or about 3°C ​​after the initial period of cell culture. In certain representative embodiments, the temperature is not allowed to fall outside of the temperature range suitable for antibody production by the cells of the cell culture. For example, the temperature is not allowed to be higher than 40°C or lower than 30°C.

[0042] In a representative embodiment, the method includes a temperature shift after an initial cell culture period. In a representative embodiment, the method includes shifting the temperature by about 1° C. to about 6° C. after the first 3 to 5 days of cell culture. In a representative embodiment, the shift is an increase in temperature. In a representative embodiment, the shift includes increasing or decreasing the temperature by 5° C. or about 1° C. to about 4° C. or about 2° C. or about 3° C. In a representative embodiment, the method includes maintaining the cell culture at an initial pH, e.g., an initial set pH value, for an initial cell culture period that is at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, about 3 days to about 6 days, about 4 days to about 6 days, or about 4 days to about 5 days. In representative embodiments, the method includes maintaining the cell culture at an initial pH, e.g., an initial set pH value, for an initial cell culture period that is at least about 72 hours, about 76 hours, about 80 hours, about 84 hours, about 88 hours, about 92 hours, about 96 hours, about 100 hours, about 104 hours, about 108 hours, about 112 hours, about 116 hours, about 120 hours, about 124 hours, about 128, about 132 hours, about 136 hours, about 140 hours, or about 144 hours.

[0043] In alternative embodiments, the method does not include a temperature shift after the initial cell culture period. In representative examples, the temperature of the cell culture is maintained at a temperature that is within ±1° C. of the initial temperature throughout the entire cell culture period. In representative examples, the temperature of the cell culture is maintained at a temperature that is within ±1° C. of the initial temperature throughout the entire cell culture period, and the method includes maintaining the cell culture at an initial pH, e.g., an initial set pH value, for an initial cell culture period that is at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, or at least about 6 days. In representative embodiments, the temperature of the cell culture is maintained at a temperature within ±1° C. of the initial temperature for the entire cell culture period, and the method includes maintaining the cell culture at an initial pH, e.g., an initial set pH value, for an initial cell culture period that is about 24 hours, about 28 hours, about 32 hours, about 36 hours, about 40 hours, about 44 hours, about 48 hours, about 52 hours, about 56 hours, about 60 hours, about 64 hours, about 68 hours, about 72 hours, about 76 hours, about 80 hours, about 84 hours, about 88 hours, about 92 hours, about 96 hours, about 100 hours, about 104 hours, about 108 hours, about 112 hours, about 116 hours, about 120 hours, about 124 hours, about 128, about 132 hours, about 136 hours, about 140 hours, or about 144 hours.

[0044] Recombinant Proteins In an exemplary embodiment, the recombinant protein has the formula: Asn-Xaa 1 -Xaa 2 (In the formula, Xaa 1 is any amino acid except Pro, and Xaa 2 is Ser or Thr).

[0045] In a representative embodiment, the recombinant protein comprises a fragment crystallizable (Fc) polypeptide. The term "Fc polypeptide" as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Also included are truncated forms of such polypeptides that contain the hinge region that promotes dimerization. Fusion proteins (and oligomers formed therefrom) that include an Fc portion offer the advantage of easy purification by affinity chromatography on Protein A or Protein G columns. In a representative embodiment, the recombinant protein comprises an Fc of an IgG, such as a human IgG. In a representative embodiment, the recombinant protein comprises an Fc of an IgG1 or IgG2. In a representative embodiment, the recombinant protein is an antibody, a peptibody, or an Fc-fusion protein.

[0046] In a representative embodiment, the recombinant glycosylated protein is an antibody. As used herein, the term "antibody" refers to a protein having a conventional immunoglobulin format, including heavy and light chains, and including variable and constant regions. For example, an antibody can be an IgG, which is a "Y-shaped" structure of two identical pairs of polypeptide chains, each pair having one "light" (generally with a molecular weight of about 25 kDa) and one "heavy" chain (generally with a molecular weight of about 50-70 kDa). An antibody has a variable region and a constant region. In the IgG format, the variable region is generally about 100-110 or more amino acids, includes three complementarity determining regions (CDRs), and is primarily responsible for antigen recognition and varies substantially among other antibodies that bind to different antigens. The constant region allows the antibody to recruit cells and molecules of the immune system. The variable region is composed of the N-terminal regions of each light and heavy chain, while the constant region is composed of the C-terminal portions of each of the heavy and light chains. (Janeway et al., “Structure of the Antibody Molecule and the Immunoglobulin Genes”, Immunobiology: The Immune System in Health and Disease, 4 thed.Elsevier Science Ltd. / Garland Publishing, (1999)).

[0047] The general structure and properties of antibody CDRs have been described in the art. Briefly, in an antibody scaffold, the CDRs are embedded within frameworks in the heavy and light chain variable regions and constitute the regions largely responsible for antigen binding and recognition. The variable region comprises at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; see also Chothia et al., 1989, Nature 342:877-883) within framework regions (referred to as framework regions 1-4, FR1, FR2, FR3 and FR4 by Kabat et al., 1991; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917).

[0048] Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, defining the antibody's isotype 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 invention include all such classes or isotypes of antibodies. The light chain constant region can be, for example, a kappa or lambda type light chain constant region, such as a human kappa or lambda type light chain constant region. The heavy chain constant region can be, for example, an alpha, delta, epsilon, gamma, or mu type heavy chain constant region, such as a human alpha, delta, epsilon, gamma, or mu type heavy chain constant region. Thus, in representative embodiments, the antibody is of the isotype IgA, IgD, IgE, IgG or IgM, including any one of IgG1, IgG2, IgG3 or IgG4.

[0049] The antibody may be a monoclonal or polyclonal antibody. In some embodiments, the antibody comprises a sequence that is substantially similar to a natural antibody produced by a mammal, such as a mouse, rabbit, goat, horse, chicken, hamster, human, etc. In this respect, the antibody may be considered a mammalian antibody, such as a mouse antibody, a rabbit antibody, a goat antibody, a horse antibody, a chicken antibody, a hamster antibody, a human antibody, etc. In certain embodiments, the recombinant protein is a human antibody. In certain embodiments, the recombinant protein is a chimeric or humanized antibody. The term "chimeric antibody" is used herein to refer to an antibody that contains a constant domain from one species and a variable domain from a second species, or more commonly contains a stretch of amino acid sequence from at least two species. The term "humanized", when used in reference to an antibody, refers to an antibody with at least the CDR regions from a non-human source that have been engineered to have a structure and immunological function that is more similar to a true human antibody than the original source antibody. For example, humanization may involve grafting CDRs from a non-human antibody, such as a mouse antibody, onto a human antibody. Humanization can also include the selection of amino acid substitutions to make a non-human sequence appear more human.

[0050] For example, antibodies can be cleaved into fragments by enzymes such as papain and pepsin. Papain cleaves an antibody to generate two Fab fragments and one Fc fragment. Pepsin cleaves an antibody to generate an F(ab') fragment. 2 In an exemplary embodiment, the recombinant glycosylated protein is an antibody fragment, e.g., Fab, Fc, F(ab') fragment, or pFc' fragment, that retains at least one glycosylation site. 2 or pFc'.

[0051] The structure of antibodies has been exploited to generate an expanding range of alternative antibody formats spanning the molecular weight range of at least 12-150 kDa and the range of possible valencies (n) from monomers (n=1), dimers (n=2) and trimers (n=3) to tetramers (n=4) and higher; such alternative antibody formats are referred to herein as "antibody protein products."

[0052] Antibody protein products include those based on antibody fragments, such as scFv, Fab and VHH / VH, that retain the complete antigen-binding ability. The smallest antigen-binding fragment that retains its complete antigen-binding site is the Fv fragment, which consists entirely of the variable (V) region. Either soluble flexible amino acid peptide linkers are used to link the V region to the scFv (single-chain fragment variable) fragment for molecular stability, or a constant (C) domain is added to the V region to generate a Fab fragment. Both scFv and Fab are widely used fragments that can be easily made in prokaryotic hosts. Other antibody protein products include disulfide bond stabilized scFv (ds-scFv), single chain Fab (scFab) and dimeric and multimeric antibody formats such as dia-, tria- and tetrabodies or minibodies (miniAbs) that contain different formats consisting of scFv linked to oligomerization domains. The smallest fragments are the VHH / VH of camelid heavy chain Abs and single domain Abs (sdAbs). The most frequently used basic unit for generating novel antibody formats is the single variable (V) domain antibody fragment (scFv), which contains V domains (VH and VL domains) from heavy and light chains linked by a peptide linker of ~15 amino acid residues. Peptibodies or peptide-Fc fusions are another antibody protein product. The structure of a peptibody consists of a biologically active peptide grafted onto an Fc domain. Peptibodies have been described in detail in the art. See, for example, Shimamoto et al., mAbs 4(5):586-591 (2012).

[0053] Other antibody protein products include single chain antibodies (SCAs); diabodies; triabodies; tetrabodies; bispecific or trispecific antibodies, etc. Bispecific antibodies can be divided into five major classes: BsIgG, adduct IgG, BsAb fragments, bispecific fusion proteins, and BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology 67(2)Part A:97-106(2015).

[0054] In representative embodiments, the recombinant protein comprises any one of these antibody protein products. In representative embodiments, the recombinant glycosylated protein is any one of scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g. diabody, triabody, tetrabody), miniAb, camelid heavy chain antibody peptibody VHH / VH, sdAb, diabody; triabody; tetrabody; bispecific or trispecific antibody, BsIgG, adduct IgG, BsAb fragment, bispecific fusion protein, and BsAb conjugate.

[0055] The recombinant protein may be an antibody protein product in monomeric or multimeric, oligomeric or multimeric form. In certain embodiments, where an antibody comprises two or more distinct antigen-binding region fragments, the antibody is considered to be bispecific, trispecific or multispecific or bivalent, trivalent or multivalent, depending on the number of distinct epitopes recognized and bound by the antibody.

[0056] For the methods of the invention, the antibody protein product may lack certain portions of the antibody, but generally the fragment will contain at least a portion of the Fc region of the antibody that is post-translationally glycosylated in eukaryotic cells.

[0057] Advantageously, the method is not limited by the antigen specificity of the antibody. Thus, the antibody has any binding specificity for virtually any antigen. In representative embodiments, the antibody binds to a hormone, growth factor, cytokine, cell surface receptor or any of its ligands. In representative embodiments, the antibody binds to a protein expressed on the cell surface of an immune cell. In representative embodiments, the antibody binds to CD1a, CD1b, CD1c, CD1d, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11A, CD11B, CD11C, CDw12, CD13, CD14, CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD2 8, 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, CD5 0, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65 , CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD79α, CD7 9β, CD80, CD81, CD82, CD83, CDw84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CDw92, CD93, CD94, CD95, CD96, C D97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDw108, CD109, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CDw121b, CD122, CD123, CD124, CD125, CD126, C D127, CDw128, CD129, CD130, CDw131, CD132, CD134, CD135, CDw136, CDw137, CD138, CD139, CD140a, CD140b, It binds to a cluster of differentiation molecule selected from the group consisting of 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.

[0058] In exemplary embodiments, the antibody may be any of the antibodies described in U.S. Pat. No. 7,947,809 and U.S. Patent Publication No. 20090041784 (glucagon receptor), U.S. Pat. No. 7,939,070, U.S. Pat. No. 7,833,527, U.S. Pat. No. 7,767,206, and U.S. Pat. No. 7,786,284 (IL-17 receptor A), U.S. Pat. No. 7,872,106, and U.S. Pat. No. 7,592,429 (sclerostin), U.S. Pat. No. 7,871,611, U.S. Pat. No. 7,815,907, U.S. Pat. No. 7,037,499, U.S. Pat. No. 8, U.S. Pat. No. 7,700,742 and U.S. Patent Application Publication No. 20100255538 (IGF-1 receptor), U.S. Pat. No. 7,868,140 (B7RP1), U.S. Pat. No. 7,807,159 and U.S. Patent Application Publication No. 20110091455 (myostatin), U.S. Pat. No. 7,736,644, U.S. Pat. No. 7,628,986, U.S. Pat. No. 7,524,496 and U.S. Patent Application Publication No. 20100111979 (deletion mutants of epidermal growth factor receptor), U.S. Pat. 8110 (SARS coronavirus), U.S. Pat. No. 7718776 and U.S. Pat. App. Pub. No. 20100209435 (OPGL), U.S. Pat. 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No. 7,199,224 (TNF-α 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. 7579186 (TGF-β type II receptor), US Patent No. 7569387 (TNF receptor-like molecule), US Patent No. 7541438 (Connective tissue growth factor), US Patent No. 7521048 (TRAIL receptor-2), US Patent No. 6319499, US Patent No. 7081523 and U.S. Patent Application Publication No. 20080182976 (erythropoietin receptor), U.S. Patent Application Publication No. 20080166352 and U.S. Patent No. 7,435,796 (B7RP1), U.S. Patent No. 7,423,128 (properdin), U.S. Patent No. 7,422,742 and U.S. Patent No. 7,141,653 (interleukin-5), U.S. Patent No. 6,740,522 and U.S. Patent No. 7,411,050 (RANKL), U.S. Patent No. 7,378,091 (carbonic anhydrase IX (CA IX) Tumor antigens), U.S. Pat. Nos. 7,318,925 and 7,288,253 (parathyroid hormone), 7,285,269 (TNF), 6,692,740 and 7,270,817 (ACPL), 7,202,343 (monocyte chemoattractant protein-1), 7,144,731 (SCF), 6,355,779 and 7,138,500 (4-1BB), 7,135,174 (PDGFD), 6,630,143 and No. 7,045,128 (Flt-3 ligand), U.S. Pat. No. 6,849,450 (metalloproteinase inhibitors), U.S. Pat. No. 6,596,852 (LERK-5), U.S. Pat. No. 6,232,447 (LERK-6), U.S. Pat. No. 6,500,429 (brain-derived neurotrophic factor), U.S. Pat. No. 6,184,359 (epithelial-derived T-cell factor), U.S. Pat. No. 6,143,874 (neurotrophic factor NNT-1), U.S. Patent Application Publication No. 20110027287 (proprotein convertase subtilisin kexin type 9 (PCSK9)),and US Patent Publication No. 20110014201 (IL-18 receptor) and US Patent Publication No. 20090155164 (C-FMS). The above patents and published patent applications are incorporated herein by reference in their entirety for the purposes of their disclosure of variable domain polypeptides, nucleic acids encoding variable domains, host cells, vectors, methods of making polypeptides encoding said variable domains, pharmaceutical compositions and methods of treating diseases associated with the respective targets of the variable domain-containing antigen binding proteins or antibodies.

[0059] In representative embodiments, the antibody is muromonab-CD3 (product marketed under the trade name Orthoclone Okt3®), abciximab (product marketed under the trade name Reopro®), rituximab (product marketed under the trade name MabThera®, Rituxan®), basiliximab (product marketed under the trade name Simulect®), daclizumab (product marketed under the trade name Zenapax®), palivizumab (product marketed under the trade name Synagis®), infliximab (product marketed under the trade name Remicade®), trachomatis (product marketed under the trade name Telomerase®), telomerase ... rastuzumab (product marketed under the trade name Herceptin®), alemtuzumab (product marketed under the trade name MabCampath®, Campath-1H®), adalimumab (product marketed under the trade name Humira®), tositumomab-I131 (product marketed under the trade name Bexxar®), efalizumab (product marketed under the trade name Raptiva®), cetuximab (product marketed under the trade name Erbitux®), ibritumomab tiuxetan (products marketed under the trade name Zevalin®), omalizumab (products marketed under the trade name Xolair®), bevacizumab (products marketed under the trade name Avastin®), natalizumab (products marketed under the trade name Tysabri®), ranibizumab (products marketed under the trade name Lucentis®), panitumumab (products marketed under the trade name Vectibix®), eculizumab (products marketed under the trade name Soliris®), certolizumab pegol (products marketed under the trade name product marketed under the trade name Cimzia®), golimumab (product marketed under the trade name Simponi®), canakinumab (product marketed under the trade name Ilaris®), catumaxomab (product marketed under the trade name Removab®), ustekinumab (product marketed under the trade name Stelara®), tocilizumab (product marketed under the trade name RoActemra®, Actemra®), ofatumumab (product marketed under the trade name Arzerra®),The antibody is one of denosumab (product marketed under the trade name Prolia®), belimumab (product marketed under the trade name Benlysta®), raxibacumab, ipilimumab (product marketed under the trade name Yervoy®), and pertuzumab (product marketed under the trade name Perjeta®). In representative embodiments, the antibody is one of anti-TNF alpha antibodies, such as adalimumab, infliximab, etanercept, golimumab, and certolizumab pegol; anti-IL1.beta antibodies, such as canakinumab; anti-IL12 / 23 (p40) antibodies, such as ustekinumab and briakinumab; and anti-IL2R antibodies, such as daclizumab. Examples of suitable anti-cancer antibodies include, but are not limited to, anti-BAFF antibodies, such as belimumab; anti-CD20 antibodies, such as rituximab; anti-CD22 antibodies, such as epratuzumab; anti-CD25 antibodies, such as daclizumab; anti-CD30 antibodies, such as iratumumab, anti-CD33 antibodies, such as gemtuzumab, anti-CD52 antibodies, such as alemtuzumab; anti-CD152 antibodies, such as ipilimumab; anti-EGFR antibodies, such as cetuximab; anti-HER2 antibodies, such as trastuzumab and pertuzumab; anti-IL6 antibodies, such as siltuximab; and anti-VEGF antibodies, such as bevacizumab; anti-IL6 receptor antibodies, such as tocilizumab.

[0060] cell The present invention relates to a method for adjusting the levels of different glycosylation forms of a protein during production by a glycosylation-competent cell. In an exemplary embodiment, the glycosylation-competent cell is a eukaryotic cell, including but not limited to a yeast cell, a filamentous fungal cell, a protist cell, an algae cell, an insect cell, or a mammalian cell. Such host cells are described in the art. See, for example, Frenzel, et al., Front Immunol 4:217 (2013). In an exemplary embodiment, the eukaryotic cell is a mammalian cell. In an exemplary embodiment, the mammalian cell is a non-human mammalian cell. In some embodiments, the cells are selected from the group consisting of Chinese hamster ovary (CHO) cells and derivatives thereof (e.g., CHO-K1, CHO pro-3), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), cells engineered to lack dihydrofolate reductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or derivatives thereof (e.g., HEK293T, HEK293-EBNA), African green monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human bone osteosarcoma epithelial cells U2-OS, adenocarcinoma human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonal carcinoma cells P19, mouse embryonic fibroblast cells NIH 3T3, mouse fibroblast L929, mouse neuroblastoma N2a, human breast cancer MCF-7, retinoblastoma Y79, human retinoblastoma SO-Rb50, human hepatocellular carcinoma Hep G2, mouse B myeloma J558L or baby hamster kidney (BHK) cells (Gaillet et al., 2007; Khan, Adv Pharm Bull 3(2):257-263(2013)).

[0061] In representative embodiments, the glycosylation competent cell is a eukaryotic cell. In representative embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a non-human mammalian cell. In representative embodiments, the non-human mammalian cell is selected from the group consisting of CHO cells, CHO derivatives (e.g., CHO-K1, CHO pro-3), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), cells engineered to lack dihydrofolate reductase (DHFR) activity (e.g., DUKX-X11, DG44), African green monkey kidney cells (e.g., COS cells, VERO cells), mouse brain tumor cells CAD, mouse embryonic fibroblast cells NIH3T3, mouse fibroblast cells L929, mouse neuroblastoma cells N2a, human breast cancer cells MCF-7, retinoblastoma cells Y79, human retinoblastoma cells SO-Rb50, human liver cancer cells HepG2, mouse B myeloma cells J558L, or baby hamster kidney (BHK) cells. Cells that are not competent for glycosylation can also be transformed into glycosylation-competent cells, for example, by transfecting them with genes encoding the relevant enzymes required for glycosylation. Exemplary enzymes include, but are not limited to, oligosaccharyltransferase, glycosidase, glucosidase I, glucosidease II, calnexin / calreticulin, glycosyltransferase, mannosidase, GlcNAc transferase, galactosyltransferase, and sialyltransferase.

[0062] Methods for preparing compositions The invention also provides a method for preparing a composition comprising a TAF glycoform of a protein produced by cells in cell culture. In an exemplary embodiment, the method comprises (i) maintaining the cell culture at an initial pH for an initial cell culture period as described herein and optionally (ii) expanding the cell culture and (iii) harvesting a cell culture supernatant comprising the protein produced by the cells. In an exemplary aspect, the method can comprise any one of the steps described herein for the method of the invention for modulating (increasing or decreasing) the level of a TAF glycoform of a protein produced by cells in cell culture.

[0063] The method may include one or more steps for purifying the protein from the cell culture or its supernatant, and preferably recovering the purified protein. In a representative embodiment, the method includes one or more chromatography steps, such as affinity chromatography (e.g., Protein A affinity chromatography), ion exchange chromatography, hydrophobic interaction chromatography. In a representative embodiment, the method includes purifying the protein using a Protein A affinity chromatography resin.

[0064] In an exemplary embodiment, the method further includes a step of formulating the purified protein, thereby obtaining a formulation comprising the purified protein. Such steps are described in Formulation and Process Development Strategies for Manufacturing, eds. Jameel and Hershenson, John Wiley & Sons, Inc. (Hoboken, NJ), 2010.

[0065] The method may also include one or more upstream steps before the cell culture step.In a representative embodiment, the method includes a step for producing a host cell that expresses the protein.For example, the method includes, in some cases, introducing a vector that includes a nucleic acid that includes a nucleotide sequence that codes for the protein into the host cell.

[0066] composition Compositions comprising TAF glycoforms of proteins are provided herein. In representative embodiments, the compositions are prepared by the methods of the present invention for preparing compositions comprising TAF glycoforms of proteins produced by cells in cell culture, as described herein. In representative embodiments, at least about 10% of the proteins in the composition are TAF glycoforms. In representative embodiments, at least about 20% of the proteins in the composition are TAF glycoforms. In representative embodiments, at least about 30% of the proteins in the composition are TAF glycoforms. In representative embodiments, at least about 40% of the proteins in the composition are TAF glycoforms. In representative embodiments, at least about 50% of the proteins in the composition are TAF glycoforms. In representative embodiments, at least about 60% of the proteins in the composition are TAF glycoforms. In representative embodiments, at least about 70% of the proteins in the composition are TAF glycoforms. In representative embodiments, at least about 80% of the proteins in the composition are TAF glycoforms. In representative embodiments, at least about 90% of the proteins in the composition are TAF glycoforms, hi representative embodiments, greater than about 90% or greater than about 95% of the proteins in the composition are TAF glycoforms.

[0067] In representative embodiments, compositions of the invention have a glycoform profile that is about 10% or more TAF glycoforms. In representative embodiments, compositions of the invention have a glycoform profile that is about 20% or more TAF glycoforms. In representative embodiments, compositions of the invention have a glycoform profile that is about 30% or more TAF glycoforms. In representative embodiments, compositions of the invention have a glycoform profile that is about 40% or more TAF glycoforms. In representative embodiments, compositions of the invention have a glycoform profile that is about 50% or more TAF glycoforms. In representative embodiments, compositions of the invention have a glycoform profile that is about 60% or more TAF glycoforms. In representative embodiments, compositions of the invention have a glycoform profile that is about 70% or more TAF glycoforms. In representative embodiments, compositions of the invention have a glycoform profile that is about 80% or more TAF glycoforms. In representative embodiments, compositions of the invention have a glycoform profile that is about 90% or more TAF glycoforms. In representative embodiments, compositions of the invention have a glycoform profile that is about 95% or more TAF glycoforms.

[0068] The composition of the present invention is a pharmaceutical composition in a representative embodiment. In a representative embodiment, the pharmaceutical composition includes a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsions such as oil / water or water / oil emulsions, and various types of wetting agents. This term also includes any of the agents approved by the regulatory agency of the US Federal Government or listed in the United States Pharmacopeia for use in animals, including humans.

[0069] The pharmaceutical composition may contain, for example, an acidifier, an additive, an adsorbent, an aerosol propellant, an air displacement agent, The composition may comprise any pharma- ceutically acceptable ingredient, including an aqueous solution of a medicament for use in a pharmaceutical composition, such as a pharmaceutical composition for use in ... See, e.g., Handbook of Pharmaceutical Excipients, Third Edition, A.H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety. See, Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated by reference in its entirety.

[0070] In a representative embodiment, the pharmaceutical composition comprises a formulation material that is non-toxic to a recipient at the dosage and concentration used. In a specific embodiment, the pharmaceutical composition comprises a therapeutically effective amount of a TAF glycoform of a protein and one or more pharma- ceutically acceptable salts; polyols; surfactants; osmotic balancing agents; isotonicity agents; antioxidants; antibiotics; antifungal agents; bulking agents; lyoprotectants; antifoaming agents; chelating agents; preservatives; coloring agents; analgesics; or additional pharmaceutical agents. In a representative embodiment, the pharmaceutical composition comprises one or more polyols and / or one or more surfactants, optionally in addition to one or more excipients, including, but not limited to, pharma- ceutically acceptable salts; osmotic balancing agents (isotonicity agents); antioxidants; antibiotics; antifungal agents; bulking agents; lyoprotectants; antifoaming agents; chelating agents; preservatives; coloring agents; and analgesics.

[0071] In certain embodiments, the pharmaceutical compositions may contain formulation materials to alter, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeability of the composition. In such embodiments, suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); injectables; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as bicnzalkonium chloride). chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide; solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbatc, triton, tromethamine, lecithin, cholesterol, tyloxapal); stabilization enhancers (such as sucrose or sorbitol); isotonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.See REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company.

[0072] The pharmaceutical composition may be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the pharmaceutical composition may be, for example, about 4, or about 5 to about 8.0, or about 4.5 to about 7.5, or about 5.0 to about 7.5. In a representative embodiment, the pH of the pharmaceutical composition is 5.5 to 7.5.

[0073] The following examples are provided merely to illustrate the present invention and are not intended to limit its scope. EXAMPLES

[0074] Example 1 This example demonstrates that the initial cell culture pH regulates the levels of specific glycoforms of a recombinant glycosylated protein produced by cells in cell culture, and that any changes in pH made after the initial cell culture period have little effect on glycoform levels.

[0075] Materials and Methods Cell lines, cell cultures and media Cells of a CHO cell line expressing a recombinant antibody of isotype IgG1 were maintained in 3 L Erlenmeyer shake flasks (Corning Life Sciences, Lowell, MA) in a working volume of 1 L. At 36°C and 5% CO 2 Culture the cells under standard humidified conditions with automated CO 2The cells were shaken at 70 rpm in an incubator (Thermo Fisher Scientific, Waltham, MA). Every 3 days, all cells were subcultured, transferred, inoculated in culture medium containing various concentrations of methotrexate (MTX), and cultured in culture medium for 4 days before inoculating the bioreactor. Cell culture production medium was used as the control medium in this study. Components of cell culture medium include growth factors, amino acids, buffers, nutrients, trace elements, vitamins, surfactants, salts, nucleotides, hormones, lipids, and other organic compounds.

[0076] Bioreactor perfusion process Cultivation is carried out at standard temperature and dissolved oxygen conditions by batch or perfusion processes.

[0077] Cell proliferation, metabolite and antibody titer analysis Viable cell density and viability were determined using a Nova CDV (Nova Biomedical, Waltham, MA). For bioreactor samples, metabolic products including glucose, lactate, ammonia, glutamine, and glutamate were obtained from a Nova Flex (Nova Biomedical, Waltham, MA).

[0078] Hydrophilic interaction liquid chromatography (HILIC) glycan map HILIC was used to determine the glycan map of enzymatically released N-linked glycans. Briefly, glycans were incubated with a solution containing PNGase F and sodium phosphate buffer (pH 7.5) for 2 hours at 37°C. A 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 minutes. After incubation, the mixture was centrifuged to pellet precipitated proteins. The supernatant was collected and placed in a vial.

[0079] Glycans were separated by HILIC based on fluorescence detection: glycans were injected and bound to the column under highly organic conditions (mobile phase A and mobile phase B were ammonium formate and acetonitrile, respectively), and then eluted with an increasing gradient of aqueous ammonium formate buffer. High resolution was achieved using a 1.7 μm small particle column format and a 150 mm column length. Total run time, including column re-equilibration, was 155 min.

[0080] Experimental design Populations of CHO cells producing IgG1 antibodies were cultured at one of three initial pH values: 6.85, 6.95 or 7.1. The pH was maintained for the first 6 days after inoculation (inoculation occurred on day 0). For at least two populations of cells, the pH was shifted around day 6. The pH of one population of cells was shifted from 6.85 to 6.95 and for at least one other population, the pH was shifted from 7.1 to 6.95. For at least three populations of cells, the pH was not shifted and the pH was maintained at 6.85, 6.95 or 7.10.

[0081] The cells were cultured for a total of 12 days. Viable cell density (VCD) was measured throughout the 12-day culture period and the results are shown in Table 1.

[0082] [Table 1]

[0083] TAF levels, high mannose (HM) glycan levels and nonfucosylated (AF) glycan levels were measured after several days of cell culture, and the observed and predicted model results are shown in Table 2.

[0084] [Table 2]

[0085] Unexpectedly, initial pH was positively associated with TAF levels (Figure 3). As pH increased, TAF levels also increased. As shown in Figure 4, TAF levels in cell cultures maintained at a pH of 7.1 or maintained at 7.1 and then shifted to 6.95 around day 6 were higher than the TAF levels in the other groups maintained at lower pH, 6.95 or 6.85. Approximately a two-fold increase was observed. Also shown in Figure 4, TAF levels in cell cultures maintained at a pH of 6.85 or maintained at a pH of 6.85 and then shifted to 6.95 were lower than the TAF levels in the other groups maintained at higher pH, 6.95 and 7.1.

[0086] A statistical analysis of these results was performed. Figure 5 shows various correlations of initial pH values ​​to TAF during the cell culture period, days 0-6, days 6-9, days 9-12, and day 12. The correlation of initial pH to TAF during each period was strong. As shown in Figure 6, the correlation of final pH (pH after the initial cell culture period) to TAF (TAF level after the initial cell culture period) was weak.

[0087] The effect of final pH (pH after the initial cell culture period) was evaluated. Two groups of cells were cultured for 12 days. For both groups, cells were cultured at a pH of 6.95 for the first 5 days of cell culture. Group 1 was maintained at this pH for the following days, while the pH of group 2 was shifted to 6.85 over days 5-9, and then to 7.1 over days 9-12. As shown in FIG. 7, both groups showed very similar TAF levels. These results suggest that adjusting the pH after day 5 does not affect TAF levels.

[0088] This example shows the effect of pH during the initial cell culture period on the levels of TAF.

[0089] Example 2 This example provides another example of how cell culture pH during the initial cell culture period affects the levels of TAF.

[0090] Cell culture in a bioreactor with a perfusion process was performed essentially as described in Example 1, except that the cells expressed a recombinant IgG4 antibody. The working volume of the culture was 1.5 L and the duration of the culture was 15 days. The initial temperature was either 36°C or 37°C, and the initial pH ranged from 6.65 to 6.9. After day 5 (day 5 to day 8), the temperature was shifted from 34 to 36°C. Table 3 outlines the various experimental conditions of this study.

[0091] [Table 3]

[0092] The levels of HM and nonfucosylated glycoforms were measured as described in Example 1. The sum of the two levels was calculated and labeled as total nonfucosylated glycoforms. HM ranged from 1.4 to 2.6%, and nonfucosylated glycoforms ranged from 0.7 to 1.6. TAF levels ranged from 2.1 to 3.9. As shown in Figure 8, both pH and final temperature affected TAF. pH showed a positive correlation and final temperature showed a negative correlation.

[0093] Example 3 This example shows the effect of initial pH during the initial cell culture period on TAF levels, and the effect of a pH shift after the initial cell culture period on TAF levels.

[0094] After the perfusion step, the cells expressing IgG1 antibodies were cultured in a bioreactor essentially as described in Example 1. The cells were cultured for a total of 12 days and maintained at a temperature of about 36° C. The cells were cultured at one of three default pH values ​​(6.85, 6.95 or 7.05) and the pH was shifted (either increased or decreased) during the initial cell culture period (day 1) or after the initial cell culture period (day 5). Group A cells were cultured at a default pH value of 7.05, group B cells were cultured at a default pH value of 6.85, while group C cells were cultured at a default pH value of 6.95. The cells of group A were divided into two subgroups: subgroup A1 and subgroup A2. The cells of subgroup A1 were cultured at an initial pH value of 7.05 and the pH was shifted to 6.95 during the initial cell culture period (day 1), while the cells of subgroup A2 were cultured at an initial pH value of 7.05 and the pH was shifted to 6.95 after the initial cell culture period (day 5). For the cells of group B the pH was increased to 6.95 after the initial cell culture period (day 5) and for the cells of group C the pH was decreased to 6.85 after the initial cell culture period (day 5). In a series of control cultures the pH was set to 6.95 and maintained throughout the entire culture period (i.e. no pH shift).

[0095] After a 12 day culture period, the TAF glycoforms of IgG1 antibodies produced by each of the groups were measured via HILIC essentially as described in Example 1. The %TAF of antibodies produced by the control cultures ranged from about 3.1% to about 4.14%. Compared to antibodies produced by the control cultures, the average %TAF of antibodies produced by Group A was higher while the average %TAF of antibodies produced by Group B was lower. A higher initial pH correlated with a higher %TAF and a lower initial pH correlated with a lower %TAF.

[0096] Interestingly, the %TAF of IgG1 antibodies produced by cells of subgroup A2 was very similar to that of antibodies produced by subgroup A2, suggesting that the pH shift after the initial cell culture period did not affect the TAF levels. This observation was further supported by the observation that the TAF levels of antibodies produced by cells of group C were very similar to that of the control cell culture. Even though cells of group C were subjected to a downward pH shift, the TAF was almost the same as the control culture, since the shift occurred after the initial cell culture period (day 5).

[0097] This example revealed that pH shifts occurring after the initial cell culture period did not significantly affect TAF levels, with higher set pH values ​​tending to higher TAF levels, while lower set pH values ​​tending to lower TAF levels.

[0098] All references cited herein, including publications, patent applications, and patents, are hereby incorporated by reference to the same extent as if each reference was incorporated by reference and was set forth in its entirety herein.

[0099] The use of the terms "a" and "an" and "the" and similar references in the context of describing this disclosure (particularly in the context of the claims which follow) are to be construed to encompass both the singular and the plural, unless otherwise stated in the specification or clearly contradicted by the context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including but not limited to"), unless otherwise indicated.

[0100] Recitation of ranges of values ​​herein is merely a shorthand method of referring individually to each separate value falling within the range and each endpoint, unless otherwise stated herein, and each separate value and endpoint is incorporated herein as if it were individually recited herein.

[0101] All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by the content. The use of any examples or representative language (e.g., "such as") provided herein is merely intended to more fully clarify the disclosure and does not pose limitations on the scope of the disclosure unless otherwise claimed. No language in the present application should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0102] Preferred embodiments of the present disclosure are described herein, including the best mode known to the inventors for carrying out the present disclosure. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that those skilled in the art will use such variations as necessary, and the inventors intend that the present disclosure should also be practiced other than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter set forth in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present disclosure, unless otherwise indicated herein or clearly contradicted by the content.

Claims

1. 1. A method for adjusting a level of total nonfucosylated (TAF) glycoforms of a recombinant glycosylated protein produced by a glycosylation-competent cell in a cell culture, comprising: (i) maintaining said cell culture at an initial pH value for an initial cell culture period; and (ii) allowing the pH of said cell culture to shift by 0.05 to 0.15 from said initial pH value after said initial cell culture period; the initial pH value is selected from a pH greater than 6.50 and less than 7.5, (a) a higher pH than the control pH of the control cell culture leads to an increase in TAF glycoforms compared to the control cell culture, or (b) a lower pH than the control pH of the control cell culture leads to a decrease in TAF glycoforms compared to the control cell culture, and The initial cell culture period is 4 to 6 days; the recombinant glycosylated protein is an IgG1 or IgG4 antibody; said glycosylation competent cell is CHO; The cell culture is carried out by a perfusion method, The method of claim 1, wherein the temperature of the cell culture during the initial cell culture period is 36-37°C.

2. 1. A method for modulating the level of total nonfucosylated (TAF) glycoforms of a recombinant glycosylated protein produced by a glycosylation-competent cell in a cell culture, comprising: (i) culturing said cell culture to produce 8.2×10 ６ ~1.94x10 ７ , or 1.21 x 10 ７ ~3.46 x 10 ７ and (ii) after said initial cell culture period, allowing the pH of said cell culture to shift by 0.05-0.15 from said initial pH value; the initial pH value is greater than 6.50 and less than 7.5, and (a) a higher pH than the control pH of the control cell culture leads to an increase in TAF glycoforms compared to the control cell culture, or (b) a lower pH than the control pH of the control cell culture leads to a decrease in TAF glycoforms compared to the control cell culture; the recombinant glycosylated protein is an IgG1 or IgG4 antibody; said glycosylation competent cell is CHO; The cell culture is carried out by a perfusion method, The method of claim 1, wherein the temperature of the cell culture during the initial cell culture period is 36-37°C.

3. 3. The method of claim 2, wherein the initial cell culture period is between 4 and 6 days.

4. A method according to any one of claims 1 to 3, comprising maintaining the cell culture at the initial set pH value for four or five days.

5. The method according to any one of claims 1 to 4, wherein the initial pH value is greater than 6.55 and less than 7.

2.

6. 6. The method of claim 5, wherein the initial pH value is greater than 6.60 and less than 7.

2.

7. 7. The method of claim 6, wherein the initial pH value is greater than 6.60 and less than 7.

2.

8. 8. The method of claim 7, wherein the initial pH value is greater than 6.65 and less than 7.

2.

9. The initial pH value is (a) greater than 6.70 and less than 7.2; (b) greater than 6.75 and less than 7.2; (c) greater than 6.80 and less than 7.2; (d) greater than 6.85 and less than 7.2; (e) 7.0 to 7.1, or (f) greater than or equal to 6.85 and less than 6.95; The method according to claim 8.

10. 10. The method of any one of claims 1 to 9, further comprising maintaining the cell culture at an initial temperature during the initial cell culture period, wherein the initial temperature is selected from a temperature between 30°C and 40°C or between 32°C and 38°C.

11. 11. The method of claim 10, wherein maintaining the cell culture at an initial temperature comprises maintaining the cell culture within ±1° C. of the initial temperature during the initial cell culture period, or wherein the temperature of the cell culture does not shift by more than 1° C. from the initial temperature during the initial cell culture period.

12. 12. The method of any one of claims 1 to 11, wherein the initial set pH value is higher than a control pH of a control cell culture, and the level of TAF glycoforms of the recombinant glycosylated protein is increased after the initial cell culture period compared to the control cell culture.

13. the level of high mannose (HM) glycoforms of the recombinant glycosylated protein after the initial cell culture period is increased compared to the control cell culture; The method according to any one of claims 1 to 12.

14. 14. The method of claim 13, wherein the levels of one or more of Man5, Man6, Man7, Man8 and / or Man9 of the recombinant glycosylated proteins after the initial cell culture period are increased compared to the control cell culture.

15. the level of non-fucosylated glycoforms of the recombinant glycosylated protein after the initial cell culture period is increased compared to the control cell culture; 15. The method according to claim 13 or 14.

16. 16. The method of claim 15, wherein the levels of one or more of the recombinant glycosylated proteins A1G0, A2G0, A2G1a, A2G1b, A2G2 and A1G1M5 after the initial cell culture period are elevated compared to the control cell culture.

17. 17. The method of any one of claims 1 to 16, wherein the initial setup pH value is lower than a control pH of a control cell culture, and the level of TAF glycoforms of the recombinant glycosylated protein after the initial cell culture period is reduced compared to the control cell culture.

18. 18. The method of claim 17, wherein the level of high mannose (HM) glycoforms of the recombinant glycosylated protein after the initial cell culture period is reduced compared to the control cell culture.

19. The method of claim 18, wherein (a) the level of one or more of Man5, Man6, Man7, Man8 and / or Man9 of the recombinant glycosylated protein after the initial cell culture period is reduced compared to the control cell culture, and / or (b) the level of non-fucosylated glycoforms of the recombinant glycosylated protein after the initial cell culture period is reduced compared to the control cell culture.

20. the level of one or more of the recombinant glycosylated proteins A1G0, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5 after the initial cell culture period is reduced compared to the control cell culture; 20. The method of claim 19.

21. 21. The method of any one of claims 1 to 20, comprising allowing the pH of the cell culture to shift by 0.05 after the initial cell culture period.

22. A method according to any one of claims 1 to 21, wherein the pH is allowed to shift to a lower pH or to a higher pH.

23. 23. The method of any one of claims 1 to 22, comprising: (a) allowing the temperature to shift by more than 2°C after the initial cell culture period; and / or (c) shifting the temperature by more than 2°C after the initial cell culture period.

24. 24. The method of claim 23, wherein the shift in temperature comprises a decrease in temperature or an increase in temperature.

25. 25. The method of claim 24, wherein the temperature is decreased by 2°C to 4°C or the temperature is increased by 2°C to 4°C.

26. 26. The method of any one of claims 1 to 25, comprising maintaining the cell culture in a medium comprising manganese at a concentration of less than 1 μM during the initial cell culture period and / or copper at a concentration of 30 ppb or less during the initial cell culture period.

27. 27. The method of claim 26, wherein the medium comprises copper at a concentration of 5 ppb or greater.

28. maintaining a dissolved oxygen (DO) level of said cell culture within the range of 50 mmHg to 100 mmHg during the initial cell culture period.

28. The method according to any one of claims 1 to 27.

29. The recombinant glycosylated protein has the formula: ____________________ １ ︁ ２ (In the formula, Xaa １ is any amino acid other than Pro, ２ The method of any one of claims 1 to 28, wherein the N-glycosylation consensus sequence is one or more of the following:

30. 30. The method of claim 29, wherein the recombinant glycosylated protein comprises a portion of a fragment crystallizable (Fc) region that retains an N-linked glycosylation site.

31. 31. The method of claim 30, wherein the Fc region is an IgG Fc region.

32. The method of claim 29 or 30, wherein the recombinant glycosylated protein is an antibody, a peptibody or an Fc fusion protein.

33. The method of any one of claims 1 to 32, further comprising recovering the cell culture supernatant containing the recombinant glycosylated protein, wherein the total cell culture period is at least 10 days.

34. 34. The method of claim 33, comprising purifying the recombinant glycosylated protein from the supernatant, wherein the recombinant glycosylated protein comprises an Fc, and wherein purifying the recombinant glycosylated protein may comprise the use of a Protein A affinity chromatography resin.

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