Methods and compositions for modulating the glycosylation profile of a protein

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 the use of 5-thio-L-fucose to regulate the glycosylation profile of cell culture media and antibodies or other Fc-containing proteins containing 5-thio-L-fucose, and thereby to regulate the binding affinity of said proteins.

[0002] Recombinant proteins such as therapeutic antibodies and Fc-fusion proteins are generally produced in mammalian cell lines. Monoclonal antibodies and Fc-fusion proteins produced in mammalian host cells can have various post-translational modifications, including glycosylation. Monoclonal antibodies such as IgG typically have N-linked glycosylation sites at asparagine 297 (Asn297) of each heavy chain (two per intact antibody). The glycans attached to Asn297 on the antibody are typically complex bi-branched structures with very low or no bisecting N-acetylglucosamine (bisecting GlcNAc) along with low amounts of terminal sialic acid and variable amounts of galactose. The glycans also generally have high levels of core fucosylation. Reduction of core fucosylation in antibodies has been shown to alter the effector functions of Fc, particularly Fc gamma receptor binding and ADCC activity. This finding has led to interest in the modification of cell lines that produce antibodies with reduced core fucosylation.

[0003] The secret to some antibody-based immunotherapies lies in the binding of the antibody's Fc portion to cell surface receptors called Fc gamma receptors (FcγR). Depending on the cytoplasmic domain, FcγR triggers various immune responses. What is common are the immunoreceptor activation tyrosine motifs (ITAM) for activating receptors (FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) and the immunoreceptor inhibitory tyrosine 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, the receptors can be classified into high-affinity FcγRI (CD64) as well as low-affinity FcγRII (CD32) and FcγRIII (CD16). Bruhns et al. described the binding of all known antibody subclasses to the various receptor classes. IgG1 has been reported to bind to receptors of all classes with different affinities. The increasing affinity of IgG1 was observed for FcγRIIb, FcγRIIIb, FcyRIIIa, 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 cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Natural killer (NK) cells that express FcγRIIIa are responsible for ADCC activity. In general, receptor affinity and thus the biological activity of an antibody depend on post-translational modifications such as the glycosylation of the antibody [Harris RJ, Chin ET, Macchi F, Keck RG, Shyong B-J, 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 exhibit distinct N-linked glycosylation at position 297 (Asn297) on each heavy chain in the Fc portion. The presence of core fucosylation on the Fc portion of recombinant proteins is known to reduce the ADCC response due to the 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]. Thus, reduction of core fucosylation leads to enhanced ADCC. This has been verified by in vivo studies of afucosylated anti-EGFR and anti-CS1 antibodies that exhibit 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 anti-tumor 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. Alternative means of modifying cell lines include the use of small molecule inhibitors against enzymes in the glycosylation pathway. Another option is the provision of small molecule fucose analogs for use in producing recombinant antibodies that have 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 proteins produced 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 fucose analogs.

[0008] The present invention thus provides a method of making an antibody or other Fc-containing protein having reduced fucosylation, comprising: 5-thio-L-fucose and / or selected from partially or fully acetylated 5-thio-L-fucose, 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 and 5-thio-L-fucose phosphonate the 5- Culturing a host cell in a culture medium containing a 5-thio-L-fucose derivative, wherein the host cell expresses an antibody or other Fc-containing protein having at least one N-glycosidic-linked sugar chain bound to the Fc domain through 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 in the sugar chain compared to the 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, and the derivatives are 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, directed to a method comprising.

[0009] In a preferred embodiment, the host cell is 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 cell is cultured in a fed-batch cell culture or a perfusion cell culture.

[0010] In a preferred embodiment, the host cell is - filling a bioreactor with the host cells and an aqueous cell culture medium - incubating the cells in the bioreactor - adding the cell culture medium to the bioreactor continuously throughout the total time of incubation of the cells in the bioreactor, or one or several times during the incubation time, whereby the cell culture medium contains 5-thio-L-fucose, cultured by. Preferably, the added medium has a pH of less than 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 relates to a method for producing a thiophucosylated antibody or other Fc-containing protein, comprising: 5-thio-L-fucose and / or culturing a host cell in a culture medium containing a -thio-L-fucose derivative selected from partially or fully acetylated 5-thio-L-fucose, 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 and 5-thio-L-fucose phosphonate, wherein the host cell expresses an antibody or other Fc-containing protein having at least one N-glycosidic-linked sugar chain bound to the Fc domain through N-acetylglucosamine at the reducing end of the sugar chain, and isolating the antibody or other Fc-containing protein from the cells. the 5 The present invention is further directed to a method comprising.

[0012] The present invention further relates to 5-thio-L-fucose and / or a dry powder cell culture medium containing a -thio-L-fucose derivative selected from partially or fully acetylated 5-thio-L-fucose, 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 and 5-thio-L-fucose phosphonate. the 5 In one embodiment, the cell culture medium is a fed-batch medium.

[0013] In another aspect, when the cell culture medium is dissolved to provide a liquid medium, the concentration of 5-thio-L-fucose and / or its derivatives in the liquid medium is between 10 nmol / l and 100 mmol / l, preferably between 0.1 μmol / l and 10 mmol / l, and the cell culture medium contains 5-thio-L-fucose and / or its derivatives in an amount. The liquid medium, for example, a fed-batch medium or a perfusion medium, is typically designed such 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 aspect, the cell culture medium contains at least one or more saccharide 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 the cell culture medium described in the present invention, comprising: a) 5-thio-L-fucose and / or Selecting a 5-thio-L-fucose derivative selected from partially or fully acetylated 5-thio-L-fucose, 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, and 5-thio-L-fucose phosphonate, the 5 Mixing the other components of the cell culture medium with the -thio-L-fucose derivative; b) Subjecting the mixture of step a) to milling; The method is directed to the method by. In a preferred aspect, step b) is carried out in a pin mill, a Fitz mill or a jet mill. In another preferred aspect, the mixture from step a) is cooled to a temperature below 0 °C before milling. Brief Description of the Drawings

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[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 components of a complex mixture, 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 contain, 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] The cell culture medium according to the present invention is a mixture of any of the components that maintain and / or support the growth of cells in vitro. It may be a complex medium or a chemically defined medium. The cell culture medium may contain all of the components necessary to maintain and / or support the growth of cells in vitro, or may contain only some of the components such that additional components are added individually. An example of a cell culture medium according to the present invention is a complete medium that contains all of the components necessary to maintain and / or support the growth of cells in vitro, as well as medium additives or feeds. In a preferred embodiment, the cell culture medium is a complete medium, a perfusion medium or a fed-batch medium. A complete medium, also called a basal medium, typically has a pH between 6.8 and 7.8. A fed-batch medium preferably has a pH of less than 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 fed-batch medium is not the basal medium that supports the initial growth and production in cell culture, but rather a cell culture medium that is added to the medium at a later stage to prevent nutrient depletion and sustain the production phase. The fed-batch medium may have higher concentrations of some components compared to the basal culture medium. For example, some components such as nutrients including amino acids or carbohydrates may be present in the fed-batch medium at concentrations 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] A mammalian cell culture medium is a mixture of components that maintains and / or supports 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 undefined chemical substances. This means that the chemical composition of all the chemicals used in the medium is known. Chemically defined media do not contain any yeast, animal or plant tissues; they do not contain feeder cells, serum, extracts or digests or other components that would provide to the medium poorly defined proteins. Components that are not chemically defined or are poorly chemically defined are those whose chemical composition and structure are not known, those that exist in various compositions, or those that can only be defined with enormous experimental effort - comparable to the evaluation of 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 a milling process, a lyophilization process, or a dry or wet granulation process. It is to be understood that powdered cell culture media means granular or particulate media and does not mean 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 combined or aggregated solvent unless otherwise indicated. Dry powder media result from a milling or lyophilization process and typically have a particle size of less than 0.5 mm, for example between 0.05 and 0.5 mm.

[0025] Dry powder media resulting from a dry or wet granulation process, for example by spray drying, wet granulation or dry compaction, typically have a particle size greater than 0.5 mm, for example between 0.5 and 5 mm. Dry compaction is typically carried out in a roll press. US 6,383,810 B2 discloses a method for producing an aggregated eukaryotic cell culture media powder. The method comprises wetting a dry powder cell culture media with a solvent and then redrying the wetted media to obtain an aggregated dry cell culture media. In one aspect, the dry powder media according to the invention are produced by dry compaction.

[0026] Cells cultured in the media according to the 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 can be any of normal cells, immortalized cells, diseased cells, transformed cells, mutant cells, somatic cells, germ cells, stem cells, progenitor cells or embryonic cells, established or transformed cell lines or those obtained from a natural source. The host cells are cells into which a gene for expressing an antibody having an Fc domain or another Fc-containing protein has been incorporated.

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

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

[0029] Various types of mills are known to those skilled in the art. The pin mill, also called a centrifugal impact mill, pulverizes solids by means of protruding pins on a high-speed rotating disk that provides breaking energy. Pin mills are sold, for example, by Munson Machinery (USA), Premium Pulman (India) or Sturtevant (USA).

[0030] The jet mill uses compressed gas to accelerate the particles and cause them to collide with each other within the 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 executed process is a process that is not executed batchwise. If the milling process is continuously executed, it means that the medium components are continuously and steadily supplied into the mill over a period of time.

[0032] Cell culture media, particularly the complete media according to the present invention, typically contain at least one or more saccharide 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, plant proteins, fatty acids and / or fatty acid derivatives and / or pluronic acid and / or chemically prepared non-ionic surfactants. An example of a suitable non-ionic surfactant is, for example, a surfactant of a bifunctional block copolymer terminated with primary hydroxyl groups, also called a poloxamer, which is available under the trade name pluronic® from BASF, Germany.

[0034] The saccharide components 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 present invention are tyrosine, amino acids constituting proteins, particularly essential amino acids, leucine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine, and amino acids not constituting proteins 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, hydroxocobalamin, 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 components containing inorganic ions such as bicarbonate, calcium, chloride, magnesium, phosphate, potassium, and sodium, or 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), and 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 mononucleotide and derivatives, glutathione, heme nucleotide phosphate and derivatives.

[0039] The nucleic acid components according to the present invention are nucleobases 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] The fed-batch medium may have a different composition compared to the complete medium. They typically contain amino acids, trace elements and vitamins. They may also contain saccharide components, although sometimes the saccharide components are added in separate feeds for production reasons.

[0041] Suitable fed-batch media may contain one or more of the following compounds: [Table 1-1] [Table 1-2] Freezing according to the present invention means cooling the temperature below 0°C.

[0042] The term "antibody" refers to (a) an immunoglobulin polypeptide and an immunologically active portion of the immunoglobulin polypeptide, i.e., an antigen-binding site that immunospecifically binds to a particular antigen (e.g., CD70) and an Fc domain containing a complex-type N-glycoside-linked sugar chain(s), or a fragment thereof, or (b) a conservatively substituted derivative of such an immunoglobulin polypeptide or fragment that immunospecifically binds 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 containing a complex-type N-glycoside-linked sugar chain.

[0043] The term "monoclonal antibody" refers to an antibody derived from a single cell clone, including any eukaryotic or prokaryotic cell clone, or phage clone, and does not refer to 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, for example, the C H 1-hinge-C H 2-C H 3 domain, optionally C H 4 domain, or a conservatively substituted derivative of such an Fc region. The term "Fc domain" refers to the constant region domain of an antibody, for example, 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 the Fab domain of an antibody, specifically binds. The terms "specifically bind" and "specifically binds to" mean that an antibody or antibody derivative binds to a corresponding target antigen in a highly selective manner and does not bind to the majority of other antigens. Typically, an antibody or other Fc-containing protein binds with an affinity of at least about 1x10 -7 M, and preferably 10 -8 M to 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M, and binds to a predetermined antigen with an affinity greater than twofold that for binding to non-specific antigens (e.g., BSA, casein) other than the predetermined antigen or closely related antigens. The term "inhibit" or "inhibition of" means to reduce by a measurable amount or to prevent altogether, particularly to reduce by a measurable amount or to prevent altogether the activity of a specific enzyme that catalyzes a reaction.

[0046] The term "afucosylated" or "afucosylated with" refers to a glycan structure that does not have fucose in its internal core structure. As used herein, "thiogalactosylated" or "thiogalactosylated with" refers to a glycan structure that contains at least one thiogalactose. Often in a "thiogalactosylated" glycan structure, the galactose is replaced by thiogalactose.

[0047] As used herein, "5-thio-L-galactose" or "ThioGal" refers to a galactose derivative in which oxygen in the sugar ring is replaced by a sulfur atom. "5-thio-L-galactose" means the alpha and / or beta anomers.

Chem.

[0048] As used herein, "acetylated 5-thio-L-galactose" or "AcThioGal" means 5-thio-L-galactose that bears from 1 to 4 acetyl groups, referring to all forms of the acetyl groups on thiogalactose. Figure 1 shows, for example, peracetylated 5-thio-L-galactose that bears 4 acetyl groups. Derivatives of 5-thio-L-galactose are 2-F-5-thio-L-galactose, 5-alkynyl-5-thio-L-galactose, 6,6,6-trifluoro-5-thio-L-galactose, 5-thio-L-galactose phosphonate in non-, partially or fully acetylated forms.

[0049] 2-F-5-thio-L-galactose in non-, partially or fully acetylated forms is of formula I:

Chem.

[0050] 5-Alkynyl-5-thio-L-fucose in non-, partially or fully acetylated form has the formula II:

Chemical formula

[0051] 6,6,6-Trifluoro-5-thio-L-fucose in non-, partially or fully acetylated form has the formula III:

Chemical formula

[0052] 5-Thio-L-fucose phosphonate in non-, partially or fully acetylated form has the formula IV:

Chemical formula

[0053] The following images show the non-acetylated form. In the acetylated form, one or more OH groups are acetylated.

Chemical formula

[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, "N-glycosidically linked sugar chain" or "N-glycosidically linked glycan" is typically attached to asparagine 297 (according to the Kabat numbering), although complex N-glycosidically linked sugar chains can be linked to other asparagine residues. As used herein, a complex N-glycosidically linked sugar chain has the following structure:

Chemical formula

[0056] "Complex N-glycosidically linked sugar chain" excludes high-mannose type sugar chains in which only mannose is incorporated at the non-reducing end of the core structure, and includes the following: 1) a complex type in which the non-reducing end side of the core structure has one or more branches of galactose-N-acetylglucosamine (also referred to as "gal-GlcNAc"), and the non-reducing end side of Gal-GlcNAc optionally has sialic acid, bisecting N-acetylglucosamine or the like; or 2) a hybrid type in which the non-reducing end side of the core structure has branches of both high-mannose type N-glycosidically linked sugar chains and complex N-glycosidically linked sugar chains. In some embodiments, a "complex N-glycosidic linked glycan" includes a complex type in which the non-reducing end side of the core structure has zero, 1 or more branches of galactose-N-acetylglucosamine (also referred to as "gal-GlcNAc"), and the non-reducing end 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 fucosylation of core fucosylation, i.e., N-glycosidic linked glycans of Fc-containing proteins or antibodies. In one embodiment, fucosylation is inhibited and thus reduced or completely suppressed. In another embodiment, the N-glycosidic linked glycans of Fc-containing proteins or antibodies are instead thiofucosylated. Both effects can also occur in parallel in either different antibodies or one antibody carrying two different glycan structures.

[0058] Also provided are antibodies produced by such methods. In other aspects, a culture medium 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, 2-F-5-thio-L-fucose in non, partial or fully acetylated form, 5-alkynyl-5-thio-L-fucose, 6,6,6-trifluoro-5-thio-L-fucose and 5-thio-L-fucose phosphonate is provided. In the following, the derivative is not always specified. When 5-thio-L-fucose is mentioned, it is obvious that it also encompasses the derivatives identified above. Nevertheless, preferred are 5-thio-L-fucose and / or partially or fully acetylated 5-thio-L-fucose.

[0059] In some embodiments, the fucosylation of the complex N-glycosidically linked glycans attached to the Fc region (or domain) is completely suppressed or otherwise reduced compared to an antibody or other Fc-containing protein from the same host cell cultured under the same culture conditions except that 5-thio-L-fucose is absent. "Under the same culture conditions except that 5-thio-L-fucose is absent" typically means that the cell culture is carried out with the same components and under the same conditions, except that 5-thio-L-fucose is not present.

[0060] To suppress or reduce core fucosylation, the host cell is 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 cell is cultured does not contain L-fucose.

[0061] The amount of 5-thio-L-fucose contained in the liquid cell culture medium in which the host cell is cultured is an amount effective to reduce the incorporation of fucose. In this context, "effective amount" refers to the amount of 5-thio-L-fucose that is sufficient to reduce the incorporation of fucose into the complex N-glycan-linked glycans 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 fed-batch media are added to the cell culture, they may contain a greater amount of 5-thio-L-fucose.

[0062] The amount of 5-thio-L-fucose that is effective can be determined by standard cell culture methodologies. For example, cell culture assays may be used to help identify the optimal dosing range. The exact amount used also depends on the time of administration, the host cell line, the cell density, etc. The fucosylation and / or thiofucosylation pattern of the antibody may be adjusted by changing 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 the components and milling them. Mixing of the components is known to those skilled in the art for producing a dry powdered cell culture medium by milling. Preferably, all the components are thoroughly mixed so that all parts of the mixture have substantially the same composition. The higher the uniformity of the composition, the higher the quality of the resulting medium with respect to 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 ball mills, pin mills, Fitz mills or jet mills. Preferred are pin mills, Fitz mills or jet mills, and most preferred is a pin mill. Those skilled in the art know how to operate such mills.

[0065] A mill of large-scale equipment with a disk diameter of about 40 cm is, for example, typically operated at 1 to 6500 revolutions per minute, preferably 1 to 3000 revolutions per minute in the case of a pin mill. Milling can be carried out under standard milling conditions to yield 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 to be milled are dry. This means that if they contain water, they contain only water of crystallization and contain it by weight of unbound or uncoordinated water molecules of 10% or less, preferably 5% or less, most preferably 2% or less. In a preferred embodiment, milling is carried out in an inert atmosphere. A preferred inert protective gas is nitrogen.

[0067] In another preferred embodiment, all components of the mixture are frozen before milling. Freezing the components before milling can be done by any means that ensures cooling the components 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 components are treated by pouring liquid nitrogen into the container in which the components are stored, for example, before introduction into the mill, using liquid nitrogen. 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 into which the components are introduced. Typically, the components are treated with liquid nitrogen for 2 - 20 seconds. Preferably, cooling the components is done such that all components entering the mill are at a temperature below 0 °C, most preferably below -20 °C.

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

[0069] The metering screw feeder, also called a dosage snail, typically runs at a speed of 10 - 200 revolutions per minute, and preferably it runs at 40 - 60 revolutions per minute. Typically, the temperature of the mill is maintained between -50 and +30 °C. In a preferred embodiment, the temperature is maintained at approximately 10 °C. The oxygen level during milling is preferably less than 10% (v / v).

[0070] The process can be, by way of example, batchwise or continuously carried out. In a preferred embodiment, the process according to the invention is carried out continuously by permanently filling the components of the mixture into a feeder for cooling over a period of time and permanently filling 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 the particle size.

[0071] By way of example, for the use of a dry powder medium resulting from milling or wet or dry compaction, a solvent, preferably water (most specifically, 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 contain physiological saline, soluble acid or base ions providing a suitable pH range (typically within the range between pH 1.0 and pH 10.0), stabilizers, surfactants, preservatives, and alcohols or other polar organic solvents.

[0072] It is also possible to add further substances such as buffer substances for pH adjustment, fetal bovine serum, sugars, etc. to the mixture of the cell culture medium and the solvent. The resulting liquid cell culture medium is then brought into contact with cells for growth or maintenance. The dry powder or granular medium according to the invention typically contains 5-thio-L-fucose in such an amount that the resulting liquid medium after dissolution 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 provides a) providing a bioreactor; b) mixing the cells to be cultured with a cell culture medium produced by dissolving a dry powder medium according to the present invention; c) incubating the mixture of step b); which is directed to a process for culturing cells.

[0074] In one aspect, the bioreactor is a perfusion bioreactor. The bioreactor is any tank, flask or vessel in which cells can be cultured. Incubation is typically carried out under suitable conditions such as a suitable temperature. Those skilled in the art know suitable incubation conditions for supporting or maintaining cell growth / culture such as suitable temperature, pH, osmotic pressure, aeration, agitation, etc. and bioreactors that limit or ideally avoid contamination by foreign microorganisms from the environment.

[0075] A perfusion bioreactor is a bioreactor in which perfusion cell culture can be carried out. It typically includes a bioreactor vessel that is closed during cell culture, a stirrer in the vessel, a line for introducing fresh medium, a collection line for removing a collection stream containing cells, liquid medium and target product from the bioreactor, and a cell retention device in the collection line that retains cells while the liquid portion of the collection can be collected. A review of perfusion cell culture providing details of 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 - filling the bioreactor with cells and an aqueous cell culture medium; - Incubating cells in a bioreactor, - Adding cell culture medium to the bioreactor continuously throughout the incubation time of the cells in the bioreactor, or one or several times during the incubation time, directed to a process for culturing cells in a bioreactor by, wherein the medium preferably has a pH of less than 8.5 and contains at least 5 - thio - L - fucose and / or its derivatives as defined above.

[0077] In a preferred embodiment, the medium is a fed - batch medium. Typically, the fed - batch medium contains individual components dissolved in a solvent between 50 and 300 g / l. In another embodiment, the medium is a perfusion medium.

[0078] In one embodiment, in the process of the present invention, the fed - batch medium added to the bioreactor continuously or either once or several times within that number during incubation has different compositions. In another, typically preferred embodiment, in the process of the present invention, the fed - batch medium added to the bioreactor continuously or either once or several times within that number during incubation always has the same composition.

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

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

[0081] 5-Thio-L-fucose and / or its derivatives are particularly suitable for modifying core fucosylation since they typically do not have a significant impact on cell performance (VCD and titer). The glycosylation pattern of the antibodies produced by the methods of the invention sometimes shows a reduction in core fucosylation, resulting in less overall core fucosylation, and sometimes a reduction in core fucosylation due to its replacement by thiofucosylation. Sometimes both effects can be observed simultaneously. Typically, the fucosylation level can be decreased by increasing the amount of 5-thio-L-fucose used, and in parallel, the incorporation of 5-thio-L-fucose increases by increasing the amount of 5-thio-L-fucose to be incorporated, where the effects are not completely anti-parallel and fucosylation and thiofucosylation can also be modified by careful selection of the concentration of 5-thio-L-fucose added to the cell culture.

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

[0083] Furthermore, antibodies produced using the method of the present invention with a medium 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. All applications, patents, and publications cited above and below, and the entire disclosure of the corresponding EP19218172.5 filed on December 19, 2019, are hereby incorporated by reference into this specification.

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

[0086] 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 as required. Glucose and titers were detected in the supernatant using a bioprocess analyzer CEDEX Bio HT (Roche, Mannheim, Germany) based on spectrophotometry and nephelometry. Viable cell density (VCD) and viability were evaluated using a Vi-CELL™ XR 2.04 cell counter (Beckman Coulter, Fullerton, CA, USA). Generally, the 5-thio-L-fucose treatment was compared to control conditions without any additives. Acetylated 5-thio-L-fucose was compared to the DMSO control used for the dissolution of the known positive control 2F-peracetylfucose and both acetylated fucose analogs.

[0087] As shown in Figures 2 to 5, within four biological replicates, the 5-thio-L-fucose treatment, regardless of acetylation, showed no significant effect on the cell performance (VCD and titer) of these four investigated CHO cell lines compared to the two control conditions and the positive control. Slight changes were detected in both platforms for mAb2 and mAb3. The production of mAb2 resulted in a slightly reduced titer after 12 days in all conditions compared to the non-supplemented control. On day 17, the titer of 2.18 g / L in the control was reduced to 2.02 g / L and 1.99 g / L using 5-thio-L-fucose treatment and DMSO control, respectively. A further reduction to 1.82 g / L, at a level similar to that of the positive control 2F-peracetylfucose (1.78 g / L), was detected using acetylated 5-thio-L-fucose. Since both peracetylfucose analogs were dissolved in DMSO, the reduction in titer was, in part, due to the solvent.

[0088] For the production of mAb3, reduced VCDs of approximately 13% and 24% were detected (compared to the control) by calculating the area under the curve up to day 17 for 5-thio-L-fucose and acetylated 5-thio-L-fucose. The DMSO control showed a similar reduction of 13%, indicating that the solvent of acetylated 5-thio-L-fucose was likely responsible for the further reduction in VCD. Since the reduced VCD did not affect the titer (2.25 g / L to 2.33 g / L), it was explored 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 with the profile obtained in the fed-batch process supplemented with fucose analogs. The antibody was purified from the cell culture supernatant using Protein A PhyTips (registered trademark) (PhyNexus Inc, San Jose, CA).

[0090] The glycosylation pattern was analyzed by ultra-high performance liquid chromatography (UPLC)-MS coupled with a mass spectrometer. The purified antibody was denatured and the glycans were released from the antibody by digestion with peptide N-glycosidase F (PNGase F), followed by labeling of the glycans using the GlycoWorks™ RapiFluor-MS™ N-Glycan kit according to the manufacturer's protocol. For analysis, a UPLC glycan column (2.1×150 mm) that separates glycans according to their hydrophobicity was used. Chromatography was performed using a gradient of 50 mM ammonium formate (A), pH 4.4 solution and acetonitrile (B) at 45 °C. Specifically, initially at a flow rate of 0.5 ml / min, the ratio of A:B was 20:80 (0 min), and changed to 27:73 at 3 min and 37:63 at 35 min. From 36.5 min 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 from 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 structure was performed by MS using positive electrospray ionization (ESI) that was 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. A mass shift of approximately +16 Da is caused by the exchange of sulfur in the thio-L-fucose derivative for the ring oxygen in fucose (164.0684 g / mol), leading to a theoretical mass of 180.0455 g / mol for the 5-thio-L-fucose derivative. This mass is similar to that of galactose at 180.0633 g / mol but can be distinguished by a higher resolution mass spectrometer (mass difference of 115 ppm).

[0092] Furthermore, the fragment spectra were obtained using LC-MS / MS. The fragmentation patterns of the glycans containing thiofucose are similar to those of the respective fucosylated glycan fragmentation patterns but differ for each glycan fragment containing fucose. For example, the Rapifluor-GlcNAc-Fuc fragment presents a theoretical mass of 679.3304 g / mol, while Rapifluor GlcNAc-thioFuc presents a theoretical mass of 695.3076 g / mol. The difference enables the unambiguous identification of the sugar structures (Figure 6, A fucosylated G0F, B thiofucosylated G0 thioF, and C G1 fragmentation pattern).

[0093] In addition, the retention times of the thiofucosylated glycans can be distinguished from those of the fucosylated glycans. In Figure 7, a representative shift in the retention times of the fluorescence peaks for the mAb treated with 800 μM 5-thio-L-fucose is shown. The thiofucosylated G0F peak was detected at 17.19 min, while the elution of the G0 peak occurred at 17.38 min. The separation of the thiofucosylated G1F isoform peaks can be observed at 20.88 and 21.52 min as compared to the retention times of both fucosylated G1F isoforms at 22.81 and 23.51 min. Generally, this data confirms the replacement of core fucosylation by 5-thio-L-fucose in the glycan structure. Figure 7 shows the fluorescence signals obtained using UPLC after separation of the released, labeled glycans, and shows the change in the retention times of the peaks corresponding to the thiofucosylated glycans as compared to the 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 the glycosylation profile. Four different clones of Chinese hamster ovary (CHO) cells expressing three different monoclonal antibodies (mAbs) and a fusion protein were tested. The cells were cultured in spin tubes at 37 °C, 5% CO2, and 80% humidity for 14 to 20 days. As the starting cell density, 3x10 5 cells / mL in 30 mL of Cellvento® 4CHO medium (pH 7.0 ± 0.1) were used for clones 1 and 2 producing mAb1 and mAb2, respectively (Cellvento® platform). The cells were cultured with agitation at 320 rpm and fed with 3% volume of Cellvento® 4Feed (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 at 230 rpm (Ex-Cell® platform). Clones 3 and 4 were fed with 5% of Ex-Cell® Advanced Feed (v / v) on days 3, 5, 7, 10, 12, 14, and 17. The feeds were supplemented with either 400 μM of 5-thio-L-fucose, 400 μM of acetylated 5-thio-L-fucose, 400 μM of acetylated 2F-fucose as a positive control, or both acetylated fucose analogs in the respective amounts of DMSO (1.17%) used as a solvent. The pH of all Cellvento® feeds was adjusted to 7.0 ± 0.1, and the pH 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 as required. Glucose and titers were detected in the supernatant using a bioprocess analyzer CEDEX Bio HT (Roche, Mannheim, Germany) based on spectrophotometry and nephelometry.

[0095] To investigate the glycosylation profile during the fed-batch experiment, samples were centrifuged and the antibodies and Fc-fusion proteins were purified from the cell culture supernatant using Protein A PhyTips® (PhyNexus Inc, San Jose, CA). The glycosylation pattern was analyzed as described for ultra-high performance liquid chromatography (UPLC)-MS coupled to a mass spectrometer using the GlycoWorks™ RapiFluor-MS™ N-Glycan kit.

[0096] Generally, the 5-thio-L-fucose treatment with or without acetylation was compared to a control condition without any additives, a known positive control of 2F-peracetylfucose, and a DMSO control used to dissolve both acetylated fucose analogs. Since the harvest of the recombinantly produced protein on day 12 was common, the glycosylation profile on day 12 was visualized. Glycan quantification was obtained using the relative peak area of the fluorescence signal. Almost all peaks were assigned to glycan structures according to their mass. Signals with unknown glycan structures and mass detection were summarized as unknown species (not shown).

[0097] Four different clones of Chinese hamster ovary (CHO) cells expressing three different monoclonal antibodies (mAbs) and a fusion protein were tested. Core fucosylation was efficiently reduced through 5-thio-L-fucose treatment in all tested cell lines producing 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 fucose analogs. 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% of core fucosylated glycans in the control (Figure 8A). Addition of the same concentration of acetylated 2F-fucose (a known fucose analog that reduces fucosylation) was inefficient and led 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 the glycans, regardless of acetylation. Afucosylation increased only up to 60% by acetylated 2F-fucose on day 12 compared to 5% for the control and 5-thio-L-fucose.

[0099] Production of mAb2 resulted in 8% core fucosylation on 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, the reduction of fucosylation of mAb2 achieved 60% afucosylation (compared to 20% under control conditions) by incorporating 32% 5-thio-L-fucose. A known positive control achieved a fucosylation level of 14% on day 12 by increasing afucosylation up 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 and led to approximately 34% afucosylation on day 12 (Figure 8C). 5-thio-L-fucose was incorporated at 43% and led to a fucosylation level 21% lower on day 12 compared to 90% in the control and 65% in the positive control.

[0101] A glycosylation pattern similar to that of 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 the presence or absence of acetylation. 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 the fucose analog.

[0102] Generally, an increase in the amount of incorporated 5-thio-L-fucose of approximately 32%, 39%, 43%, and 70% was detected for mAb2, the fusion protein, mAb3, and mAb1, respectively, on day 12. An increase in the amount of fucosylation of approximately 5%, 34%, 52%, and 60% was detected for mAb1, mAb3, the fusion protein, and mAb2, respectively.

[0103] Figure 8 shows the effect of 5-thio-L-fucose on the fucosylation profiles using CHO cell lines producing (A) mAb1, (B) mAb2, (C) mAb3, and (D) the 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 the 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 fed-batch were investigated. Samples with a cell viability of over 60% on days 5, 7, 10, 12 and 14 were collected. After centrifugation, the antibody and Fc fusion protein were purified from the cell culture supernatant using Protein A PhyTips® (PhyNexus Inc, San Jose, CA). The glycosylation pattern was analyzed as described by ultra performance liquid chromatography-mass spectrometry (UPLC-MS) coupled to a mass spectrometer using the GlycoWorks™ RapiFluor-MS™ N-Glycan kit. Initial investigation of the glycan profile during the fed-batch experiment indicated high efficiency of 5-thio-L-fucose. Production of mAb1 using 5-thio-L-fucose treatment showed a reduction in fucosylation of 64%, 68% and 67% on days 7, 10 and 12 respectively compared to the control. In comparison, addition of the same concentration of 2F-peracetylfucose, a known fucose analog, achieved only a reduction of 11%, 50% and 58% 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 faster 5-thio-L-fucose in fed-batch, the glycosylation profile of mAb3 on day 5 was added for investigation. The detected fucosylation level obtained using 400 μM 5-thio-L-fucose treatment on day 5 was already at a low level of about 48%, while 400 μM acetylated 2F-fucose resulted in about 84% fucosylation on day 5, which was at a level similar to the 87% fucosylation detected in the control. The control fucosylation levels on days 7, 10, 12, and 14 were constant between 87% and 90%, while the 5-thio-L-fucose treatment reduced core fucosylation to about 34%, 25%, 21%, and 18%, respectively. In comparison, the known inhibitor acetylated 2F-fucose only showed a reduction in fucosylation levels to about 78%, 72%, 65%, and 57% on days 7, 10, 12, and 14, respectively. Therefore, 5-thio-L-fucose would be similarly preferred for applications with short culture times such as batch or perfusion processes. In addition, reduced amounts or various feeding regimes may be applied to reduce the required amount of fucose derivatives added throughout the process or to titrate the fucosylation level.

[0106] Figure 9 shows the accelerated effect of 5-thio-L-fucose during a fed-batch experiment 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 various 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 at 37 °C, 5% CO2, and 80% humidity between 14 and 20 days. As the starting cell density, 3 × 10 in 30 mL of Cellvento® 4CHO medium (pH 7.0 ± 0.1) 5Cells / mL were used for Clone 1 producing mAb1 on the Cellvento® platform. The cells were cultured with rocking at 320 rpm and fed on days 3, 5, 10, 12, and 14 with 3% volume of Cellvento® 4Feed (v / v) and on day 7 with 6% (v / v) feed. 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 the rocking was set at 230 rpm (Ex-Cell® platform). Clone 3 was fed on days 3, 5, 7, 10, 12, 14, and 17 with 5% of Ex-Cell® Advanced Feed (v / v).

[0108] The feeds were supplemented with DMSO (1.17%) in respective amounts used as solvents for increasing concentrations of 5-thio-L-fucose, acetylated 5-thio-L-fucose, or acetylated 5-thio-L-fucose. The pH of all Cellvento® feeds was adjusted to 7.0 ± 0.1 and the Ex-Cell® feed 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 as required. Glucose and titers were detected in the supernatant using a bioprocess analyzer CEDEX Bio HT (Roche, Mannheim, Germany).

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

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

[0111] Figure 10 shows the dose response of 5-thio-L-fucose to the glycosylation profile. (A) Effect of acetylated 5-thio-L-fucose on the CHO cell line producing mAb1. Data represent the mean ± SEM on days 7, 10, and 12. (B) Effect of 5-thio-L-fucose on the CHO cell line producing mAb3. Data represent the mean ± 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 part is to investigate whether the antibodies produced in a process containing thiofucose exhibit high binding affinity to FcyRIIIa and thus potentially increased ADCC. Indeed, the increase in the binding of afucosylated antibodies to FcyRIIIa and thereby 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 a higher affinity for IgG1 compared to the F isoform and was used for these studies.

[0113] The binding of samples after 5-thio Fuc 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. Using the His capture Kit from GE Healthcare and the S Sensor Chip CM5 series, anti-His antibodies were conjugated in the active and reference flow cells according to the manufacturer's instructions. For all experiments, the immobilization level was 12185 ± 436 resonance units (RU; 1RU ≒ 1pg / mm 2) was used as the range. Furthermore, 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 injected at a flow rate of 10 μL / min at 0.011 μg / mL for 60 s, resulting in a capture level of 4.5 ± 0.2 RU. Subsequently, mAb3 was injected at a flow rate of 30 μL / min for 150 s at five increasing concentrations in the range of 1.9 to 5.6, 16.7, 50, 150, and 450 nM across both the reference and active flow cells. Binding was continuously recorded and non-specific binding and injection artifacts were automatically subtracted. A single-cycle kinetic theory was used with a dissociation time of 600 s at a flow rate of 30 μL / min. The data collection rate was performed at 10 Hz. After each experiment, both flow cells were regenerated for 30 s using 10 mM glycine-HCl pH 1.5 according to the kit instructions. Data from three technical replicates and two biological replicates were fitted to 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). The data are presented in Table 1 as fold change compared to the control.

[0114] Cells treated with increased concentrations of 5-thio-L-fucose produced antibodies with increased affinity for FcγRIIIa. Samples from day 5 treated with either 50 μM 5-thio-L-fucose or 400 μM 2F-PerAcFuc 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, an increase in affinity for FcγRIIIa was observed for all treated samples. On day 12, 2.0-, 3.1-, 3.8-, and 4.8-fold increases in affinity were detected for 50, 200, 400, and 800 μM 5-thio-L-fucose treatment, respectively. Sensorgrams (Figure 11) resulting from injection of the same series of concentrations of control and treated antibodies illustrate higher affinity through higher responses of antibodies treated with 5-thio-L-fucose compared to the control on day 12. Additionally, treatment with 400 μM 5-thio-L-fucose showed slightly higher binding affinity compared to 400 μM 2F-PerAcFuc, a known inhibitor, which led to a 3.6-fold increase in affinity. Samples treated on day 14 showed similar KD values due to similar glycosylation patterns compared to day 12. Binding enhanced 1.7-, 3.6-, 4.1-, and 4.5-fold 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 for 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 the binding of FcγRIIIa to mAb3 control (black) or 5-thio-L-fucose-treated mAb3 (gray) on day 12. The sensorgrams show the binding levels of a series of concentrations from 6 to 486 nM to the captured FcγRIIIa (n = 3). All sensorgrams were double-referenced.

Claims

**Claim 1** A method for producing an antibody or an Fc-containing protein other than an antibody having reduced fucosylation, comprising culturing a host cell in a culture medium containing 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 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 expresses an antibody or an Fc-containing protein other than an antibody having an Fc domain having at least one N-glycosidically linked sugar chain bound to the Fc domain through N-acetylglucosamine at the reducing end of the sugar chain, and isolating the antibody or the Fc-containing protein other than an antibody from the cells, wherein the antibody or the Fc-containing protein other than an antibody has reduced fucosylation in the sugar chain as compared to the antibody or the Fc-containing protein other than an antibody from the same host cells cultured under the same culture conditions except that 5-thio-L-fucose and / or its derivative is absent, said method. **Claim 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. **Claim 3** The method according to claim 1 or 2, wherein the host cell is a Chinese hamster ovary cell. **Claim 4** The method according to any one of claims 1 to 3, wherein the host cell is cultured in fed-batch cell culture or perfusion cell culture. **Claim 5** The host cell is - filling the bioreactor with the host cells and an 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 one or several times during the incubation time, whereby the cell culture medium contains 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, A method according to any one of claims 1 to 4, characterized by being cultured thereby.

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

7. A method for producing a thiofucosylated antibody or an Fc-containing protein other than an antibody, comprising culturing host cells in a culture medium containing 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, wherein the host cells express an antibody or an Fc-containing protein other than an antibody having at least one N-glycosidically linked sugar chain bound to the Fc domain through N-acetylglucosamine at the reducing end of the sugar chain, and isolating the antibody or the Fc-containing protein other than an antibody from the cells.

8. A dry powder cell culture medium containing 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.

9. The cell culture medium according to claim 8, characterized in that the fed-batch medium is a fed-batch medium.

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

11. The cell culture medium according to any one of claims 8 to 10, characterized in that the cell culture medium contains at least one saccharide component, 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.

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