Control of total non-fucosylated glycoform of antibodies produced in cell culture

Abstract

To provide simple and efficient methods for operating and modulating the level of total afucosylated (TAF) glycoforms in recombinant production of therapeutic antibodies.SOLUTION: There is provided herein, a method of modulating the levels of total afucosylated (TAF) glycoforms of a recombinant glycosylated protein produced by glycosylation-competent cells in a cell culture, the methods comprising: (i) maintaining the cell culture at an initial set point pH for an initial cell culture period and (ii) ceasing to maintain the cell culture at the initial set point pH after the initial cell culture period, where the initial pH is selected from a pH that is greater than about 6.50 and less than 7.5, and the initial cell culture period is about 4 days to about 6 days.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Application No. 62 / 471,347, filed Mar. 14, 2017, the content of which is incorporated herein by reference.

Background Art

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

[0003] The 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). HM glycoforms not only affect the serum clearance rate of antibodies, but such glycoforms can also affect antibody effector function or antibody - mediated target cell death, also known as antibody - dependent cell - mediated cytotoxicity (ADCC), in addition to non - fucosylated (non - fuc) glycoforms.

[0004] Many factors affect the glycan structure and thus the final glycosylation form (glycoform) of the protein. For example, the cell line expressing the antibody, the cell - culture medium, the feed - medium composition, and the timing of additions during cell culture can affect the production of the protein's glycoform.

[0005] While many methods have been suggested by research groups to affect the levels of specific glycoforms of antibodies, a simple and efficient method for manipulating and regulating the levels of total afucosylated (TAF) glycoforms during the recombinant production of therapeutic antibodies is still needed in the biopharmaceutical industry.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Means for Solving the Problems

[0007] Maintaining a desired initial pH (e.g., an initial set pH value) during an initial cell culture period (e.g., the first 2, 3, 4, 5, or 6 days after inoculation) is important for adjusting the levels of TAF glycoforms of recombinantly produced glycosylated proteins, while this is the first description of data demonstrating that the cell culture pH after the initial cell culture period does not significantly affect the TAF glycoform levels. The finding that the initial pH, rather than the pH at later stages, affects the TAF levels was unexpected. Without being bound by a particular theory, by adjusting the initial pH (e.g., an initial set pH value) during an initial cell culture period (e.g., the first 2, 3, 4, 5, or 6 days after inoculation), it becomes possible for the recombinant production of glycosylated proteins to have a desired, or predetermined, or preselected TAF glycoform level. Accordingly, the present invention relates to a method for producing a recombinant glycosylated protein (glycoprotein) having a desired, or predetermined, or preselected TAF glycoform level.

[0008] The present invention provides a method for adjusting the TAF glycoform level of a recombinant glycosylated protein produced by glycosylation-competent cells in cell culture. In a representative embodiment, the method includes maintaining cell culture at an initial pH, such as an initial set pH value, over an initial cell culture period.

[0009] In a representative embodiment, the initial cell culture period is from about 4 days to about 6 days after inoculation, for example, about 4 days, about 5 days, about 6 days, or from about 2 days to about 6 days after inoculation, for example, 2 days, 3 days after inoculation, or from about 48 hours to about 144 hours, 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, 93 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 the number of hours increased in increments thereof.

[0010] In a representative embodiment, the initial cell culture period is based on a specific viable cell density (VCD) of the cell culture. In a representative embodiment, the initial cell culture period is the time after inoculation when the VCD of the cell culture is about 6.5x10 6 cells / mL or less. In a representative embodiment, the method includes maintaining the cell culture at an initial set pH value until the VCD of the cell culture reaches about 6.5x10 6 cells / mL. In a representative embodiment, the method includes maintaining the cell culture at an initial set pH value until the VCD of the cell culture reaches from about 6.9x10 6 to about 8.2x10 6 . In a representative embodiment, the method includes maintaining the cell culture at an initial set pH value until the VCD of the cell culture reaches from about 8.2x10 6 to about 1.94x10 7 . In a representative embodiment, the method includes maintaining the cell culture at an initial set pH value until the VCD of the cell culture reaches from about 1.21x10 7 to about 3.46x10 7 .

[0011] In a representative embodiment, the initial set pH value is selected from a pH that is higher than about 6.5 and less than about 7.5.

[0012] The present invention also relates to a composition comprising a glycosylated protein and its TAF glycoform.

Brief Description of the Drawings

[0013]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

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

[0015] All N-glycans have a common core sugar sequence: Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser / Thr (Man3GlcNAc2Asn) and are classified into one of three types: (A) high mannose (HM) or oligomannose (OM) type consisting of two N-acetylglucosamine (GalNAc) moieties and a large number (e.g., 5, 6, 7, 8, or 9) of mannose (Man) residues, (B) complex type containing more than two GlcNAc moieties and any number of other sugar types, or (C) hybrid type containing a Man residue on one branch and GlcNAc at the base of the complex branch. Figure 1 (from Stanley et al., Chapter 8: N-Glycans, Essentials of Glycobiology, 2 nd nd ed., Cold Spring Harbor Laboratory Press; 2009) shows the three types of N-glycans.

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

[0017] N-linked glycosylation begins in the endoplasmic reticulum (ER), and as a result of a complex series of reactions, the linkage of a core glycan structure basically composed of two GlcNAc residues and three Man residues occurs. The glycan complex formed in the ER is modified by the action of enzymes in the Golgi apparatus. When the saccharide is relatively difficult to access by the enzyme, this generally remains in the original HM form. When the enzyme can access the saccharide, many of the Man residues are cleaved off, and the saccharide is further modified, resulting in a complex-type N-glycan structure. For example, mannosidase-1 located in the cis-Golgi can cleave or hydrolyze the HM glycan, while fucosyltransferase FUT-8 located in the medial-Golgi fucosylates the glycan (Hanrue Imai-Nishiya (2007), BMC Biotechnology, 7:84).

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

[0019] The invention provided herein relates to a method for adjusting the levels of various glycosylation forms (glycoforms) of a protein during recombinant production by glycosylation-competent cells. Without being bound by a particular theory, the method of the invention is believed to provide a means for a tailor-made composition containing a particular glycoform of a certain recombinant protein in a particular amount.

[0020] In representative embodiments, the level of total afucosylated (TAF) glycoforms is adjusted. As used herein, "total afucosylated glycoform" or "TAF glycoform" or "TAF" or "final TAF" refers to the total amount of high mannose glycoforms and afucosylated glycoforms. As used herein, the terms "high mannose" or "HM" or "final HM" encompass glycoforms containing 5, 6, 7, 8, or 9 mannose residues, abbreviated as Man5, Man6, Man7, Man8, and Man9, respectively. As used herein, the terms "afucosylated glycoform" or "afu glycoform" or "afucosylated glycan" or "afu" or "AF" or "afucosylated" refer to glycoforms lacking an α1,6-linked fucose on a GlcNAc residue included in an amide bond with a core fucose, such as Asn of an N-glycosylation site. Examples of afucosylated glycoforms include, but are not limited to, A1G0, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5. Further examples of afucosylated glycans include, for example, A1G1a, G0[H3N4], G0[H4N4], G0[H5N4], FO-N[H3N3]. See, for example, Reusch and Tejada, Glycobiology 25(12):1325-1334(2015). In a representative aspect, herein, as further described in Example 1, the levels of TAF, HM glycoforms, and afucosylated glycoforms are determined via HILIC. After enzymatic cleavage of N-glycans, HILIC is performed to obtain a chromatogram with several peaks, each peak corresponding to the average distribution (amount) of various glycoforms. For these purposes, % peak area = peak area / total peak area x 100% and % total peak area = total sample area / total standard area x 100%. The calculations used for the purpose of determining %TAF can be performed as follows: % afucosylated glycoform = %A1G0 + %A2G0 + %A2G1a + %A2G1b + %A2G2 + %A1G1M5. % High mannose glycoform = % 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 a representative embodiment, the recombinant glycosylated protein is produced by glycosylation-competent cells in cell culture. In a representative embodiment, the method includes maintaining the cell culture at an initial set pH value over an initial cell culture period. As used herein, the term "maintaining" means setting the set pH value to the initial set pH value and not changing the set pH value over a specified time period. 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 "initial set pH value" refers to the set pH value set by a user during or immediately after inoculation of the cell culture. As will be appreciated by those skilled in the art, 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. Generally, the actual pH of the cell culture is within ±0.05 of the set pH value, and in some embodiments, the actual pH of the cell culture is within ±0.03 or ±0.02 of the set pH value. In a representative embodiment, maintaining the cell culture at an initial set pH value over an initial cell culture period means that the pH of the cell culture does not shift by more than 0.05 from the initial set pH value during the initial cell culture period. In a representative embodiment, the method includes maintaining the cell culture at an initial set pH value over an initial cell culture period, and the initial set pH value is higher than about 6.5 and less than 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 higher.For example, the initial set 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. Further, the initial set value is 7.5 or less. For example, the initial set 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, 7.00, 6.98, 6.96, 6.94, 6.92, 6.90, 6.88, 6.86, 6.84, 6.82, 6.80, 6.78, 6.76, 6.74, 6.72, 6.70, 6.68, 6.66, 6.64, 6.62, 6.60, 6.58, 6.56, 6.54, 6.52, 6.50 or less. In a representative embodiment, the initial set pH value is higher than about 6.55 and less than about 7.5. In a representative embodiment, the initial set pH value is higher than about 6.60 and less than about 7.5. In a representative embodiment, the initial set pH value is higher than about 6.65 and less than about 7.5. In a representative embodiment, the initial set pH value is higher than about 6.7 and less than about 7.5. In a representative embodiment, the initial set pH value is higher than about 6.75 and less than about 7.5. In a representative embodiment, the initial set pH value is higher than about 6.80 and less than about 7.5. In a representative embodiment, the initial set pH value is higher than about 6.5 and less than about 7.45. In a representative embodiment, the initial set pH value is higher than about 6.5 and less than about 7.4. In a representative embodiment, the initial set pH value is higher than about 6.5 and less than about 7.35. In a representative embodiment, the initial set pH value is higher than about 6.5 and less than about 7.3. In a representative embodiment, the initial set pH value is higher than about 6.5 and less than about 7.25. In a representative embodiment, the initial set pH value is higher than about 6.5 and less than about 7.2. In a representative embodiment, the initial set pH value is higher than about 6.5 and less than about 7.15. In a representative embodiment, the initial set pH value is higher than about 6.5 and less than about 7.1. In a representative embodiment, the initial set pH value is higher than about 6.85 and less than 7.2.In a representative embodiment, the initial set pH value is from about 7.0 to 7.1. The initial set pH value is, in a representative embodiment, from about 6.85 or more to about 6.95 or less. In a representative embodiment, the initial set pH value is from about 6.95 to 7.15.

[0022] In a representative embodiment, the method includes maintaining cell culture at the initial set pH value over an initial cell culture period. As used herein, the phrase "initial cell culture period" refers to the time after inoculation or subsequent time when glycosylation competent cells are added to the cell culture medium for the purpose of culturing cells for recombinant protein production. In a representative embodiment, the initial cell culture period is from about 4 days to about 6 days (e.g., about 4 days, 5 days or about 6 days), or an increment at about 96 hours to about 144 hours or increments thereof. In a representative embodiment, the initial cell culture period is from 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 defined for a specific viable cell density (VCD) of the cell culture. In a representative embodiment, the initial cell culture period is the time after inoculation while the VCD of the cell culture is at about 6.5x10 6 cells / mL or less. In a representative embodiment, the method includes maintaining cell culture at the initial set pH value until the cell culture reaches a VCD of about 6.5x10 6 cells / mL. In a representative embodiment, the method includes maintaining cell culture at the initial set pH value until the cell culture reaches a VCD of from about 8.2x10 6 to about 1.94x10 7 . In a representative embodiment, the method includes maintaining cell culture at the initial set pH value until the cell culture reaches a VCD of from about 1.21x10 7 to about 3.46x10 7 .

[0023] In a representative embodiment, the initial pH is higher than the control pH of the control cell culture. Without being bound by a particular theory, maintaining a higher pH (compared to the control pH) leads to an increase in the TAF glycoforms of the protein produced by the cells in the cell culture. Thus, in a representative embodiment, the method of the present invention relates to increasing the level of the TAF glycoforms of the protein produced by the cells in the cell culture. In a representative embodiment, the level of the HM glycoforms of the 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 the recombinant glycosylated protein is increased compared to the control cell culture. In a representative embodiment, the level of the non-fucosylated glycoforms of the 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 the recombinant glycosylated protein is increased compared to the control cell culture. In a representative embodiment, the level of one or more of A1G1a, G0[H3N4], G0[H4N4], G0[H5N4], and FO-N[H3N3] of the recombinant glycosylated protein is increased compared to the control cell culture.

[0024] As used herein, the term "increase" and derivatives thereof need not be a 100% or complete increase. Rather, the degree of increase that a person skilled in the art would recognize as having a potential benefit varies. In this regard, the method of the present invention can increase the level of TAF, HM, or non-fucosylated glycoform to some degree or level as compared to a control cell culture. In representative embodiments, the increase provided by the method of the present invention is at least or about a 10% increase (e.g., at least or about a 20% increase, at least or about a 30% increase, at least or about a 40% increase, at least or about a 50% increase, at least or about a 60% increase, at least or about a 70% increase, at least or about an 80% increase, at least or about a 90% increase, at least or about a 95% increase, at least or about a 98% increase) as compared to a control cell culture. In representative embodiments, the increase provided by the method of the present invention is more than 100%, e.g., 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or even 1000% as compared to a control cell culture. In representative embodiments, the level of a protein, TAF, HM, or non-fucosylated glycoform increases by at least about 1.5-fold as compared to a control cell culture. In representative embodiments, the level of a protein, TAF, HM, or non-fucosylated glycoform increases by at least about 2-fold as compared to a control cell culture. In representative embodiments, the level of a protein, TAF, HM, or non-fucosylated glycoform increases by at least about 3-fold as compared to a control cell culture. In representative embodiments, the level of a protein, TAF, HM, or non-fucosylated glycoform increases by at least about 4-fold or 5-fold as compared to a control cell culture.

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

[0026] In representative embodiments, elevated levels of the TAF glycoforms of the protein are observed longer than the fourth, fifth, or sixth day of cell culture, or beyond the initial cell culture period. In representative embodiments, elevated levels of the TAF glycoforms of the protein are observed over 7, 8, 9, 10, 11, or 12 days (post-inoculation) of cell culture, or 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 glycoform of the protein are observed when the protein is recovered from the cell culture.

[0027] In a representative embodiment, the initial pH is lower than the control pH for the control cell culture. Without being bound by a particular theory, maintaining a lower pH (compared to the control pH) leads to a decrease in the TAF glycoforms of the protein produced by the cells in the cell culture. Thus, in a representative embodiment, the method of the invention relates to reducing the level of the TAF glycoforms of the protein produced by the cells in the cell culture. In a representative embodiment, the level of the HM glycoforms of the recombinant glycosylated protein is decreased 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 decreased compared to the control cell culture. In a representative embodiment, the level of the non-fucosylated glycoforms of the recombinant glycosylated protein is decreased 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 decreased compared to the control cell culture. In a representative embodiment, the level of one or more of A1G1a, G0[H3N4], G0[H4N4], G0[H5N4], and FO-N[H3N3] of the recombinant glycosylated protein is decreased compared to the control cell culture.

[0028] As used herein, the term "reduced" and derivatives thereof need not be a 100% or complete reduction. Rather, the degree of reduction that a person of ordinary skill in the art would recognize as having a potential benefit can vary. In this regard, the method of the present invention can reduce the level of TAF, HM, or non-fucosylated glycoform to some 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 a 10% reduction (e.g., at least or about a 20% reduction, at least or about a 30% reduction, at least or about a 40% reduction, at least or about a 50% reduction, at least or about a 60% reduction, at least or about a 70% reduction, at least or about an 80% reduction, at least or about a 90% reduction, at least or about a 95% reduction, at least or about a 98% reduction) compared to a control cell culture. In representative embodiments, the reduction provided by the method of the present 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 protein, TAF, HM, or non-fucosylated glycoform is reduced by at least about 1.5-fold compared to a control cell culture. In representative embodiments, the level of a protein, TAF, HM, or non-fucosylated glycoform is reduced by at least about 2-fold compared to a control cell culture. In representative embodiments, the level of a protein, TAF, HM, or non-fucosylated glycoform is reduced by at least about 3-fold compared to a control cell culture. In representative embodiments, the level of a protein, TAF, HM, or non-fucosylated glycoform is reduced by at least about 4-fold or 5-fold compared to a control cell culture.

[0029] In representative embodiments, at an early stage such as the first day after inoculation, a decrease in the level of the TAF glycoform of the protein is observed or observable or detected or detectable. In representative embodiments, at an early stage such as the second day after inoculation, a decrease in the level of the TAF glycoform of the protein is observed or observable or detected or detectable. In representative embodiments, at an early stage such as the third day after inoculation, a decrease in the level of the TAF glycoform of the protein is observed or observable or detected or detectable. In representative embodiments, at an early stage such as the fourth day after inoculation, a decrease in the level of the TAF glycoform of the protein is observed or observable or detected or detectable. In representative embodiments, after approximately the fifth day after inoculation, a decrease in the level of the TAF glycoform of the protein is observed or observable or detected or detectable. In representative embodiments, at the time when the protein is recovered from the cell culture, a decrease in the level of the TAF glycoform of the protein is observed or observable or detected or detectable.

[0030] In representative embodiments, a decrease in the level of the TAF glycoform of the protein is observed longer than the fourth, fifth, or sixth day of the cell culture or beyond the initial cell culture period. In representative embodiments, over 7, 8, 9, 10, 11, or 12 days of the cell culture (after inoculation) or longer (e.g., 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, 1 year), a decrease in the level of the TAF glycoform of the protein is observed. In representative embodiments, a decrease in the level of the TAF glycoform of the protein is observed when the protein is recovered from the cell culture.

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

[0032] To evaluate the glycoforms present in a glycoprotein containing a composition or to determine the glycoform profile of a specific sample containing a glycoprotein, various methods are known in the art. Suitable methods include cationic MALDI-TOF analysis, anionic MALDI-TOF analysis, weak anion exchange (WAX) chromatography, normal phase chromatography (NP-HPLC), exoglycosidase digestion, Bio-Gel P-4 chromatography, anion exchange chromatography, and one-dimensional n.m.r. spectroscopy and combinations thereof. See, for example, Mattu et al., JBC 273:2260-2272 (1998); Field et al., Biochem J 299 (Pt 1):261-275 (1994); Yoo et al., MAbs 2(3):320-334 (2010) Wuhrer M.et al., Journal of Chromatography B, 2005, Vol.825, Issue 2, pp. 124-133; Ruhaak L.R., Anal Bioanal Chem, 2010, Vol.397:3457-3481 and Geoffrey, R.G.et.al. Analytical Biochemistry 1996, Vol.240, 210-226. Also, the examples shown herein describe suitable methods for evaluating the glycoforms present in a glycoprotein-containing composition.

[0033] Temperature and other cell culture parameters In a representative embodiment, the method further comprises maintaining the cell culture at an initial temperature during an 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 a representative embodiment, the initial temperature is from about 32°C to about 38°C or from about 35°C to about 38°C. In a representative aspect, 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 a representative aspect, maintaining the cell culture at the initial temperature over 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] Regarding the present invention, cell culture can be maintained according to any series of conditions suitable for recombinant protein production. For example, cell culture can be maintained at a specific cell density, culture volume, dissolved oxygen level, pressure, osmotic pressure, etc. In a representative embodiment, the cell culture before inoculation is shaken (e.g., 70 rpm) with 5% CO2 under standard humidification conditions in a CO2 incubator. In a representative embodiment, the cell culture is inoculated at a seeding density of 10 6 cells / mL in 1.5 L of medium. In a representative embodiment, the method includes maintaining an osmotic pressure of about 200 mOsm / kg to about 500 mOsm / kg. In a representative embodiment, the method includes maintaining an osmotic pressure 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 osmotic pressure of about 225 mOsm / kg to about 350 mOsm / kg. In a representative embodiment, the method includes maintaining the dissolved oxygen (DO) level of the cell culture at an oxygen saturation of about 20% to about 60% during the initial cell culture period. In a representative example, the method includes maintaining the DO level of the cell culture at an oxygen saturation of about 30% to about 50% (e.g., about 35% to about 45%) during the initial cell culture period. In a representative example, the method includes maintaining the DO level of the cell culture at an oxygen saturation of about 20%, about 30%, about 40%, about 50% or about 60% during the initial cell culture period.

[0035] Cell cultures can be maintained in any one or more culture media. In a representative embodiment, the cell culture can be maintained in a medium appropriate for cell growth and / or provided in one or more feeding media according to some appropriate feeding schedule. In a representative embodiment, the method includes maintaining the cell culture in a medium containing glucose, lactate, ammonia, glutamine, and / or glutamate. In a representative embodiment, the method includes maintaining the cell culture in a medium containing manganese at a concentration of less than about 1 μM during an initial cell culture period. In a representative embodiment, the method includes maintaining the cell culture in a medium containing from about 0.25 μM to about 1 μM manganese. In a representative embodiment, the method includes maintaining the cell culture in a medium containing a negligible amount of manganese. In a representative embodiment, the method includes maintaining the cell culture in a medium containing copper at a concentration of about 50 ppb or less during an initial cell culture period. In a representative embodiment, the method includes maintaining the cell culture in a medium containing copper at a concentration of about 40 ppb or less during an initial cell culture period. In a representative embodiment, the method includes maintaining the cell culture in a medium containing copper at a concentration of about 30 ppb or less during an initial cell culture period. In a representative embodiment, the method includes maintaining the cell culture in a medium containing copper at a concentration of about 20 ppb or less during an initial cell culture period. In a representative embodiment, the medium contains copper at a concentration of about 5 ppb or more or about 10 ppb or more.

[0036] In a representative embodiment, the type of cell culture is a fed-batch culture method or a continuous perfusion culture method. However, the method of the present invention is advantageously not limited to any particular type of cell culture.

[0037] After the initial cell culture period In a representative embodiment, the method of the present invention for adjusting the TAF glycoform level of a recombinant glycosylated protein includes maintaining cell culture at an initial pH over an initial cell culture period. In a representative aspect, the method further includes stopping maintaining cell culture at the initially set pH value after the initial cell culture period. For the purposes herein, the concept of "stopping maintaining cell culture at the initially set pH value" refers to ceasing the actions required to adjust cell culture at the initially set pH value. For example, one or more settings for a pH control system can be changed to effectively stop maintaining the initially set pH value. In a representative aspect, "stopping maintaining cell culture at the initially set pH value" can refer to tolerating a pH shift or change. For example, "stopping maintaining cell culture at the initially set pH value" can refer to an active step of shifting the pH, for example, by changing the set pH value in a pH control system, or a passive step of allowing the pH to shift or ceasing the adjustment or maintenance of a specific pH or pH range. In a representative aspect, the maintenance or adjustment of cell culture pH stops after the first 4 to 6 days of cell culture. In a representative aspect, a method for adjusting the TAF glycoform level of a recombinant glycosylated protein produced by glycosylation-competent cells maintains cell culture at an initial pH over an initial cell culture period, stops maintaining cell culture at the initial pH after the initial cell culture period, and tolerates shifting the pH of the cell culture, for example, by more than 0.05. For the purposes herein, the concept of "tolerating a pH shift" can refer to an active step of shifting the pH, for example, by changing the set pH value in a pH control system, or a passive step of allowing the pH shift or ceasing the adjustment or maintenance of a specific pH or pH range. In a representative aspect, it is tolerated to shift the pH of the cell culture by about 0.05 to about 2.0 after the initial cell culture period. For example, after the initial period of cell culture, it is tolerated to shift the pH by about 0.1 to about 1.5 or about 0.5 to about 1.0. In certain representative aspects, the pH is not tolerated to be outside the pH range appropriate for antibody production by the cells of the cell culture.For example, a pH of 9 or higher or 4 or lower is not tolerated.

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

[0039] Methods for adjusting or maintaining the pH of cell cultures and performing the same using a high-throughput bioreactor fully equipped with a device including a pH monitoring system are known in the art. See, for example, the paper by Seung Joon Lee, “Dissolved Oxygen and pH Monitoring within Cell Culture Media using a Hydrogel Microarray Sensor”, December 2006, Texas A&M University; 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, pages 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., Biotechnol. Bioeng. 87(2):243 - 254 (2005); Hermann et al., Biotechnol. Bioeng. 81:178 - 186 (2002); European Patent No. 3128319; U.S. Patent Application Publication No. 2015 / 0376647. pH monitors are commercially available and include, for example, Easyferm Plus ARC 225 (Hamilton, Reno, NV). Also, methods for maintaining the pH of cell cultures are described in the examples herein.

[0040] In a representative embodiment, the method of the present invention for adjusting the TAF glycoform level of a recombinant glycosylated protein includes maintaining cell culture at an initial set pH value over an initial cell culture period, and further includes maintaining 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, wherein the initial temperature is from 30°C to 40°C. In a representative embodiment, the method further includes maintaining the initial cell temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more during the initial cell culture period and after the initial cell culture period. In a representative embodiment, the method further includes ceasing to maintain the initial temperature after the initial cell culture period. In a representative embodiment, the method includes ceasing to maintain 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 includes 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 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 a representative embodiment, a temperature shift of the cell culture is allowed after the initial cell culture period. In a representative embodiment, after the initial cell culture period, the temperature is no longer maintained within ±1°C of the selected initial temperature. In a representative embodiment, a temperature shift of about 1°C to about 6°C is allowed after the initial cell culture period. For example, after the initial period of cell culture, a temperature shift of about 1°C to about 5°C or about 1°C to about 4°C or about 2°C or about 3°C is allowed. In certain representative embodiments, the temperature is not allowed to fall outside the range of temperatures appropriate 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, such as an initial set pH value, over 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 a representative embodiment, the method includes maintaining the cell culture at an initial pH, such as an initial set pH value, over 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 an alternative embodiment, the method does not include a temperature shift after the initial cell culture period. In a representative example, the temperature of the cell culture is maintained at a temperature within ±1 °C of the initial temperature throughout the cell culture period. In a representative example, the temperature of the cell culture is maintained at a temperature within ±1 °C of the initial temperature throughout the cell culture period, and the method includes maintaining the cell culture at an initial pH, such as 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 a representative embodiment, the temperature of the cell culture is maintained 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, such as 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 protein In a representative embodiment, the recombinant protein has the formula: Asn-Xaa1-Xaa2 (wherein Xaa1 is any amino acid other than Pro and Xaa2 is Ser or Thr) and comprises an amino acid sequence comprising one or more N-glycosylation consensus sequences.

[0045] In a representative embodiment, the recombinant protein comprises a fragment crystallizable (Fc) polypeptide. As used herein, the term "Fc polypeptide" 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 a hinge region that promotes dimerization. Fusion proteins (and oligomers formed therefrom) that include an Fc portion afford the advantage of easy purification by affinity chromatography on a protein A or protein G column. In a representative embodiment, the recombinant protein includes the Fc of IgG, such as human IgG. In a representative aspect, the recombinant protein includes the Fc of IgG1 or IgG2. In a representative aspect, the recombinant protein is an antibody, a peptibody, or an Fc-fusion protein.

[0046] In a representative aspect, the recombinant glycosylated protein is an antibody. As used herein, the term "antibody" refers to a protein having the conventional immunoglobulin format, including heavy and light chains, and including variable and constant regions. For example, the antibody can be IgG, which is a "Y-shaped" structure of two identical pairs of polypeptide chains, each pair having one "light" (generally having a molecular weight of about 25 kDa) and one "heavy" chain (generally having a molecular weight of about 50-70 kDa). Antibodies have variable and constant regions. In the IgG format, the variable region is generally about 100-110 or more amino acids, contains three complementarity determining regions (CDRs), is mainly involved in antigen recognition, and varies substantially among other antibodies that bind different antigens. The constant region mobilizes cells and molecules of the immune system to the antibody. The variable region is composed of the N-terminal regions of each light and heavy chain, while the constant region is composed of the respective C-terminal portions 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 characteristics of the CDRs of antibodies are described in the art. Briefly, in the antibody backbone, the CDRs are embedded within the frameworks in the heavy and light chain variable regions, and they constitute regions that are largely involved in antigen binding and recognition. The variable regions contain at least three heavy or light chain CDRs (see also Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, Md.; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883) within the framework regions (designated 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, and define the antibody 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 present 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 a representative embodiment, the present antibody is an antibody of isotype IgA, IgD, IgE, IgG or IgM, including any one of IgG1, IgG2, IgG3 or IgG4.

[0049] The antibody can be a monoclonal antibody or a 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 regard, the antibody can be regarded as a mammalian antibody, such as a mouse antibody, rabbit antibody, goat antibody, horse antibody, chicken antibody, hamster antibody, human antibody, etc. In certain embodiments, the recombinant protein is a human antibody. In certain embodiments, the recombinant protein is a chimeric antibody or a humanized antibody. The term "chimeric antibody" is used herein to refer to an antibody that contains a constant domain from one species and a variable domain from a second species, or more generally contains stretches of amino acid sequences from at least two species. The term "humanized", when used in reference to an antibody, refers to an antibody having at least a CDR region derived from a non-human origin that has been engineered to have a structure and immunological function that is more similar to a true human antibody than the antibody of its original origin. For example, humanization can include transplanting CDRs from a non-human antibody, such as a mouse antibody, etc. into a human antibody. Humanization can also include the selection of amino acid substitutions to make the non-human sequences look more like human sequences.

[0050] The antibody can be cleaved into fragments by enzymes such as papain and pepsin. Papain cleaves the antibody to produce two Fab fragments and one Fc fragment. Pepsin cleaves the antibody to produce an F(ab’)2 fragment and a pFc’ fragment. In a representative embodiment, the recombinant glycosylated protein is an antibody fragment that retains at least one glycosylation site, such as Fab, Fc, F(ab’)2 or pFc’.

[0051] The structure of the antibody has been utilized to generate an expanded range of alternative antibody forms that span a molecular weight range of at least 12 - 150 kDa and a valence (n) range that extends from monomer (n = 1), dimer (n = 2) and trimer (n = 3) to tetramer (n = 4) and higher possibilities; such alternative antibody forms are referred to herein as "antibody protein products".

[0052] As antibody protein products, those based on antibody fragments that retain complete antigen-binding ability include, for example, scFv, Fab, and VHH / VH. The smallest antigen-binding fragment that retains its complete antigen-binding site is the Fv fragment, which consists entirely of the variable (V) region. To link the V regions for the scFv (single-chain fragment variable) fragment for molecular stability, a soluble and flexible amino acid peptide linker is used, 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 produced in prokaryotic hosts. Other antibody protein products include disulfide bond-stabilized scFv (ds-scFv), single-chain Fab (scFab), as well as dimeric and multimeric antibody forms such as diabodies, triabodies, and tetrabodies or minibodies (miniAbs) that consist of scFv linked to an oligomerization domain. 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 forms is the single-chain variable (V) domain antibody fragment (scFv), which contains the V domains (VH and VL domains) from the heavy and light chains linked by a peptide linker of ~15 amino acid residues. Peptibodies or peptide-Fc fusions are another type of antibody protein product. The structure of peptibodies consists of a biologically active peptide grafted onto the Fc domain. Peptibodies are 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 (SCA); diabodies; triabodies; tetrabodies; bispecific or trispecific antibodies, etc. Bispecific antibodies can be divided into five main classes: BsIgG, additional IgG, BsAb fragments, bispecific fusion proteins, and BsAb conjugates. See, for example, Spiess et al., Molecular Immunology 67(2) Part A:97-106 (2015).

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

[0055] The recombinant protein can be a monomeric form or a multimeric, oligomeric or multimeric antibody protein product. In certain embodiments where the antibody comprises more than one individual antigen-binding region fragment, the antibody is considered bispecific, trispecific or multispecific or bivalent, trivalent or multivalent depending on the number of individual epitopes recognized and bound by the antibody.

[0056] With respect to the methods of the present invention, the antibody protein product can lack certain portions of the antibody. However, generally, the fragment comprises at least a portion of the Fc region of the antibody that is glycosylated by post-translational modification in eukaryotic cells.

[0057] Advantageously, the method is not limited to the antigen specificity of the antibody. Thus, the antibody has some binding specificity for substantially any antigen. In representative embodiments, the antibody binds to a hormone, growth factor, cytokine, cell surface receptor or some ligand thereof. In representative embodiments, the antibody binds to a protein expressed on the cell surface of an immune cell. In representative embodiments, the antibody is CD1a, CD1b, CD1c, CD1d, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11A, CD11B, CD11C, CDw12, CD13, CD14, CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD79α, CD79β, CD80, CD81, CD82, CD83, CDw84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDw108, CD109, CD114, CDIt binds to a surface antigen classification molecule selected from the group consisting of 115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CDw121b, CD122, CD123, CD124, CD125, CD126, CD127, CDw128, CD129, CD130, CDw131, CD132, CD134, CD135, CDw136, CDw137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CD145, CD146, CD147, CD148, CD150, CD151, CD152, CD153, CD154, CD155, CD156, CD157, CD158a, CD158b, CD161, CD162, CD163, CD164, CD165, CD166 and CD182.

[0058] In representative embodiments, the antibodies are described in U.S. Patent No. 7,947,809 and U.S. Patent Application Publication No. 2009 / 0041784 (glucagon receptor), U.S. Patent No. 7,939,070, U.S. Patent No. 7,833,527, U.S. Patent No. 7,767,206 and U.S. Patent No. 7,786,284 (IL-17 receptor A), U.S. Patent No. 7,872,106 and U.S. Patent No. 7,592,429 (sclerostin), U.S. Patent No. 7,871,611, U.S. Patent No. 7,815,907, U.S. Patent No. 7,037,498, U.S. Patent No. 7,700,742 and U.S. Patent Application Publication No. 2010 / 0255538 (IGF-1 receptor), U.S. Patent No. 7,868,140 (B7RP1), U.S. Patent No. 7,807,159 and U.S. Patent Application Publication No. 2011 / 0091455 (myostatin), U.S. Patent No. 7,736,644, U.S. Patent No. 7,628,986, U.S. Patent No. 7,524,496 and U.S. Patent Application Publication No. 2010 / 0111979 (deletion mutants of epidermal growth factor receptor), U.S. Patent No. 7,728,110 (SARS coronavirus), U.S. Patent No. 7,718,776 and U.S. Patent Application Publication No. 2010 / 0209435 (OPGL), U.S. Patent No. 7,658,924 and U.S. Patent No. 7,521,053 (angiopoietin-2), U.S. Patent No. 7,601,818, U.S. Patent No. 7,795,413, U.S. Patent Application Publication No. 2009 / 0155274, U.S. Patent Application Publication No. 2011 / 0040076 (NGF), U.S. Patent No. 7,579,186 (TGF-β type II receptor), U.S. Patent No. 7,541,438 (connective tissue growth factor), U.S. Patent No. 7,438,910 (IL1-R1), U.S. Patent No. 7,423,128 (propargine), U.S. Patent No. 7,411,057, U.S. Patent No. 7,824,679, U.S. Patent No. 7,109,003, U.S. Patent No. 6,682,736, U.S. Patent No. 7,132,281 and U.S. Patent No. 7,807,797 (CTLA-4), U.S. Patent No. 7,084,257, U.S. Patent No. 7,790,859, U.S. Patent No. 7,335,743,U.S. Patent No. 7,084,257 and U.S. Patent Application Publication No. 2011 / 0045537 (Interferon-gamma), U.S. Patent No. 7,932,372 (MAdCAM), U.S. Patent No. 7,906,625, U.S. Patent Application Publication No. 2008 / 0292639 and U.S. Patent Application Publication No. 2011 / 0044986 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U.S. Patent No. 7,887,799 and U.S. Patent No. 7,879,323 (Fibroblast Growth Factor-like Polypeptide), U.S. Patent No. 7,867,494 (IgE), U.S. Patent Application Publication No. 2010 / 0254975 (Alpha-4 Beta-7), U.S. Patent Application Publication No. 2010 / 0197005 and U.S. Patent No. 7,537,762 (Activin Receptor-like Kinase 1), U.S. Patent No. 7,585,500 and U.S. Patent Application Publication No. 2010 / 0047253 (IL-13),U.S. Patent Application Publication No. 20090263383 and U.S. Patent No. 7449555 (CD148), U.S. Patent Application Publication No. 20090234106 (Activin A), U.S. Patent Application Publication No. 20090226447 (Angiopoietin-1 and Angiopoietin-2), U.S. Patent Application Publication No. 20090191212 (Angiopoietin-2), U.S. Patent Application Publication No. 20090155164 (C-FMS), U.S. Patent No. 7537762 (Activin Receptor-Like Kinase-1), U.S. Patent No. 7371381 (Galanin), U.S. Patent Application Publication No. 20070196376 (Insulin-Like Growth Factor), U.S. Patent No. 7267960 and U.S. 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hormone), U.S. Patent No. 7285269 (TNF), U.S. Patent No. 6692740 and U.S. Patent No. 7270817 (ACPL), U.S. Patent No. 7202343 (Monocyte chemoattractant protein-1), U.S. Patent No. 7144731 (SCF), U.S. Patent No. 6355779 and U.S. Patent No. 7138500 (4-1BB), U.S. Patent No. 7135174 (PDGFD), U.S. Patent No. 6630143 and U.S. Patent No. 7045128 (Flt-3 ligand), U.S. Patent No. 6849450 (Metalloproteinase inhibitor), U.S. Patent No. 6596852 (LERK-5), U.S. Patent No. 6232447 (LERK-6), U.S. Patent No. 6500429 (Brain-derived neurotrophic factor), U.S. Patent No. 6184359 (Epithelial-derived T cell factor), U.S. Patent No. 6143874 (Neurotrophic factor NNT-1), U.S. Patent Application Publication No. 20110027287 (Proprotein convertase subtilisin / kexin type 9 (PCSK9)),It is one of those described in US Patent Application Publication No. 20110014201 (IL-18 receptor) and US Patent Application 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 individual targets of variable domain-containing antigen-binding proteins or antibodies.,

[0059] In representative embodiments, the antibodies are muromonab - CD3 (product marketed under the trade name Orthoclone Okt3®), abciximab (product marketed under the trade name Reopro®), rituximab (products marketed under the trade names 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®), trastuzumab (product marketed under the trade name Herceptin®), alemtuzumab (products marketed under the trade names 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 (product marketed under the trade name Zevalin®), omalizumab (product marketed under the trade name Xolair®), bevacizumab (product marketed under the trade name Avastin®), natalizumab (product marketed under the trade name Tysabri®), ranibizumab (product marketed under the trade name Lucentis®), panitumumab (product marketed under the trade name Vectibix®), eculizumab (product marketed under the trade name Soliris®), certolizumab pegol (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 (products marketed under the trade names RoActemra®, Actemra®), ofatumumab (product marketed under the trade name Arzerra®),It is one of denosumab (product marketed under the trade name Prolia®), belimumab (product marketed under the trade name Benlysta®), lirilumab, ipilimumab (product marketed under the trade name Yervoy®), and pertuzumab (product marketed under the trade name Perjeta®). In representative embodiments, the antibody is an anti-TNF alpha antibody such as adalimumab, infliximab, etanercept, golimumab, and certolizumab pegol; an anti-IL1-beta antibody such as canakinumab; an anti-IL12 / 23 (p40) antibody such as ustekinumab and briakinumab; and an anti-IL2R antibody 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 brentuximab vedotin; anti-CD33 antibodies such as gemtuzumab ozogamicin; 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 glycosylation-competent cells. In a representative embodiment, the glycosylation-competent cells are eukaryotic cells including, but not limited to, yeast cells, filamentous fungal cells, protist cells, algal cells, insect cells, or mammalian cells. Such host cells are described in the art. See, for example, Frenzel, et al., Front Immunol 4:217 (2013). In a representative embodiment, the eukaryotic cells are mammalian cells. In a representative embodiment, the mammalian cells are non-human mammalian cells. In some embodiments, the cells are Chinese hamster ovary (CHO) cells and derivatives thereof (e.g., CHO-K1, CHO pro-3), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), cells engineered to lack dihydrofolate reductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or derivatives thereof (e.g., HEK293T, HEK293-EBNA), African green monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human 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 NIH 3T3, mouse fibroblast L929, mouse neuroblastoma cells N2a, human breast cancer cells MCF-7, retinoblastoma cells Y79, human retinoblastoma cells SO-Rb50, human liver cancer cells Hep G2, mouse B myeloma cells 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 cells are eukaryotic cells. In representative embodiments, the eukaryotic cells are mammalian cells. In some embodiments, the mammalian cells are non-human mammalian cells. In representative embodiments, the non-human mammalian cells are 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 neonatal hamster kidney (BHK) cells. Cells that are not glycosylation-competent can also be transformed into glycosylation-competent cells, for example, by transfecting them with genes encoding relevant enzymes required for glycosylation. Representative enzymes include, but are not limited to, oligosaccharyltransferase, glycosidase, glucosidase I, glucosidease II, calnexin / calreticulin, glycosyltransferase, mannosidase, GlcNAc transferase, galactosyltransferase, and sialyltransferase.

[0062] Method for preparing a composition The present invention also provides a method for preparing a composition comprising the TAF glycoform of a protein produced by cells in cell culture. In a representative embodiment, the method comprises (i) maintaining cell culture at an initial pH over an initial cell culture period as described herein and optionally (ii) expanding the cell culture and (iii) recovering the supernatant of the cell culture comprising the protein produced by the cells. In a representative aspect, the method may comprise any one of the steps described herein with respect to the method of the present invention for adjusting (increasing or decreasing) the level of the TAF glycoform of a protein produced by cells in cell culture.

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

[0064] In a representative embodiment, the method further comprises steps for formulating the purified protein and the like, 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 steps for generating a host cell that expresses the protein. For example, in some instances, the method includes introducing into a host cell a vector comprising a nucleic acid comprising a nucleotide sequence encoding the protein.

[0066] Composition Compositions are provided herein that include the TAF glycoforms of the protein. In a representative embodiment, the composition is prepared by the method of the invention for preparing a composition that includes the TAF glycoforms of the protein produced by a cell in cell culture as described herein. In a representative aspect, at least about 10% of the protein in the composition is the TAF glycoform. In a representative aspect, at least about 20% of the protein in the composition is the TAF glycoform. In a representative aspect, at least about 30% of the protein in the composition is the TAF glycoform. In a representative aspect, at least about 40% of the protein in the composition is the TAF glycoform. In a representative aspect, at least about 50% of the protein in the composition is the TAF glycoform. In a representative aspect, at least about 60% of the protein in the composition is the TAF glycoform. In a representative aspect, at least about 70% of the protein in the composition is the TAF glycoform. In a representative aspect, at least about 80% of the protein in the composition is the TAF glycoform. In a representative aspect, at least about 90% of the protein in the composition is the TAF glycoform. In a representative aspect, more than about 90% or more than about 95% of the protein in the composition is the TAF glycoform.

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

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

[0069] This pharmaceutical composition may contain any pharmaceutically acceptable components, such as acidifying agents, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anticoagulants, anticoagulants, antibacterial preservatives, antioxidants, disinfectants, bases, binders, buffers, chelating agents, coating agents, coloring agents, desiccants, surfactants, diluents, disinfectants, disintegrants, dispersants, solubilizing agents, pigments, emollients, emulsifiers, emulsion stabilizers, injectables, film-forming agents, seasonings, flavoring agents, flow promoters, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oily vehicles, organic bases, troche bases, pigments, plasticizers, brighteners, preservatives, sequestering agents, skin penetration enhancers, solubilizers, solvents, stabilizers, suppository bases, surfactants, surfactants, suspending agents, sweeteners, therapeutic agents, thickeners, isotonic agents, toxic agents, viscosity increasing agents, water absorbents, water miscible co-solvents, emollients or wetting agents. For example, refer to Handbook of Pharmaceutical Excipients, Third Edition, A.H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated herein by reference in its entirety. Refer to Remington’s Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated herein by reference in its entirety.

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

[0071] In certain embodiments, the pharmaceutical composition may contain formulation materials to change, maintain, or preserve, for example, the pH, osmotic pressure, viscosity, transparency, 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); antibacterial 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); fillers (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); injectants; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrin); proteins (such as serum albumin, gelatin or immunoglobulins); coloring agents, flavoring agents and diluents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium 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 (such as polysorbates such as pluronics, PEG, sorbitan esters, polysorbate 20, polysorbatc, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability promoters (such as sucrose or sorbitol); isotonicity promoters (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants, but are not limited thereto.See REMINGTON’S PHARMACEUTICAL SCIENCES, 18th Edition, (A.R. Genrmo, ed.), 1990, Mack Publishing Company.

[0072] The pharmaceutical composition can be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the pharmaceutical composition can 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 - 7.5.

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

Example

[0074] Example 1 This example demonstrates that the initial cell culture pH regulates the levels of specific glycoforms of recombinant glycosylated proteins produced by cells in cell culture, and that changes in pH made after the initial cell culture period have little to no effect on glycoform levels.

[0075] Materials and Methods Cell Lines, Cell Culture, and Media Cells of a CHO cell line expressing a recombinant antibody of isotype IgG1 were maintained in a 3 L Erlenmeyer shake flask (Corning Life Sciences, Lowell, MA) with a working volume of 1 L. The cells were cultured under standard humidified conditions of 36 °C and 5% CO2 and shaken at 70 rpm in an automated CO2 incubator (Thermo Fisher Scientific, Waltham, MA). Every three days, the whole cells were subcultured, transferred, inoculated, and cultured in a culture medium containing various concentrations of methotrexate (MTX) for 4 days and then inoculated into a bioreactor. A cell culture production medium was used as the control medium in this test. The components of the 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 under standard temperature and dissolved oxygen conditions by a batch process or a perfusion process.

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

[0078] Hydrophilic interaction liquid chromatography (HILIC) glycan map The glycan map of enzymatically released N-linked glycans was determined using HILIC. Briefly, the glycans were incubated with a solution containing PNGase F and sodium phosphate buffer (pH 7.5) at ~37 °C for ~2 h. Next, a labeling solution containing 2-aminobenzoic acid (2-AA) and sodium cyanoborohydride was added to the glycans treated with PNGase F, and the mixture was incubated at ~80 °C for approximately 75 min. After incubation, the mixture was centrifuged to obtain a pellet of precipitated protein. The supernatant was collected and placed in a vial.

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

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

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

[0082]

Table 1

[0083] From 4 days to several days after cell culture, TAF levels, high-mannose (HM) glycan levels, and afucosylated (AF) glycan levels were measured, and the observed results and the predicted model results are shown in Table 2.

[0084]

Table 2

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

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

[0087] The effect of the final pH (pH after the initial cell culture period) was evaluated. Two groups of cells were cultured for 12 days. For both groups, the cells were cultured at a pH of 6.95 for the first 5 days of cell culture. For the next several days, group 1 was maintained at this pH, while the pH of group 2 was shifted to 6.85 from day 5 to day 9 and then shifted to 7.1 from day 9 to day 12. As shown in Figure 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 level of TAF.

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

[0090] Cell culture in a bioreactor by a perfusion process was basically carried out 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 was in the range of 6.65 to 6.9. The temperature was shifted to a temperature of 34 to 36 °C after the 5th day (from the 5th day to the 8th day). Table 3 outlines the various experimental conditions for this test.

[0091] [Table 3]

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

[0093] Example 3 This example shows that there is an effect of the initial pH during the initial cell culture period on the TAF level and an effect of the pH shift after the initial cell culture period on the TAF level.

[0094] Basically as described in Example 1, after the perfusion step, the cells expressing the IgG1 antibody were cultured in the bioreactor. 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 initial set 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). The cells in group A were cultured at an initial set pH value of 7.05, the cells in group B were cultured at an initial set pH value of 6.85, while the cells in group C were cultured at an initial set pH value of 6.95. The cells in group A were divided into two subgroups: subgroup A1 and subgroup A2. The cells in subgroup A1 were cultured at an initial set pH value of 7.05 and the pH was shifted to 6.95 during the initial cell culture period (day 1), while the cells in subgroup A2 were cultured at an initial set pH value of 7.05 and the pH was shifted to 6.95 after the initial cell culture period (day 5). For the cells in group B, the pH was increased to 6.95 after the initial cell culture period (day 5), and for the cells in 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 at 6.95 and maintained throughout the culture period (i.e., no pH shift).

[0095] After the 12-day culture period, the TAF glycoforms of the IgG1 antibody produced by each of the groups were measured via HILIC, basically as described in Example 1. The %TAF of the antibody produced by the control culture was in the range of about 3.1% to about 4.14%. The average %TAF of the antibody produced by group A was higher compared to the antibody produced by the control culture, while the average %TAF of the antibody produced by group B was lower. The higher initial pH correlated with a higher %TAF, and the lower initial pH correlated with a lower %TAF.

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

[0097] This example revealed that a pH shift occurring after the initial cell culture period did not significantly affect the TAF level, that a higher set pH value tended towards a higher TAF level, while a lower set pH value tended towards a lower TAF level.

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

[0099] The use of the terms “a,” “an,” and “the” and similar references in the description of the present disclosure (especially in the context of the following claims) should be construed to include both the singular and the plural unless otherwise specified herein or clearly contradicted by the context. The terms “comprising,” “having,” “including,” and “containing” should be construed as open-ended terms (i.e., meaning “including but not limited to”) unless otherwise noted.

[0100] The recitation of a range of values herein is merely a shorthand way of referring individually to each separate value that falls within the range and each endpoint, and each separate value and endpoint is incorporated herein as if it were individually recited herein.

[0101] All of the methods described herein can be performed in any suitable order, unless otherwise indicated herein or otherwise clearly contradicted by the content. The use of any examples or exemplary language provided herein (e.g., "such as") is merely to clarify the disclosure in more detail and does not raise a limitation in the scope of the disclosure, unless otherwise claimed. Nothing in the language of this application should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0102] Preferred embodiments of the disclosure are described herein, including the best mode known to the inventors for practicing the disclosure. Variations of these preferred embodiments will be apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to use such variations as appropriate, and the inventors intend for the disclosure to be practiced beyond specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter shown in the claims appended hereto, as permitted by applicable law. Further, unless otherwise indicated herein or otherwise clearly contradicted by the content, any combination of the above elements in all possible variations is included by the disclosure.

Claims

1. A method for adjusting the level of total nonfucosylated (TAF) glycoforms of an IgG1 or IgG4 antibody composition produced by glycosylation-competent Chinese hamster ovary (CHO) cells in perfusion cell culture, comprising: (i) maintaining the perfusion cell culture at an initial pH value and initial temperature for an initial cell culture period of 4 to 6 days; (ii) shifting the temperature by more than 2°C after said initial cell culture period; and (iii) culturing the glycosylation-competent CHO cells for at least 10 days, and then harvesting the cell culture supernatant containing the IgG1 or IgG4 antibody composition; the initial pH value is selected from a pH greater than 6.50 and less than 7.5, and the initial temperature is between 30°C and 40°C; The method, wherein the temperature of the cell culture does not shift by more than 1° C. during the initial cell culture period.

2. A method for adjusting the level of total nonfucosylated (TAF) glycoforms of an IgG1 or IgG4 antibody composition produced by glycosylation-competent Chinese hamster ovary (CHO) cells in perfusion cell culture, comprising: (i) maintaining the perfusion cell culture at a preset pH value until the cell culture reaches a viable cell density (VCD) of 8.2×10 6 cells / mL; while maintaining the cell culture at the initial pH value, the temperature of the cell culture does not shift by more than 1° C. from the initial temperature; maintaining the initial pH value greater than 6.50 and less than 7.5, and the initial temperature between 30°C and 40°C; (ii) shifting the temperature by more than 2°C after the cell culture reaches a viable cell density (VCD) of 8.2 x 106 cells / mL; and (iii) culturing the glycosylation-competent CHO cells for at least 10 days, after which time the cell culture supernatant comprising the IgG1 or IgG4 antibody composition is harvested.

3. The method of claim 1, wherein the pH does not shift by more than 0.05 (i) during the initial cell culture period of 4 to 6 days, or (ii) before the cell culture reaches a viable cell density (VCD) of 8.2 x 10 cells / mL.

4. The method described in claim 1 or 2, wherein the initial pH value is less than 7.

2.

5. The method described in claim 4, wherein the initial pH value is less than 7.

1.

6. The method described in claim 5, wherein the initial pH value is less than 7.

0.

7. The method described in claim 1 or 2, wherein the initial temperature is about 36°C or about 37°C.

8. A method as described in claim 1 or 2, comprising lowering the temperature by 3°C, 4°C, 5°C or 6°C.

9. The method described in claim 1, wherein the initial cell culture period is 4 days.

10. The method described in claim 1, wherein the initial cell culture period is 5 days.

11. The method described in claim 1, wherein the initial cell culture period is 6 days.

12. The method described in claim 1 or 2, wherein the level of TAF glycoforms in the supernatant IgG1 or IgG4 antibody composition is less than 10%.

13. The method described in claim 12, wherein the level of TAF glycoforms in the supernatant IgG1 or IgG4 antibody composition is 2.1% to 3.9% or 2.8% to 7.5%.

14. The method described in claim 1 or 2, wherein the level of high mannose (HM) glycoforms in the supernatant IgG1 or IgG4 antibody composition is 1.4% to 2.6% or 1.8% to 2.8%.

15. The method described in claim 1 or 2, wherein the level of nonfucosylated (AF) glycoforms in the supernatant IgG1 or IgG4 antibody composition is 0.7% to 1.6% or 1.5% to 2.3%.

16. The method of claim 1 or 2, wherein (i) the cell culture medium comprises glucose, glutamine and / or glutamate, (ii) the osmolality of the cell culture is from about 200 mOsm / kg to about 500 mOsm / kg, and / or (iii) the dissolved oxygen (DO) level of the cell culture is from about 50 mmHg to about 100 mmHg or from about 20% to about 60% oxygen saturation.

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