Methods and compositions for modulating the glycosylation profile of proteins

Abstract

The present invention relates to cell culture media containing 5-thio-L-fucose and the use of 5-thio-L-fucose to modulate the glycosylation profile of antibodies or other Fc-containing proteins and thereby modulate the binding affinity of the proteins.

Description

[Technical Field]

[0001] The present invention relates to cell culture media containing 5-thio-L-fucose and the use of 5-thio-L-fucose to modulate the glycosylation profile of antibodies or other Fc-containing proteins, and thereby modulate the binding affinity of the proteins.

[0002] Recombinant proteins such as therapeutic antibodies and Fc-fusion proteins are commonly produced in mammalian cell lines. Monoclonal antibodies and Fc-fusion proteins produced in mammalian host cells can have a variety of post-translational modifications, including glycosylation. Monoclonal antibodies, such as IgG, typically have an N-linked glycosylation site at asparagine 297 (Asn297) of each heavy chain (two per intact antibody). The glycan attached to Asn297 on antibodies is typically a complex biantennary structure with very little or no bisecting N-acetylglucosamine (bisecting GIcNAc) along with low amounts of terminal sialic acid and variable amounts of galactose. The glycans also often have high levels of core fucosylation. Reduction of core fucosylation in antibodies has been shown to alter Fc effector functions, particularly Fc gamma receptor binding and ADCC activity. This observation has led to interest in engineering cell lines that produce antibodies with reduced core fucosylation.

[0003] The secret to some antibody-based immunotherapies is the binding of the antibody Fc portion to cell surface receptors called Fcγ receptors (FcγRs). Depending on the cytoplasmic domain, FcγRs trigger various immune responses. Common features include immunoreceptor tyrosine-based activation motifs (ITAMs) for activating receptors (FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) and immunoreceptor tyrosine-based inhibitory motifs (ITIMs) for inhibitory receptors such as FcγRIIb [Lu J and Sun PD, Structural mechanism of high-affinity FcgammaRI recognition of immunoglobulin G. Immunol Rev 2015, 268, 192-200]. Furthermore, receptors can be classified into high-affinity FcγRI (CD64) and low-affinity FcγRII (CD32) and FcγRIII (CD16). Bruhns et al. described binding to all known antibody subclasses of various receptor classes. IgG1 has been reported to bind with different affinities to all classes of receptors. Increasing affinity of IgG1 was observed for FcγRIIb, FcγRIIIb, FcγRIIIa, and FcγRIIa, with the highest affinity for FcγRI [Bruhns P, Iannascoli B, England P, Mancardi DA, Fernandez N, Jorieux S, and Daeron M, Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses. Blood 2009, 113, 3716-25].

[0004] The two major Fc effector functions are antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Natural killer (NK) cells expressing FcγRIIIa are responsible for ADCC activity. In general, receptor affinity and, therefore, antibody biological activity depend on post-translational modifications such as antibody glycosylation [Harris RJ, Chin ET, Macchi F, Keck RG, Shyong BJ, Ling VT, Cordoba AJ, Marian M, Sinclair D, Battersby JE, and Jones AJS, Analytical Characterization of Monoclonal Antibodies: Linking Structure to Function. In Current Trends in Monoclonal Antibody Development and Manufacturing, ed.; Shire SJ, et al.; Springer New York: New York, NY, 2010, 193-205].

[0005] Antibodies display distinct N-linked glycosylation at position 297 (Asn297) on each heavy chain in the Fc region. The presence of core fucosylation on the Fc region of recombinant proteins is known to reduce ADCC responses due to steric hindrance of fucose [Mizushima T, Yagi H, Takemoto E, Shibata-Koyama M, Isoda Y, Iida S, Masuda K, Satoh M, and Kato K, Structural basis for improved efficacy of therapeutic antibodies on defucosylation of their Fc glycans. Genes to cells 2011, 16, 1071-1080]. Therefore, reducing core fucosylation leads to enhanced ADCC. This was validated by in vivo studies of afucosylated anti-EGFR and anti-CS1 antibodies, which showed enhanced ADCC and antitumor activity compared to their fucosylated counterparts [Gomathinayagam S, Laface D, Houston-Cummings NR, Mangadu R, Moore R, Shandil I, Sharkey N, Li H, Stadheim TA, and Zha D, In vivo antitumor efficacy of afucosylated anti-CS1 monoclonal antibody produced in glycoengineered Pichia pastoris. Journal of Biotechnology 2015, 208, 13-21]. In conclusion, modulation of Fc glycosylation is a strategy for enhancing the biological activity of therapeutic antibodies.

[0006] Methods for reducing fucosylation include methods for modifying cell lines. An alternative to modifying cell lines involves the use of small molecule inhibitors of enzymes in the glycosylation pathway. Another option is to provide small molecule fucose analogs for use in producing recombinant antibodies with complex N-linked glycans but reduced fucosylation. This is suggested, for example, in WO09135181. However, there is still room for improvement in the modulation of glycosylation patterns, and thus the biological activity, of therapeutic proteins.

[0007] It has been discovered that the addition of 5-thio-L-fucose and / or certain derivatives thereof to cell culture media and feeds results in reduced fucosylation of antibodies / Fc-containing proteins. The resulting proteins exhibit enhanced FcγRIIIa binding, which correlates with increased ADCC. Surprisingly, the use of 5-thio-L-fucose and / or certain derivatives thereof led to high incorporation of the fucose analog.

[0008] The present invention therefore provides a method for making an antibody or other Fc-containing protein with reduced fucosylation, comprising: The method includes culturing host cells in a culture medium containing 5-thio-L-fucose and / or a 5-thio-L-fucose derivative selected from partially or fully acetylated 5-thio-L-fucose, non-, partially or fully acetylated 2-F-5-thio-L-fucose, 5-alkynyl-5-thio-L-fucose, 6,6,6-trifluoro-5-thio-L-fucose, and 5-thio-L-fucose phosphonate, wherein the host cells express an antibody or other Fc-containing protein having an Fc domain with at least one N-glycoside-linked sugar chain attached to the Fc domain through an N-acetylglucosamine at the reducing end of the sugar chain. and isolating an antibody or other Fc-containing protein from the cell, wherein the antibody or other Fc-containing protein has reduced fucosylation on the glycosylation chain compared to an antibody or other Fc-containing protein from the same host cell cultured under the same culture conditions but in the absence of 5-thio-L-fucose and / or a derivative thereof, the derivative being selected from partially or fully acetylated 5-thio-L-fucose, non-, partially or fully acetylated forms of 2-F-5-thio-L-fucose, 5-alkynyl-5-thio-L-fucose, 6,6,6-trifluoro-5-thio-L-fucose and 5-thio-L-fucose phosphonate; The present invention is directed to a method, including:

[0009] In a preferred embodiment, the host cells are cultured in a cell culture medium containing 5-thio-L-fucose and / or partially or fully acetylated 5-thio-L-fucose. In a preferred embodiment, the host cell is a Chinese hamster ovary cell. In another preferred embodiment, the host cells are cultured in fed-batch cell culture or perfusion cell culture.

[0010] In a preferred embodiment, the host cell - loading the bioreactor with host cells and aqueous cell culture medium; - Incubating the cells in the bioreactor - Adding cell culture medium to the bioreactor continuously throughout the incubation of the cells in the bioreactor or once or several times during said incubation period, wherein the cell culture medium contains 5-thio-L-fucose; It is cultivated by Preferably, the medium to be added has a pH of less than pH 8.5 and contains 5-thio-L-fucose at a concentration between 10 nmol / l and 100 mmol / l, preferably between 0.1 μmol / l and 10 mmol / l.

[0011] The present invention further provides a method for producing a thiofucosylated antibody or other Fc-containing protein, comprising: culturing host cells in a culture medium containing 5-thio-L-fucose and / or a 5-thio-L-fucose derivative selected from partially or fully acetylated 5-thio-L-fucose, non-, partially or fully acetylated 2-F-5-thio-L-fucose, 5-alkynyl-5-thio-L-fucose, 6,6,6-trifluoro-5-thio-L-fucose and 5-thio-L-fucose phosphonate, wherein the host cells express an antibody or other Fc-containing protein having an Fc domain with at least one N-glycoside-linked sugar chain attached to the Fc domain through an N-acetylglucosamine at the reducing end of the sugar chain; and isolating the antibody or other Fc-containing protein from the cells; The present invention is directed to a method comprising:

[0012] The present invention is further directed to a dry powder cell culture medium comprising 5-thio-L-fucose and / or a 5-thio-L-fucose derivative selected from partially or fully acetylated 5-thio-L-fucose, non-, partially or fully acetylated forms of 2-F-5-thio-L-fucose, 5-alkynyl-5-thio-L-fucose, 6,6,6-trifluoro-5-thio-L-fucose, and 5-thio-L-fucose phosphonate. In one embodiment, the cell culture medium is a feed medium.

[0013] In another embodiment, the cell culture medium comprises 5-thio-L-fucose and / or a derivative thereof in an amount such that, when dissolved to provide a liquid medium, the concentration of 5-thio-L-fucose and / or a derivative thereof in the liquid medium is between 10 nmol / l and 100 mmol / l, preferably between 0.1 μmol / l and 10 mmol / l. Liquid media, such as feed or perfusion media, are typically designed so that the final concentration of 5-thio-L-fucose and / or its derivatives in the cell culture is between 0.1 and 1 mmol / l. In one embodiment, the cell culture medium comprises at least one or more sugar components, one or more amino acids, one or more vitamins or vitamin precursors, one or more salts, one or more buffer components, one or more cofactors, and one or more nucleic acid components.

[0014] The present invention further provides a method for producing a cell culture medium according to the present invention, comprising the steps of: a) mixing 5-thio-L-fucose and / or a 5-thio-L-fucose derivative selected from partially or fully acetylated 5-thio-L-fucose, non-, partially or fully acetylated 2-F-5-thio-L-fucose, 5-alkynyl-5-thio-L-fucose, 6,6,6-trifluoro-5-thio-L-fucose and 5-thio-L-fucose phosphonate with other components of a cell culture medium; b) subjecting the mixture of step a) to milling; It is directed towards methods using In a preferred embodiment, step b) is carried out in a pin mill, a Fitz mill or a jet mill. In another preferred embodiment, the mixture from step a) is cooled to a temperature below 0° C. before milling. [Brief explanation of the drawings]

[0015] [Figure 1]FIG. 1 shows the structural similarities between acetylated and non-acetylated L-fucose and 5-thio-L-fucose. [Figure 2] Figures 2-5 show the effect of 5-thio-L-fucose on cell performance using four different CHO cell lines producing three different IgG1s (A, B, and C) and a fusion protein (D). Viable cell density (VCD) is visualized on the left, and titer is visualized on the right. The control feed contained no additives, and the DMSO control contained 1.17% DMSO, the same concentration used as the solvent for both peracetylated fucose analogs. For further details, see Example 1. [Figure 3] Figures 2-5 show the effect of 5-thio-L-fucose on cell performance using four different CHO cell lines producing three different IgG1s (A, B, and C) and a fusion protein (D). Viable cell density (VCD) is visualized on the left, and titer is visualized on the right. The control feed contained no additives, and the DMSO control contained 1.17% DMSO, the same concentration used as the solvent for both peracetylated fucose analogs. For further details, see Example 1.

[0016] [Figure 4] Figures 2-5 show the effect of 5-thio-L-fucose on cell performance using four different CHO cell lines producing three different IgG1s (A, B, and C) and a fusion protein (D). Viable cell density (VCD) is visualized on the left, and titer is visualized on the right. The control feed contained no additives, and the DMSO control contained 1.17% DMSO, the same concentration used as the solvent for both peracetylated fucose analogs. For further details, see Example 1. [Figure 5]Figures 2-5 show the effect of 5-thio-L-fucose on cell performance using four different CHO cell lines producing three different IgG1s (A, B, and C) and a fusion protein (D). Viable cell density (VCD) is visualized on the left, and titer is visualized on the right. The control feed contained no additives, and the DMSO control contained 1.17% DMSO, the same concentration used as the solvent for both peracetylated fucose analogs. For further details, see Example 1.

[0017] [Figure 6] Figure 6 shows the sugar structures (A fucosylated GOF, B thiofucosylated GOthioF and C G1 fragmentation pattern). Details for Figure 6 can be found in Example 2. [Figure 7] Figure 7 shows the fluorescence signal obtained after separation of the released, labeled glycans using UPLC, showing the altered retention times of the peaks corresponding to thiofucosylated glycans compared to fucosylated glycans. Details about Figure 7 can be found in Example 2. [Figure 8] Figure 8 shows the effect of 5-thio-L-fucose on fucosylation profiles using CHO cell lines producing (A) mAb1, (B) mAb2, (C) mAb3, and (D) fusion proteins. Details can be found in Example 3.

[0018] [Figure 9] Figure 9 shows the accelerated effect of 5-thio-L-fucose during fed-batch experiments producing (A) mAb1 or (B) mAb3. Details can be found in Example 4. [Figure 10] Figure 10 shows the dose response of 5-thio-L-fucose on glycosylation profiles. Further details can be found in Example 5. [Figure 11] Figure 11 shows overlaid plots of FcγRIIIa binding to mAb3 control (black) or 5-thio-L-fucosylated mAb3 (gray) on day 12. Further details can be found in Example 6.

[0019] Cell culture media support and maintain the growth of cells in an artificial environment. Depending on the type of organism whose growth will be supported, cell culture media contain a complex mixture of components, sometimes more than 100 different components. Cell culture media required for the growth of mammalian, insect, or plant cells are typically much more complex than media that support the growth of bacteria and yeast. Chemically defined media often include, but are not limited to, amino acids, vitamins, metal salts, antioxidants, chelators, growth factors, buffers, hormones, and many more substances known to those skilled in the art.

[0020] A cell culture medium according to the present invention is any mixture of components that maintain and / or support cell growth in vitro. It may be a complex medium or a chemically defined medium. A cell culture medium may contain all components necessary to maintain and / or support cell growth in vitro, or only some components so that additional components are added separately. An example of a cell culture medium according to the present invention is a complete medium, which contains all components necessary to maintain and / or support cell growth in vitro, as well as medium additives or feeds. In preferred embodiments, the cell culture medium is a complete medium, a perfusion medium, or a feed-batch medium. A complete medium, also called a basal medium, typically has a pH between 6.8 and 7.8. A feed-batch medium preferably has a pH below 8.5, preferably between 6.5 and 8.5. The cell culture medium may be a liquid medium or a dry powder medium.

[0021] Typically, the cell culture medium according to the present invention is used to maintain and / or support the growth of cells in a bioreactor. A feed or feed medium is a cell culture medium that is not a basal medium that initially supports growth and production in a cell culture, but is added to the medium at a later stage to prevent nutrient depletion and sustain the production phase. Compared to the basal culture medium, the feed medium may have higher concentrations of some components. For example, some components, such as nutrients including amino acids or carbohydrates, may be present in the feed medium at about 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100X, 200X, 400X, 600X, 800X, or even about 1000X the concentration in the basal medium.

[0022] Mammalian cell culture media is a mixture of components that maintain and / or support the growth of mammalian cells in vitro. Examples of mammalian cells are human or animal cells, preferably CHO cells, COS cells, I VERO cells, BHK cells, AK-1 cells, SP2 / 0 cells, L5.1 cells, hybridoma cells or human cells.

[0023] A chemically defined cell culture medium is a cell culture medium that does not contain any chemically undefined substances. This means that the chemical composition of all chemicals used in the medium is known. Chemically defined media do not contain any yeast, animal, or plant tissue; they do not contain feeder cells, serum, extracts or digests, or other components that would contribute to the medium with poorly defined chemical proteins. Chemically undefined or poorly defined components are those whose chemical composition and structure are unknown, exist in various compositions, or can only be defined with extensive experimental effort—comparable to evaluating the chemical composition and structure of proteins such as albumin or casein.

[0024] Powdered cell culture media or dry powder media are typically cell culture media resulting from milling, freeze-drying, or dry or wet granulation processes. Powdered cell culture media refer to granular, particulate media, not liquid media. The term "dry powder" may be used interchangeably with the term "powder." However, as used herein, "dry powder" simply refers to the macroscopic form of the granular material and is not intended to mean that the material is completely free of complexed or aggregated solvents, unless otherwise indicated. Dry powder media result from milling or freeze-drying processes and typically have a particle size of less than 0.5 mm, e.g., between 0.05 and 0.5 mm.

[0025] Dry powder media resulting from dry or wet granulation processes, such as spray drying, wet granulation, or dry compaction, typically have particle sizes greater than 0.5 mm, for example, between 0.5 and 5 mm. Dry compaction is typically performed in a roll press. US 6,383,810 B2 discloses a method for producing an agglomerated eukaryotic cell culture medium powder. The method includes wetting a dry powder cell culture medium with a solvent and then drying the wetted medium again to obtain an agglomerated dry cell culture medium. In one embodiment, the dry powder medium according to the present invention is produced by dry compaction.

[0026] The cells cultured in the medium according to the present invention may be prokaryotic cells, such as bacterial cells, or eukaryotic cells, such as plant or animal cells. Preferably, the cells are mammalian cells. The cells may be normal, immortalized, diseased, transformed, mutant, somatic, germ, stem, progenitor, or embryonic cells, established or transformed cell lines, or those obtained from natural sources. The host cells may be cells into which a gene for expressing an antibody or another Fc-containing protein having an Fc domain has been introduced.

[0027] Particle size refers to the average diameter of the particles. When a particle size is given, it means that at least 80%, preferably at least 90%, of the particles have the given particle size or fall within the given particle size range. Particle diameter is determined by laser light scattering.

[0028] The inert atmosphere is created by filling each container or device with an inert gas. Suitable inert gases are argon or, preferably, noble gases such as nitrogen. These inert gases are non-reactive and prevent undesired chemical reactions from occurring. In the process according to the present invention, creating an inert atmosphere means that the oxygen concentration is reduced to an absolute value of less than 10% (v / v), for example, by introducing liquid nitrogen or nitrogen gas.

[0029] Various types of mills are known to those skilled in the art. Pin mills, also called centrifugal impact mills, pulverize solids by means of protruding pins on a rapidly rotating disc that provide the crushing energy. Pin mills are sold, for example, by Munson Machinery (USA), Premium Pulman (India) or Sturtevant (USA).

[0030] Jet mills use compressed gas to accelerate particles and cause them to collide with each other in a process chamber. Jet mills are sold, for example, by Sturtevant (USA) or PMT (Austria). The Fitzmill, commercialized by Fitzpatrick (USA), uses a rotor with blades for milling.

[0031] A continuously run process is a process that is not run in a batch mode. When a milling process is continuously run, it means that the media components are permanently and steadily fed into the mill over a period of time.

[0032] Cell culture media, particularly complete media according to the present invention, typically include at least one or more sugar components, one or more amino acids, one or more vitamins or vitamin precursors, one or more salts, one or more buffer components, one or more cofactors, and one or more nucleic acid components.

[0033] The medium may also contain surface-active components such as sodium pyruvate, insulin, vegetable proteins, fatty acids and / or fatty acid derivatives and / or pluronic acid and / or chemically prepared non-ionic surfactants. One example of a suitable non-ionic surfactant is a difunctional block copolymer surfactant terminated in primary hydroxyl groups, also called poloxamer, available, for example, under the trade name pluronic® from BASF, Germany.

[0034] The sugar building blocks are all monosaccharides or disaccharides such as glucose, galactose, ribose or fructose (examples of monosaccharides) or sucrose, lactose or maltose (examples of disaccharides).

[0035] Examples of amino acids according to the invention are tyrosine, proteinogenic amino acids, in particular the essential amino acids leucine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine, as well as non-proteinogenic amino acids such as D-amino acids, preferably L-amino acids. Tyrosine means L- or D-tyrosine, preferably L-tyrosine. Cysteine ​​means L- or D-cysteine, preferably L-cysteine.

[0036] Examples of vitamins are vitamin A (retinol, retinal, various retinoids, and four carotenoids), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin, niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxamine, pyridoxal), vitamin B7 (biotin), vitamin B9 (folic acid, folinic acid), vitamin B 12 (cyanocobalamin, hydroxycobalamin, methylcobalamin), vitamin C (ascorbic acid), vitamin D (ergocalciferol, cholecalciferol), vitamin E (tocopherol, tocotrienol) and vitamin K (phylloquinone, menaquinone). Vitamin precursors are also included.

[0037] Examples of salts are inorganic ions such as bicarbonate, calcium, chloride, magnesium, phosphate, potassium, and sodium, or components containing trace elements such as Co, Cu, F, Fe, Mn, Mo, Ni, Se, Si, Ni, Bi, V, and Zn. Examples are copper(II) sulfate pentahydrate (CuSO4·5H2O), sodium chloride (NaCl), calcium chloride (CaCl2·2H2O), potassium chloride (KCl), iron(II) sulfate, anhydrous monobasic sodium phosphate (NaH2PO4), anhydrous magnesium sulfate (MgSO4), anhydrous dibasic sodium phosphate (Na2HPO4), magnesium chloride hexahydrate (MgCl2·6H2O), zinc sulfate heptahydrate.

[0038] Examples of buffers are CO2 / HCO3 (carbonate), phosphate, HEPES, PIPES, ACES, BES, TES, MOPS and TRIS. Examples of cofactors are thiamine derivatives, biotin, vitamin C, NAD / NADP, cobalamin, flavin mononucleotides and derivatives, glutathione, heme nucleotide phosphates and derivatives.

[0039] Nucleic acid building blocks according to the invention are nucleic acid bases such as cytosine, guanine, adenine, thymine or uracil, nucleosides such as cytidine, uridine, adenosine, guanosine and thymidine, and nucleotides such as adenosine monophosphate or adenosine diphosphate or adenosine triphosphate.

[0040] Fed-batch media may have a different composition compared to complete media. They typically contain amino acids, trace elements and vitamins. They may also contain sugar components, although sometimes for production reasons the sugar components are added in a separate feed.

[0041] A suitable feed medium may contain one or more of the following compounds: [Table 1-1] [Table 1-2] Freezing according to the present invention means cooling to a temperature below 0°C.

[0042] The term "antibody" refers to (a) immunoglobulin polypeptides and immunologically active portions of immunoglobulin polypeptides, i.e., polypeptides of the immunoglobulin family that contain an antigen-binding site that immunospecifically binds to a particular antigen (e.g., CD70) and an Fc domain containing complex N-glycoside-linked carbohydrate chain(s), or fragments thereof, or (b) conservatively substituted derivatives of such immunoglobulin polypeptides or fragments that immunospecifically bind to an antigen (e.g., CD70). Antibodies are generally described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988). An Fc-containing protein is any protein that contains an Fc domain or a region that contains complex N-glycoside-linked carbohydrate chains.

[0043] The term "monoclonal antibody" refers to an antibody derived from a single cell clone, including any eukaryotic or prokaryotic cell clone, or a phage clone, and not the method by which it is produced. Thus, the term "monoclonal antibody" is not limited to antibodies produced through hybridoma technology.

[0044] The term "Fc region" refers to the constant region of an antibody, e.g., C H 1-hinge-C H 2-C H 3 domains, optionally C H It refers to a four domain Fc region or a conservatively substituted derivative of such an Fc region. The term "Fc domain" refers to the constant region domain of an antibody, e.g., C H 1. Hinge, C H 2. C H 3 or C H 4 domain, or a conservatively substituted derivative of such an Fc domain. The term "Fab region" refers to the variable region of an antibody that binds to an antigen.

[0045] An "antigen" is a molecule to which an antibody, typically an antibody Fab domain, specifically binds. The terms "specifically bind" and "specifically bind" mean that an antibody or antibody derivative binds to its corresponding target antigen in a highly selective manner and does not bind to a majority of other antigens. Typically, an antibody or other Fc-containing protein binds to at least about 1 x 10 -7 M, and preferably 10 -8 M~10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 It binds with an affinity of M and binds to a predetermined antigen with an affinity that is more than two-fold greater than its affinity for binding to a nonspecific antigen other than the predetermined antigen or a close antigen (e.g., BSA, casein). The term "inhibit" or "inhibition of" means to reduce by a measurable amount or to prevent entirely, particularly to reduce by a measurable amount or to prevent entirely, the activity of a particular enzyme that catalyzes a reaction.

[0046] The term "afucosylation" or "afucosylated" refers to a glycan structure that does not have fucose in the internal core structure. As used herein, "thiofucosylation" or "thiofucosylated" refers to a glycan structure containing at least one thiofucose. Often, in a "thiofucosylated" glycan structure, a fucose is replaced by a thiofucose.

[0047] As used herein, "5-thio-L-fucose" or "thioFuc" refers to a fucose derivative in which an oxygen atom in the sugar ring is replaced with a sulfur atom. "5-thio-L-fucose" refers to the alpha and / or beta anomers. [ka]

[0048] As used herein, "acetylated 5-thio-L-fucose" or "ActhioFuc" refers to any form of acetyl group on the thiofucose, meaning 5-thio-L-fucose bearing one to four acetyl groups. Figure 1, for example, shows peracetylated 5-thio-L-fucose bearing four acetyl groups. Derivatives of 5-thio-L-fucose are 2-F-5-thio-L-fucose in non-, partially or fully acetylated form, 5-alkynyl-5-thio-L-fucose, 6,6,6-trifluoro-5-thio-L-fucose, 5-thio-L-fucose phosphonate.

[0049] 2-F-5-thio-L-fucose in non-, partially or fully acetylated form can be represented by formula I: [ka] where R are, independently of each other, H or acetyl.

[0050] The non-, partially or fully acetylated form of 5-alkynyl-5-thio-L-fucose can be represented by formula II: [ka] where R are, independently of each other, H or acetyl.

[0051] 6,6,6-trifluoro-5-thio-L-fucose in non-, partially or fully acetylated form can be represented by formula III: [ka] where R are, independently of each other, H or acetyl.

[0052] The non-, partially or fully acetylated form of 5-thio-L-fucose phosphonate can be represented by formula IV: [ka] where R are, independently of each other, H or acetyl.

[0053] The image below shows the non-acetylated form: In the acetylated form, one or more OH groups are acetylated. [ka]

[0054] As used herein, "core fucosylation" or "fucosylation" refers to the addition of fucose to N-acetylglucosamine ("GIcNAc") at the reducing end of an N-linked glycan.

[0055] As used herein, an "N-glycoside-linked glycan" or "N-glycoside-linked glycan" is typically linked to asparagine 297 (according to Kabat numbering), although complex N-glycoside-linked glycans can be linked to other asparagine residues. As used herein, a complex N-glycoside-linked glycan has the following structure: [ka] They have predominantly biantennary complex-type glycans with a + / - sign, where + / - indicates whether a sugar molecule may or may not be present, and the number indicates the position of the linkage between the sugar molecules. In the above structure, the sugar chain end attached to asparagine is called the reducing end (on the right), and the opposite end is called the non-reducing end. Fucose is usually attached to N-acetylglucosamine ("GIcNAc") at the reducing end, typically by an α1,6 linkage (the 6-position of GIcNAc is linked to the 1-position of fucose). "Gal" refers to galactose, and "Man" refers to mannose.

[0056] "Complex N-glycosidic glycans" exclude high-mannose glycans in which only mannose is incorporated at the non-reducing end of the core structure, but include the following: 1) a complex type in which the non-reducing end of the core structure has one or more branches of galactose-N-acetylglucosamine (also referred to as "gal-GlcNAc") and the non-reducing end of Gal-GIcNAc optionally has sialic acid, a bisecting N-acetylglucosamine, or the like; or 2) A hybrid type in which the non-reducing end of the core structure has both high-mannose N-glycosidic and complex N-glycosidic branches. In some embodiments, "complex N-glycoside-linked glycans" include complex types in which the non-reducing terminal side of the core structure has zero, one, or more galactose-N-acetylglucosamine (also referred to as "gal-GlcNAc") branches, and the non-reducing terminal side of Gal-GlcNAc optionally further has a structure such as sialic acid, bisecting N-acetylglucosamine, or the like.

[0057] The gist of the present invention is to provide reagents and methods for reducing core fucosylation, i.e., fucosylation of N-glycosidic sugar chains of Fc-containing proteins or antibodies. In one embodiment, fucosylation is inhibited, thereby reducing or completely suppressing. In another embodiment, the N-glycosidic sugar chains of Fc-containing proteins or antibodies are instead thiofucosylated. Both effects can also occur in parallel, either in different antibodies or in a single antibody bearing two different glycan structures.

[0058] Also provided are antibodies produced by such methods. In another aspect, a culture medium is provided containing an effective amount of a 5-thio-L-fucose derivative selected from 5-thio-L-fucose and / or partially or fully acetylated 5-thio-L-fucose, non-, partially, or fully acetylated 2-F-5-thio-L-fucose, 5-alkynyl-5-thio-L-fucose, 6,6,6-trifluoro-5-thio-L-fucose, and 5-thio-L-fucose phosphonate. In the following, the derivative is not always specified. It is clear that when 5-thio-L-fucose is mentioned, it also encompasses the derivatives identified above. Nevertheless, preferred is 5-thio-L-fucose and / or partially or fully acetylated 5-thio-L-fucose.

[0059] In some embodiments, fucosylation of complex N-glycoside-linked sugar chains attached to the Fc region (or domain) is completely suppressed or reduced compared to antibodies or other Fc-containing proteins from the same host cells cultured under the same culture conditions except for the absence of 5-thio-L-fucose. "Under the same culture conditions except for the absence of 5-thio-L-fucose" typically means that the cell culture is carried out under the same conditions containing the same components, but with the difference that 5-thio-L-fucose is not present.

[0060] To suppress or reduce core fucosylation, host cells are cultured in a cell culture medium containing 5-thio-L-fucose and / or its derivatives. In a preferred embodiment, the cell culture medium in which the host cells are cultured does not contain L-fucose.

[0061] The amount of 5-thio-L-fucose included in the liquid cell culture medium in which the host cells are cultured is an amount effective to reduce fucose incorporation. In this context, "effective amount" refers to an amount of 5-thio-L-fucose sufficient to reduce the incorporation of fucose into complex N-glycan-linked sugar chains of an antibody or another Fc-containing protein by at least 10 percent, at least 20 percent, at least 30 percent, at least 40 percent, or at least 60 percent. Typically the amount is between 0.1 μM and 1 mM. When feed media are added to cell cultures, they may contain greater amounts of 5-thio-L-fucose.

[0062] The amount of 5-thio-L-fucose that is effective can be determined by standard cell culture methodology. For example, cell culture assays can be used to help identify optimal dosage ranges. The exact amount used will also depend on the time of administration, the host cell line, cell density, etc. The fucosylation and / or thiofucosylation pattern of an antibody may be modulated by altering the concentration of 5-thio-L-fucose in the culture medium and / or the duration of exposure to 5-thio-L-fucose.

[0063] The powdered cell culture medium of the present invention is preferably produced by mixing all components and milling them. Mixing components is known to those skilled in the art of producing dry powdered cell culture media by milling. Preferably, all components are thoroughly mixed so that all parts of the mixture have roughly the same composition. The more uniform the composition, the higher the quality of the resulting medium for uniform cell growth.

[0064] Milling can be carried out using any type of mill suitable for producing a powdered cell culture medium. Typical examples are a ball mill, a pin mill, a Fitz mill, or a jet mill. Preferred are a pin mill, a Fitz mill, or a jet mill, and most preferred is a pin mill. Those skilled in the art know how to implement such a mill.

[0065] Large scale equipment mills with disc diameters of about 40 cm, for example, typically run at 1 to 6500 rpm in the case of pin mills, preferably 1 to 3000 rpm. Milling may be done under standard milling conditions, resulting in a powder having a particle size between 10 and 300 μm, most preferably between 25 and 100 μm.

[0066] Preferably, all components of the mixture subjected to milling are dry, meaning that if they contain water, they contain only water of crystallization, but not more than 10%, preferably not more than 5%, and most preferably not more than 2% by weight of unbound or uncoordinated water molecules. In a preferred embodiment, milling is carried out in an inert atmosphere, the preferred inert protective gas being nitrogen.

[0067] In another preferred embodiment, all components of the mixture are frozen before milling. Freezing the ingredients before milling can be done by any means that ensures that the ingredients are cooled to a temperature below 0°C, and most preferably below -20°C. In a preferred embodiment, freezing is done using liquid nitrogen. This means that the ingredients are treated with liquid nitrogen, for example, by pouring liquid nitrogen into a container in which the ingredients are stored before being introduced into the mill. In a preferred embodiment, the container is a feeder. When the container is a feeder, the liquid nitrogen is preferably introduced at or near the side of the feeder where the ingredients are introduced. Typically, the components are treated with liquid nitrogen for 2 to 20 seconds. Preferably, the cooling of the ingredients is done so that all ingredients entering the mill are at a temperature below 0°C, most preferably below -20°C.

[0068] In a preferred embodiment, all ingredients are placed in a container, from which the mixture is transferred to a feeder, most preferably a metering screw feeder. In the feeder, the ingredients are sometimes further mixed—and additionally cooled, depending on the type of feeder. The frozen mixture is then transferred from the feeder to a mill so that the milled mixture still has a temperature preferably below 0°C, more preferably below -20°C. Typically, the blending time, which refers to the residence time of the components of the mixture in the feeder, is greater than 1 minute, preferably between 15 and 60 minutes.

[0069] Metering screw feeders, also known as dosage snails, typically operate at speeds of 10 to 200 rpm, preferably 40 to 60 rpm. Typically, the temperature of the mill is kept between -50 and +30°C. In a preferred embodiment, the temperature is kept at approximately 10°C. The oxygen level during milling is preferably less than 10% (v / v).

[0070] The process can be carried out batchwise or continuously, for example, In a preferred embodiment, the process according to the invention is carried out continuously by continuously charging the ingredients of the mixture into a feeder for cooling over a period of time, and continuously charging the cooled mixture from the feeder into the mill. After milling, the resulting dry powder medium may be further compressed, for example, by dry compaction in a roll press, to increase particle size.

[0071] For example, for use of a dry powdered medium resulting from milling or wet or dry compaction, a solvent, preferably water (most particularly distilled water and / or deionized water or purified water or water for injection) or an aqueous buffer, is added to the medium and the components are mixed until the medium is completely dissolved in the solvent, and a ready-to-use liquid medium is produced. The solvent may also include saline, soluble acid or base ions to provide a suitable pH range (typically within the range between pH 1.0 and pH 10.0), stabilizers, surfactants, preservatives, and alcohol or other polar organic solvents.

[0072] It is also possible to add additional substances to the cell culture medium and solvent mixture, such as buffer substances for adjusting the pH, fetal bovine serum, sugars, etc. The resulting liquid cell culture medium is then contacted with cells for growth or maintenance. A dry powder or granular medium according to the present invention typically contains 5-thio-L-fucose in an amount such that after dissolution the resulting liquid medium contains an effective amount of 5-thio-L-fucose, typically between 10 nmol / L and 100 mmol / L, preferably between 0.1 μmol / L and 10 mmol / L. The dry powder or granular medium preferably does not contain any L-fucose.

[0073] The present invention further comprises: a) providing a bioreactor; b) mixing the cells to be cultured with a cell culture medium produced by dissolving the dry powder medium according to the present invention; c) incubating the mixture of step b); The present invention is directed to a process for culturing cells according to the present invention.

[0074] In one embodiment, the bioreactor is a perfusion bioreactor. A bioreactor is any vessel, flask, or tank in which cells can be cultured. Incubation is typically under suitable conditions, such as a suitable temperature, etc. Those skilled in the art will know suitable incubation conditions to support or maintain cell growth / culture, such as suitable temperature, pH, osmolality, aeration, agitation, etc., and bioreactors that limit or ideally avoid contamination by adventitious microorganisms from the environment.

[0075] A perfusion bioreactor is a bioreactor in which perfusion cell culture can be performed. It typically includes a bioreactor vessel that is closed during cell culture, an agitator in the vessel, a line for introducing fresh medium, a harvest line for removing a harvest stream containing cells, liquid medium, and target product from the bioreactor, and a cell retention device in the harvest line that retains cells while the liquid portion of the harvest can be collected. A review of perfusion cell culture providing details on preferred configurations can be found in "Perfusion mammalian cell culture for recombinant protein manufacturing - A critical review," Jean-Marc Bielser et al., Biotechnology Advances 36 (2018) 1328-1340.

[0076] The present invention also provides - loading the cells and aqueous cell culture medium into a bioreactor; - incubating the cells in a bioreactor; - adding cell culture medium to the bioreactor constantly throughout the incubation of the cells in the bioreactor or once or several times during said incubation period; The present invention is directed to a process for culturing cells in a bioreactor according to the method of claim 1, wherein the culture medium preferably has a pH of less than pH 8.5 and contains at least 5-thio-L-fucose and / or a derivative thereof as defined above.

[0077] In a preferred embodiment, the medium is a feed medium. Typically, the feed medium contains between 50 and 300 g / l of solid components dissolved in solvent. In another embodiment, the medium is a perfusion medium.

[0078] In one embodiment, in the process of the invention, the feed media that are added to the bioreactor either constantly during incubation or at one or several times within said incubation have different compositions. In another, typically preferred embodiment, in the process of the invention, the feed medium that is added to the bioreactor either constantly during incubation or at one or several times within said incubation always has the same composition.

[0079] Antibodies and antibody derivatives produced by the methods of the invention can be isolated from cell culture and purified using, for example, gel electrophoresis, filtration, dialysis, and chromatography such as affinity chromatography and / or ion exchange chromatography.

[0080] In some embodiments, antibodies or antibody derivatives produced by the methods of the present invention have increased effector function (e.g., ADCC activity) compared to antibodies or antibody derivatives produced in the absence of 5-thio-L-fucose. ADCC activity may be measured using assays known in the art, and in exemplary embodiments, is increased by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or 20-fold compared to the core-fucosylated parent antibody.

[0081] 5-Thio-L-fucose and / or its derivatives are particularly suitable for modifying core fucosylation, as they typically do not significantly affect cellular performance (VCD and titer). The glycosylation patterns of antibodies produced by the methods of the present invention sometimes show reduced core fucosylation, resulting in less overall core fucosylation, and sometimes core fucosylation is reduced due to its replacement by thiofucosylation. Sometimes both effects can be observed simultaneously. Typically, the fucosylation level can be reduced by using increasing amounts of 5-thio-L-fucose, and in parallel, the incorporation of 5-thio-L-fucose is increased by using increasing amounts of 5-thio-L-fucose, where the effects are not completely antiparallel, and afucosylation and thiofucosylation can also be modified by carefully selecting the concentration of 5-thio-L-fucose added to the cell culture.

[0082] 5-Thio-L-fucose exhibits higher efficiency compared to the known inhibitor 2F-peracetylfucose. As seen in Example 4, antibody production using 5-thio-L-fucose treatment showed a 64%, 68%, and 67% reduction in fucosylation compared to the control on days 7, 10, and 12, respectively. In comparison, addition of the same concentration of 2F-peracetylfucose, a known fucose analog, was only able to achieve a reduction of 11%, 50%, and 58%, on days 7, 10, and 12, respectively.

[0083] Furthermore, antibodies produced using the methods of the present invention using media containing 5-thio-L-fucose can be shown to exhibit higher binding affinity to their receptors.

[0084] The present invention is further illustrated by, but not limited to, the following figures and examples. The entire disclosures of all applications, patents, and publications cited above and below, and corresponding EP19218172.5 filed December 19, 2019, are hereby incorporated by reference.

[0085] example The following examples represent practical applications of the present invention. Example 1: Expression of recombinant protein in the presence of 5-thio-L-fucose. A typical fed-batch process was used to determine the effect of 5-thio-L-fucose on the glycosylation profile of recombinant proteins (Figure 1). Three different monoclonal antibodies (mAbs) and four different clones of Chinese hamster ovary (CHO) cells expressing the fusion protein were tested. Cells were cultured in spin tubes for 14 to 20 days at 37°C, 5% CO2, and 80% humidity. The starting cell density was 3 x 10 in 30 mL of Cellvento® 4 CHO medium (pH 7.0 ± 0.1). 5Cells / mL were used for clones 1 and 2 producing mAb1 and mAb2, respectively (Cellvento® platform). Cells were cultured at a rocking speed of 320 rpm and fed with 3% volume of Cellvento® 4 Feed (v / v) on days 3, 5, 10, 12, and 14, and with 6% (v / v) feed on day 7. Clones 3 and 4 producing mAb3 and the fusion protein were seeded at the same cell density in 30 mL of Ex-Cell® Advanced Media (pH 7.2 ± 0.1), while rocking was set to 230 rpm. Clones 3 and 4 were fed with 5% Ex-Cell® Advanced Feed (v / v) on days 3, 5, 7, 10, 12, 14, and 17. The feeds were supplemented with either 400 μM 5-thio-L-fucose, 400 μM acetylated 5-thio-L-fucose, 400 μM acetylated 2F-fucose as a positive control, or the respective amount of DMSO (1.17%) used as a solvent for both acetylated fucose analogs. The pH of all Cellvento® feeds was adjusted to 7.0±0.1, and that of the Ex-Cell® feeds was adjusted to 8.5±0.1.

[0086] Glucose levels were maintained above 4 g / L by adding a specific amount of 400 g / L glucose stock solution up to 6 g / L during the week and up to 13 g / L over the weekend on demand. Glucose and titer were detected in the supernatant using a bioprocess analyzer CEDEX Bio HT (Roche, Mannheim, Germany) based on spectrophotometry and turbidimetry. Viable cell density (VCD) and viability were assessed using a Vi-CELL™ XR 2.04 cell counter (Beckman Coulter, Fullerton, CA, USA). In general, 5-thio-L-fucose treatments were compared to a control condition without any additives. Acetylated 5-thio-L-fucose was compared to the known positive control, 2F-peracetylfucose, and a DMSO control used to dissolve both acetylated fucose analogs.

[0087] As shown in Figures 2-5, within four biological replicates, 5-thio-L-fucose treatment, with or without acetylation, showed no significant effect on the cellular performance (VCD and titer) of the four investigated CHO cell lines compared with the two control conditions and the positive control. Minor changes were detected in both platforms for mAb2 and mAb3. mAb2 production resulted in slightly reduced titers after 12 days in all conditions compared with the unsupplemented control. By day 17, the titer of 2.18 g / L in the control was reduced to 2.02 g / L and 1.99 g / L with 5-thio-L-fucose treatment and the DMSO control, respectively. A further reduction to 1.82 g / L, a level similar to that of the positive control, 2F-peracetylfucose (1.78 g / L), was detected with acetylated 5-thio-L-fucose. Because both peracetylated fucose analogs were dissolved in DMSO, the reduction in titer was partially attributable to the solvent.

[0088] For the production of mAb3, a reduced VCD of approximately 13% and 24% was detected (compared to the control) by calculating the area under the curve for 5-thio-L-fucose and acetylated 5-thio-L-fucose up to day 17. The DMSO control showed a similar reduction of 13%, indicating that the solvent for acetylated 5-thio-L-fucose likely led to a further reduction in VCD. Because the reduced VCD did not affect titer (2.25 g / L to 2.33 g / L), it is considered to be within the biological variability of the experiment.

[0089] Example 2: 5-thio-L-fucose is incorporated into the glycan structure The glycosylation profile of the recombinant protein produced in the control fed-batch was compared to the profile obtained in a fed-batch process supplemented with a fucose analog. Antibodies were purified from cell culture supernatants using Protein A PhyTips® (PhyNexus Inc, San Jose, CA).

[0090] Glycosylation patterns were analyzed by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS). Purified antibodies were denatured, and glycans were released from the antibodies by digestion with peptide N-glycosidase F (PNGase F). The glycans were then labeled using the GlycoWorks™ RapiFluor-MS™ N-Glycan Kit according to the manufacturer's protocol. For analysis, a UPLC glycan column (2.1 x 150 mm) was used, which separates glycans according to their hydrophobicity. Chromatography was performed at 45°C using a 50 mM ammonium formate (A), pH 4.4 solution, and acetonitrile (B) gradient. Specifically, the A:B ratio was initially 20:80 (0 min), changing to 27:73 at 3 min and 37:63 at 35 min at a flow rate of 0.5 ml / min. From 36.5 to 39.5 min, only A was used as the mobile phase, and the flow rate was reduced to 0.2 ml / min. Subsequently, the ratio was changed to 20% A:80% B, and the flow rate was increased to 0.5 ml / min at 43.1 min until the end of the process at 55 min. Detection was performed by a fluorescence detector using an excitation wavelength of 265 nm and an emission wavelength of 425 nm. Identification of the glycan structures was performed by MS using positive electrospray ionization (ESI), calibrated with standards every 30 s.

[0091] Glycans containing unnatural 5-thio-L-fucose were identified through retention time shifts, mass shifts, and fragmentation patterns. The mass shift of approximately +16 Da is caused by the exchange of the ring oxygen in fucose (164.0684 g / mol) with sulfur in the thio-L-fucose derivative, leading to a theoretical mass of 180.0455 g / mol for the 5-thio-L-fucose derivative. This mass is similar to the mass of galactose (180.0633 g / mol), but can be distinguished by a higher-resolution mass spectrometer (115 ppm mass difference).

[0092] Furthermore, fragment spectra were obtained using LC-MS / MS. The fragmentation patterns of thio-fucose-containing glycans were similar to those of the respective fucosylated glycans, but differed among the fucose-containing glycan fragments. For example, the Rapifluor-GlcNAc-Fuc fragment exhibited a theoretical mass of 679.3304 g / mol, whereas the Rapifluor GlcNAc-thioFuc exhibited a theoretical mass of 695.3076 g / mol. The differences allowed for unambiguous identification of the sugar structures (Figure 6, A. Fucosylated GOF, B. Thiofucosylated GOthioF, and C. G1 fragmentation patterns).

[0093] Additionally, the retention times of thiofucosylated glycans can be differentiated from fucosylated glycans. Figure 7 shows an exemplary shift in the retention time of the fluorescence peak for a mAb treated with 800 μM 5-thio-L-fucose. The thiofucosylated G0F peak was detected at 17.19 min, while the elution of the G0 peak occurs at 17.38 min. Separation of the thiofucosylated G1F isoform peak can be observed at 20.88 and 21.52 min, compared with the retention times of both fucosylated G1F isoforms at 22.81 and 23.51 min. Overall, this data confirms the replacement of core fucosylation by 5-thio-L-fucose in the glycan structure. FIG. 7 shows the fluorescence signal obtained using UPLC after separation of the released, labeled glycans, showing the change in retention time of the peak corresponding to thiofucosylated glycans compared to fucosylated glycans.

[0094] Example 3: 5-thio-L-fucose can reduce fucosylation through the incorporation of 5-thio-L-fucose. A general fed-batch process was used to determine the effect of 5-thio-L-fucose on glycosylation profiles. Three different monoclonal antibodies (mAbs) and four different clones of Chinese hamster ovary (CHO) cells expressing the fusion protein were tested. Cells were cultured in spin tubes for 14 to 20 days at 37°C, 5% CO2, and 80% humidity. The starting cell density was 3 x 10 in 30 mL of Cellvento® 4 CHO medium (pH 7.0 ± 0.1). 5 Cells / mL were used for clones 1 and 2 producing mAb1 and mAb2, respectively (Cellvento® platform). Cells were cultured with agitation at 320 rpm and fed with 3% volume of Cellvento® 4 Feed (v / v) on days 3, 5, 10, 12, and 14, and with 6% (v / v) feed on day 7. Clones 3 and 4 producing mAb3 and the fusion protein were seeded at the same cell density in 30 mL of Ex-Cell® Advanced Media (pH 7.2 ± 0.1) while the agitation was set to 230 rpm (Ex-Cell® platform). Clones 3 and 4 were fed with 5% Ex-Cell® Advanced Feed (v / v) on days 3, 5, 7, 10, 12, 14, and 17. The feeds were supplemented with either 400 μM 5-thio-L-fucose, 400 μM acetylated 5-thio-L-fucose, 400 μM acetylated 2F-fucose as a positive control, or the respective amount of DMSO (1.17%) used as a solvent for both acetylated fucose analogs. The pH of all Cellvento® feeds was adjusted to 7.0 ± 0.1, and that of Ex-Cell® feeds was adjusted to 8.5 ± 0.1. Glucose levels were maintained above 4 g / L by adding specific amounts of a 400 g / L glucose stock solution up to 6 g / L during the week and up to 13 g / L over the weekend on demand. Glucose and titer were detected in the supernatant using a bioprocess analyzer CEDEX Bio HT (Roche, Mannheim, Germany) based on spectrophotometry and turbidimetry.

[0095] To investigate the glycosylation profile during fed-batch experiments, samples were centrifuged and antibodies and Fc-fusion proteins were purified from cell culture supernatants using Protein A PhyTips® (PhyNexus Inc, San Jose, CA). Glycosylation patterns were analyzed by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) using the GlycoWorks™ RapiFluor-MS™ N-Glycan kit as described.

[0096] In general, 5-thio-L-fucose treatments with or without acetylation were compared with a control condition without any additives, a known positive control, 2F-peracetylfucose, and a DMSO control used to dissolve both acetylated fucose analogs. Because harvesting recombinantly produced proteins on day 12 is typical, glycosylation profiles were visualized on day 12. Quantification of glycans was obtained using the relative peak areas of the fluorescent signals. Nearly all peaks were assigned to glycan structures according to their mass. Signals with unknown glycan structures and no mass detection were summarized as unknown species (not shown).

[0097] We tested three different monoclonal antibodies (mAbs) and four different clones of Chinese hamster ovary (CHO) cells expressing the fusion proteins. Core fucosylation was efficiently reduced through 5-thio-L-fucose treatment in all tested cell lines producing the different recombinant proteins.

[0098] The reduction of fucosylation of mAb1 produced in clone 1 by 5-thio-L-fucose was achieved through high incorporation of the fucose analog. Addition of 400 μM 5-thio-L-fucose, with or without acetylation, resulted in a reduction of core-fucosylated glycans to approximately 26% on day 12, compared to 94% in the control (Figure 8A). Addition of the same concentration of acetylated 2F-fucose (a known fucose analog that reduces fucosylation) was less efficient, leading to 36% core-fucosylation on day 12. In contrast to acetylated 2F-fucose, the reduction of native fucosylation by 5-thio-L-fucose was achieved through high incorporation of 5-thio-L-fucose into approximately 70% of glycans, regardless of acetylation. Afucosylation was only increased by 60% on day 12 with acetylated 2F-fucose, compared to 5% for the control and 5-thio-L-fucose.

[0099] Production of mAb2 resulted in 8% core fucosylation at day 12 after 5-thio-L-fucose treatment, with or without acetylation, compared to 77% fucosylation in the control (Figure 8B). In contrast to mAb1, whose afucosylation level did not change in response to 5-thio-L-fucose treatment, reduced fucosylation in mAb2 achieved 60% afucosylation (compared to 20% in the control condition) by incorporating 32% 5-thio-L-fucose. The known positive control achieved a 14% fucosylation level at day 12 by increasing afucosylation to 78%.

[0100] The same amount of afucosylation compared to the known fucose analog, acetylated 2F-fucose, was achieved only by clone 3 expressing mAb3, leading to approximately 34% afucosylation at day 12 (Figure 8C). 5-Thio-L-fucose was incorporated at 43%, leading to a 21% lower fucosylation level at day 12 compared to 90% in the control and 65% in the positive control.

[0101] A glycosylation pattern similar to that for mAb2 was observed for the fusion protein produced in clone 4 on day 12 (Figure 8D). Compared to the control, core fucosylation was reduced by approximately 63% via 5-thio-L-fucose treatment, regardless of whether acetylation was present. For both, incorporation of 5-thio-L-fucose was observed in 39% of the glycans, and 52% were afucosylated. The positive control reached 82% afucosylation without detectable incorporation of fucose analogs.

[0102] Overall, at day 12, increases in the amount of incorporated 5-thio-L-fucose of approximately 32%, 39%, 43%, and 70% were detected for mAb2, the fusion protein, mAb3, and mAb1, respectively. Increased amounts of fucosylation of approximately 5%, 34%, 52% and 60% were detected for mAb1, mAb3, the fusion protein and mAb2.

[0103] Figure 8 shows the effect of 5-thio-L-fucose on fucosylation profiles using CHO cell lines producing (A) mAb1, (B) mAb2, (C) mAb3, and (D) fusion protein. Data represent the mean ± SEM of two biological replicates on day 12.

[0104] Example 4: Addition of 5-thio-L-fucose to the process leads to a more rapid reduction in fucosylation compared to addition of 2F-peracetylfucose. To determine the time required for 5-thio-L-fucose supplementation to affect the glycosylation profile of recombinantly produced proteins, samples taken at various time points during the feed-batch were examined. Samples with cell viabilities greater than 60% were collected on days 5, 7, 10, 12, and 14. After centrifugation, antibodies and Fc-fusion proteins were purified from cell culture supernatants using Protein APhyTips® (PhyNexus Inc, San Jose, CA). Glycosylation patterns were analyzed by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) using the GlycoWorks™ RapiFluor-MS™ N-Glycan kit as described. Initial investigation of the glycan profile during fed-batch experiments indicated a high efficiency of 5-thio-L-fucose. Production of mAb1 using 5-thio-L-fucose treatment showed a 64%, 68%, and 67% reduction in fucosylation compared to the control on days 7, 10, and 12, respectively. In comparison, addition of the same concentration of 2F-peracetylfucose, a known fucose analog, only achieved an 11%, 50%, and 58% reduction on days 7, 10, and 12, respectively. This indicates a higher efficiency of 5-thio-L-fucose compared to the known inhibitor 2F-peracetylfucose.

[0105] To detect the effect of 5-thio-L-fucose earlier in the fed-batch procedure, the glycosylation profile of mAb3 on day 5 was also investigated. The detected fucosylation level obtained with 400 μM 5-thio-L-fucose treatment on day 5 was already low at approximately 48%, whereas 400 μM acetylated 2F-fucose resulted in approximately 84% fucosylation on day 5, a level similar to the 87% fucosylation detected in the control. While the fucosylation level of the control remained constant between 87% and 90% on days 7, 10, 12, and 14, 5-thio-L-fucose treatment reduced core fucosylation to approximately 34%, 25%, 21%, and 18%, respectively. In comparison, the known inhibitor acetylated 2F-fucose only reduced fucosylation levels by approximately 78%, 72%, 65%, and 57% on days 7, 10, 12, and 14, respectively. Therefore, 5-thio-L-fucose may be preferable for applications with short culture times, such as batch or perfusion processes. In addition, reduced amounts or different feeding regimes may be applied to reduce the required amount of fucose derivative added throughout the process or to titrate the fucosylation level.

[0106] Figure 9 shows the accelerated effect of 5-thio-L-fucose during fed-batch experiments producing (A) mAb1 or (B) mAb3. Data represent the mean ± SEM of two biological replicates.

[0107] Example 5: Increasing the concentration of 5-thio-L-fucose leads to higher incorporation. The ability to achieve different fucosylation levels through the addition of 5-thio-L-fucose during fed-batch was investigated using two platforms and two different clones producing either mAb1 or mAb3. Cells were cultured in spin tubes for 14 and 20 days at 37°C, 5% CO2, and 80% humidity. The starting cell density was 3 x 10 in 30 mL of Cellvento® 4CHO medium (pH 7.0 ± 0.1). 5Cells / mL were used for clone 1 producing mAb1 (Cellvento® platform). Cells were cultured with agitation at 320 rpm and fed with 3% volume of Cellvento® 4 Feed (v / v) on days 3, 5, 10, 12, and 14, and with 6% (v / v) feed on day 7. Clone 3 producing mAb3 was seeded at the same cell density in 30 mL of Ex-Cell® Advanced Media (pH 7.2 ± 0.1), while agitation was set to 230 rpm (Ex-Cell® platform). Clone 3 was fed with 5% Ex-Cell® Advanced Feed (v / v) on days 3, 5, 7, 10, 12, 14, and 17.

[0108] The feeds were supplemented with increasing concentrations of 5-thio-L-fucose, acetylated 5-thio-L-fucose, or DMSO (1.17%), which was used as a solvent for acetylated 5-thio-L-fucose. The pH of all Cellvento® feeds was adjusted to 7.0±0.1, and that of the Ex-Cell® feeds was adjusted to 8.5±0.1. Glucose levels were maintained above 4 g / L by adding specific amounts of a 400 g / L glucose stock solution up to 6 g / L during the week and up to 13 g / L over the weekend on demand. Glucose and titer were detected in the supernatant using a bioprocess analyzer, CEDEX Bio HT (Roche, Mannheim, Germany).

[0109] A dose response using clone 1 producing mAb1 was performed with 200, 400, and 800 μM acetylated 5-thio-L-fucose (Figure 10A). Glycosylation profiles were examined on days 7, 10, and 12. Because little change was detected over time, the glycan structures of the produced IgG are expressed as the average values ​​for all three days. The data indicated that the addition of 200 μM, 400 μM, and 800 μM acetylated 5-thio-L-fucose increased the incorporation of the non-natural fucose analog from 50% to 81% and 87%, respectively. This reduced the fucosylation level by 46%, 16%, and 9%, compared to 96% fucosylation in both control conditions (containing 1.17% DMSO as a solvent for acetylated 5-thio-L-fucose).

[0110] Furthermore, the effect of increasing the concentration of 5-thio-L-fucose was investigated for clone 3 producing mAb3. Concentrations of 5-thio-L-fucose ranging from 50 μM to 800 μM were calculated to increase the amount of 5-thio-L-fucose incorporation and the amount of afucosylation. The highest applied concentration, 800 μM 5-thio-L-fucose, resulted in approximately 36% afucosylation and approximately 48% incorporation of fucose analogs, leading to approximately 15% remaining native fucosylated glycans on day 12. Consequently, the addition of 800 μM 5-thio-L-fucose allowed for a reduction of fucosylation by approximately 75% on day 12 compared to the control. Stepwise reduction of the 5-thio-L-fucose concentration to 400 μM, 200 μM, and 50 μM resulted in approximately 44%, 36%, and 11% incorporation of the fucose analog, respectively, on day 12. This data indicates that the amount of 5-thio-L-fucose incorporation can be targeted by careful selection of the concentration.

[0111] Figure 10 shows the dose response of 5-thio-L-fucose on glycosylation profiles. (A) Effect of acetylated 5-thio-L-fucose on a CHO cell line producing mAb1. Data represent mean values ​​± SEM on days 7, 10, and 12. (B) Effect of 5-thio-L-fucose on a CHO cell line producing mAb3. Data represent mean values ​​± SEM of two biological replicates on day 12.

[0112] Example 6: Effect of 5-thio-L-fucose treatment on FcγRIIIa binding The purpose of this section is to investigate whether antibodies produced in a process containing thiofucose would exhibit high binding affinity to FcyRIIIa and thus potentially increase ADCC. Indeed, increased binding of afucosylated antibodies to FcyRIIIa, and thus enhanced ADCC activity, has been described in the literature. Two isoforms of FcγRIIIa are known, with either a phenylalanine (F158) or valine (V158) residue at position 158. The latter isoform has been reported to have higher affinity for IgG1 compared to the F isoform and was used for these studies.

[0113] The binding of samples after 5-thioFuc treatment was investigated by surface plasmon resonance technology using a Biacore T200 system (GE Healthcare, Uppsala, Sweden). The analysis temperature was set at 25°C and the sample compartment temperature at 15°C. The His capture kit from GE Healthcare was used to couple anti-His antibodies to the active and reference flow cells using an S Sensor Chip CM5 series according to the manufacturer's instructions. For all experiments, the immobilization level was 12185 ± 436 resonance units (RU; 1 RU ≈ 1 pg / mm 2) range. Additionally, an anti-His antibody was used to capture His-tagged FcγRIIIa V176 (CD8-H52H4) from AcroBiosystems (Newark, DE, USA). The receptor was expressed in HEK293 cells, and 0.011 μg / mL was injected for 60 s at a flow rate of 10 μL / min, resulting in a capture level of 4.5 ± 0.2 RU. Subsequently, mAb3 was injected for 150 s at five increasing concentrations ranging from 1.9 to 5.6, 16.7, 50, 150, and 450 nM at a flow rate of 30 μL / min across both the reference and active flow cells. Binding was continuously recorded, and nonspecific binding and injection artifacts were automatically subtracted. Single-cycle kinetics was used with a dissociation time of 600 s at a flow rate of 30 μL / min. Data collection was performed at a 10 Hz rate. After each experiment, both flow cells were regenerated for 30 s with 10 mM glycine-HCl pH 1.5 according to the kit instructions. Data from three technical replicates and two biological replicates were fitted with a 1:1 binding model. The global dissociation constant, KD, was determined from the ratio of the values ​​of the kinetic rate constant for dissociation (kd) and the kinetic rate constant for association (ka). Data are presented in Table 1 as fold change compared to the control.

[0114] Cells treated with increasing concentrations of 5-thio-L-fucose produced antibodies with increased affinity for FcγRIIIa. Samples treated with either 50 μM 5-thio-L-fucose or 400 μM 2F-PerAcFuc from day 5 achieved 1.2- and 1.3-fold enhanced binding compared to the control. Samples treated with 200, 400, and 800 μM 5-thio-L-fucose showed 2.0-, 2.9-, and 2.6-fold increased affinity, respectively, compared to the control on day 5. At later time points, increased affinity for FcγRIIIa was observed for all treated samples. At day 12, 2.0-, 3.1-, 3.8-, and 4.8-fold increased affinity was detected with 50, 200, 400, and 800 μM 5-thio-L-fucose treatment, respectively. Sensorgrams (Figure 11) resulting from injection of the same concentration series of control and treated antibody demonstrate higher affinity through a higher response of the 5-thio-L-fucose-treated antibody compared to the control on day 12. In addition, treatment with 400 μM 5-thio-L-fucose showed slightly higher binding affinity compared to the known inhibitor, 400 μM 2F-PerAcFuc, leading to a 3.6-fold increase in affinity. Samples treated on day 14 showed similar KD values ​​compared to day 12, due to similar glycosylation patterns. 1.7-, 3.6-, 4.1-, and 4.5-fold enhanced binding through 5-thio-L-fucose treatment was detected at 50, 200, 400, and 800 μM, respectively.

[0115] Table 1: Effect of 5-thio-L-fucose treatment on the affinity of mAb3 to FcγRIIIa on days 5 (A), 12 (B) and 14 (C). Data represent two biological replicates. [Table 2] [Table 3] [Table 4]

[0116] Figure 11 shows an overlay plot of FcγRIIIa binding to mAb3 control (black) or 5-thio-L-fucosylated mAb3 (gray) on day 12. Sensorgrams show binding levels to captured FcγRIIIa at a concentration range from 6 to 486 nm (n=3). All sensorgrams were double-referenced.

Claims

1. 1. A method for producing an antibody or other Fc-containing protein with reduced fucosylation, comprising culturing a host cell in a culture medium comprising 5-thio-L-fucose and / or a 5-thio-L-fucose derivative selected from partially or fully acetylated 5-thio-L-fucose, non-, partially or fully acetylated forms of 2-F-5-thio-L-fucose, 5-alkynyl-5-thio-L-fucose, 6,6,6-trifluoro-5-thio-L-fucose and 5-thio-L-fucose phosphonate, wherein the host cell the cells express an antibody or other Fc-containing protein having an Fc domain with at least one N-glycoside-linked sugar chain attached to the Fc domain through an N-acetylglucosamine at the reducing end of the sugar chain, and isolating the antibody or other Fc-containing protein from the cells, wherein the antibody or other Fc-containing protein has reduced fucosylation on the sugar chain compared to an antibody or other Fc-containing protein from the same host cells cultured under the same culture conditions except for the absence of 5-thio-L-fucose and / or its derivatives.

2. 2. The method according to claim 1, wherein the culture medium contains 5-thio-L-fucose and / or partially or fully acetylated 5-thio-L-fucose.

3. 3. The method according to claim 1 or 2, characterized in that the host cell is a Chinese hamster ovary cell.

4. The method according to any one of claims 1 to 3, characterized in that the host cells are cultured in fed-batch cell culture or perfusion cell culture.

5. The host cell - loading the bioreactor with host cells and aqueous cell culture medium; - Incubating the cells in the bioreactor - adding cell culture medium to the bioreactor continuously throughout the entire incubation time of the cells in the bioreactor or once or several times during said incubation time, whereby the cell culture medium comprises 5-thio-L-fucose and / or 5-thio-L-fucose derivatives selected from partially or fully acetylated 5-thio-L-fucose, 2-F-5-thio-L-fucose, 5-alkynyl-5-thio-L-fucose, 6,6,6-trifluoro-5-thio-L-fucose and non-, partially or fully acetylated forms of 5-thio-L-fucose phosphonate, The method according to any one of claims 1 to 4, characterized in that the culture is carried out by

6. 6. The method according to claim 1, wherein the culture medium to be added has a pH of less than pH 8.5 and contains 5-thio-L-fucose and / or a derivative thereof at a concentration between 10 nM and 100 mmol / l.

7. A method for producing a thiofucosylated antibody or other Fc-containing protein, the method comprising culturing host cells in a culture medium containing 5-thio-L-fucose and / or a 5-thio-L-fucose derivative selected from partially or fully acetylated 5-thio-L-fucose, non-, partially or fully acetylated forms of 2-F-5-thio-L-fucose, 5-alkynyl-5-thio-L-fucose, 6,6,6-trifluoro-5-thio-L-fucose, and 5-thio-L-fucose phosphonate, wherein the host cells express an antibody or other Fc-containing protein having an Fc domain with at least one N-glycoside-linked sugar chain attached to the Fc domain through an N-acetylglucosamine at the reducing end of the sugar chain, and isolating the antibody or other Fc-containing protein from the cells.

8. A dry powder cell culture medium comprising 5-thio-L-fucose and / or a 5-thio-L-fucose derivative selected from partially or fully acetylated 5-thio-L-fucose, non-, partially or fully acetylated forms of 2-F-5-thio-L-fucose, 5-alkynyl-5-thio-L-fucose, 6,6,6-trifluoro-5-thio-L-fucose, and 5-thio-L-fucose phosphonate.

9. 9. The cell culture medium of claim 8, wherein the cell culture medium is a feed medium.

10. 10. The cell culture medium according to claim 8 or 9, comprising 5-thio-L-fucose and / or a derivative thereof in an amount such that, when the cell culture medium is dissolved to provide a liquid medium, the concentration of 5-thio-L-fucose in the liquid medium is between 10 nM and 100 mmol / L.

11. 11. The cell culture medium according to any one of claims 8 to 10, characterized in that the cell culture medium comprises at least one or more sugar components, one or more amino acids, one or more vitamins or vitamin precursors, one or more salts, one or more buffer components, one or more cofactors and one or more nucleic acid components.

12. a) mixing 5-thio-L-fucose and / or 5-thio-L-fucose derivatives selected from partially or fully acetylated 5-thio-L-fucose, non-, partially or fully acetylated forms of 2-F-5-thio-L-fucose, 5-alkynyl-5-thio-L-fucose, 6,6,6-trifluoro-5-thio-L-fucose and 5-thio-L-fucose phosphonate with other components of a cell culture medium; b) subjecting the mixture of step a) to milling; 12. A method for producing a cell culture medium according to any one of claims 8 to 11, by

13. 13. The method for producing a cell culture medium according to claim 12, characterized in that step b) is carried out in a pin mill, a Fitz mill or a jet mill.

14. 14. The method for producing a cell culture medium according to claim 12 or 13, wherein the mixture from step a) is cooled to a temperature below 0° C. before milling.

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