Methods and compositions for modulating the glycosylation profile of proteins

Abstract

The present invention relates to a cell culture medium comprising 5-thio-L-fucose, and the use of 5-thio-L-fucose for modulating the glycosylation profile of antibodies or other Fc-containing proteins and thereby modulating the binding affinity of the proteins.

Description

Technical Field

[0001] The present invention relates to a cell culture medium comprising 5-thio-L-fucose, and the use of 5-thio-L-fucose for modulating the glycosylation profile of antibodies or other Fc-containing proteins and thereby modulating the binding affinity of the proteins. Background Art

[0002] Recombinant proteins, such as therapeutic antibodies and Fc fusion proteins, are typically produced in mammalian cell lines.

[0003] The 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) usually have N-connected glycosylation sites (two for each complete antibody) at the asparagine 297 (Asn297) of each heavy chain. The polysaccharide attached to Asn297 on the antibody is typically a composite biantennary structure with very low or no bisection N-acetylglucosamine (bisection GlcNAc), with a low amount of terminal sialic acid and a variable amount of galactose. Polysaccharide also usually has a high level of core fucosylation. It has been shown that the reduction of core fucosylation in the antibody changes Fc effector functions, particularly Fc gamma receptor binding and ADCC activity. This observation results in the interest of the through engineering approaches of producing cell lines with reduced core fucosylated antibodies.

[0004] Key to some antibody-based immunotherapies is the binding of the Fc portion of the antibody to cell surface receptors called Fcγ receptors (FcγRs). Depending on the intracytoplasmic domain, FcγRs trigger different immune responses. Common are immunoreceptor tyrosine-based activation motifs (ITAMs) for activating receptors (FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) and immunoreceptor tyrosine-based inhibition motifs (ITIMs) for inhibitory receptors (such as FcγRIIb) [Lu J and Sun PD, Structural mechanism of high affinity FcgammaRI recognition ofimmunoglobulin G .Immunol Rev2015, 268, 192-200]. In addition, receptors can be divided into high-affinity FcγRI (CD64) and low-affinity FcγRII (CD32) and FcγRIII (CD16). Bruhns et al. described the binding of different receptor classes to all known antibody subclasses. IgG1 was reported to bind to all types of receptors with varying affinities. Increased 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].

[0005] 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, the receptor affinity and thus the biological activity of an antibody depends on post-translational modifications of the antibody, such as 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 Editors; Shire SJ et al; Springer New York: New York, NY, 2010, 193–205].

[0006] Antibodies exhibit different N-linked glycosylation at position 297 (Asn297) of each heavy chain in the Fc portion. The presence of core-fucosylation on the Fc portion of recombinant proteins is known to reduce ADCC response 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]. Thus, reduction in core-fucosylation leads to enhanced ADCC. This was confirmed by in vivo studies of afucosylated anti-EGFR and anti-CS1 antibodies, which showed enhanced ADCC and anti-tumor activity when 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 to enhance the biological activity of therapeutic antibodies.

[0007] Methods for reducing fucosylation include methods for engineering cell lines.

[0008] Alternatives to engineered cell lines include the use of small molecule inhibitors of enzymes in the glycosylation pathway. Another option is to provide small molecule fucose analogs for the production of recombinant antibodies with complex N-linked glycans but reduced fucosylation. This has been proposed, for example, in WO 09135181.

[0009] However, there remains a need for improved modulation of the glycosylation patterns of therapeutic proteins, and thus of their biological activities. Summary of the Invention

[0010] It has been found 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 is associated with increased ADCC. Surprisingly, the use of 5-thio-L-fucose and / or certain derivatives thereof results in high incorporation of fucose analogs.

[0011] Therefore, the present invention relates to a method for producing an antibody or other Fc-containing protein with reduced fucosylation, the method comprising:

[0012] culturing a host cell in a medium comprising 5-thio-L-fucose and / or a 5-thio-L-fucose derivative selected from the group consisting of 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-acetylated, partially acetylated, or fully acetylated forms of 5-thio-L-fucosephosphonate, wherein the host cell expresses an antibody or other Fc-containing protein having an Fc domain having at least one N-glycoside-linked sugar chain bound to the Fc domain via an N-acetylglucosamine at the reducing terminus of the sugar chain, and

[0013] The antibody or other Fc-containing protein is isolated from the cell, wherein the antibody or other Fc-containing protein has reduced fucosylation at the sugar chain compared to the antibody or other Fc-containing protein from the same host cell cultured under otherwise identical culture conditions but in the absence of 5-thio-L-fucose and / or its derivatives selected from the group consisting of 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-acetylated, partially acetylated or fully acetylated forms of 5-thio-L-fucosephosphonate.

[0014] In a preferred embodiment, the host cell is cultured in a cell culture medium comprising 5-thio-L-fucose and / or partially or fully acetylated 5-thio-L-fucose.

[0015] In a preferred embodiment, the host cell is a Chinese hamster ovary cell.

[0016] In another preferred embodiment, the host cells are cultured in fed-batch cell culture or perfusion cell culture.

[0017] In a preferred embodiment, the host cell is cultured as follows:

[0018] - filling a bioreactor with said host cells and aqueous cell culture medium,

[0019] - incubating said cells in said bioreactor,

[0020] adding cell culture medium to the bioreactor continuously throughout the incubation time of the cells in the bioreactor or once or several times during the incubation time, wherein the cell culture medium comprises 5-thio-L-fucose.

[0021] Preferably, the pH of the added culture medium is below pH 8.5 and comprises 5-thio-L-fucose in a concentration between 10 nmol / l and 100 mmol / l, preferably between 0.1 µmol / l and 10 mmol / l.

[0022] The present invention further relates to a method for preparing a thiofucosylated antibody or other Fc-containing protein, the method comprising:

[0023] culturing a host cell in a medium comprising 5-thio-L-fucose and / or a 5-thio-L-fucose derivative selected from the group consisting of 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-acetylated, partially acetylated, or fully acetylated forms of 5-thio-L-fucosephosphonate, wherein the host cell expresses an antibody or other Fc-containing protein having an Fc domain having at least one N-glycoside-linked sugar chain bound to the Fc domain via an N-acetylglucosamine at the reducing terminus of the sugar chain, and

[0024] The antibody or other Fc-containing protein is isolated from the cells.

[0025] The present invention further relates to a dry powder cell culture medium comprising 5-thio-L-fucose and / or a 5-thio-L-fucose derivative selected from the group consisting of 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-acetylated, partially acetylated or fully acetylated forms of 5-thio-L-fucosephosphonate.

[0026] In one embodiment, the cell culture medium is a feed medium.

[0027] In another embodiment, the cell culture medium comprises 5-thio-L-fucose and / or its derivatives in an amount such that, when dissolved to provide a liquid culture medium, the concentration of 5-thio-L-fucose and / or its derivatives in the liquid culture medium is between 10 nmol / l and 100 mmol / l, preferably between 0.1 µmol / l and 10 mmol / l.

[0028] Liquid culture medium (eg feed medium or perfusion medium) is usually designed so that the final concentration of 5-thio-L-fucose and / or its derivatives in the cell culture is between 0.1-1 mmol / L.

[0029] In one embodiment, the cell culture medium comprises at least one or more carbohydrate 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.

[0030] The present invention further relates to a method for producing a cell culture medium according to the invention, said method being carried out as follows:

[0031] a) mixing 5-thio-L-fucose and / or a 5-thio-L-fucose derivative selected from the group consisting of 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-acetylated, partially acetylated or fully acetylated forms of 5-thio-L-fucosephosphonate with other components of the cell culture medium,

[0032] b) subjecting said mixture of step a) to milling.

[0033] In a preferred embodiment, step b) is carried out in a pin mill, a fitz mill or a jet mill.

[0034] In another preferred embodiment, the mixture from step a) is cooled to a temperature below 0° C. before grinding. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Showing the structural similarity of L-fucose and 5-thio-L-fucose with and without acetylation.

[0036] Figure 2-5The effect of 5-thio-L-fucose on cell performance is shown using four different CHO cell lines producing three different IgG1 (A, B and C) and fusion protein (D). Viable cell density (VCD) is visualized on the left, and titer is visualized on the right. The control feed does not contain supplements, while the DMSO control contains 1.17% DMSO, which is the same concentration as the solvent used for the two fully acetylated fucose analogs. Further details are shown in Example 1.

[0037] Figure 6 Shows the sugar structure (A fucosylated G0F, B thiofucosylated G0 thioF and C G1 fragmentation pattern). Figure 6 Details can be found in Example 2.

[0038] Figure 7 Shown are the fluorescent signals obtained after separation of released, labeled glycans using UPLC, showing the shifted retention times of the peaks corresponding to sulfofucosylated glycans compared to fucosylated glycans. Figure 7 Details can be found in Example 2.

[0039] Figure 8 The effect of 5-thio-L-fucose on the fucosylation profile is shown using CHO cell lines producing (A) mAb1, (B) mAb2, (C) mAb3, and (D) fusion proteins. Details can be found in Example 3.

[0040] Figure 9 Shown is the accelerating effect of 5-thio-L-fucose during fed-batch experiments producing (A) mAb1 or (B) mAb3. Details can be found in Example 4.

[0041] Figure 10 The dose response of 5-thio-L-fucose on the glycosylation profile is shown. Further details can be found in Example 5.

[0042] Figure 11 Overlays are shown for FcγRIIIa binding to mAb3 control (black) or to 5-thio-L-fucose treated mAb3 (grey) at day 12. Further details can be found in Example 6. DETAILED DESCRIPTION

[0043] Cell culture media support and sustain the growth of cells in an artificial environment. Depending on the type of organism whose growth is to be supported, cell culture media contain a complex mixture of components, sometimes exceeding one hundred different components. The cell culture media required for the propagation of mammalian, insect, or plant cells are typically much more complex than those used to support the growth of bacteria and yeast. Chemically defined media typically contain, but are not limited to, amino acids, vitamins, metal salts, antioxidants, chelating agents, growth factors, buffers, hormones, and many more substances known to those skilled in the art.

[0044] The cell culture medium according to the present invention is a mixture of any components that maintain and / or support the in vitro growth of cells. It can be a complex medium or a chemically defined medium. The cell culture medium may contain all or only some of the components necessary to maintain and / or support the in vitro growth of cells, so that additional components are added separately. An example of a cell culture medium according to the present invention is a full culture medium, which contains all the components necessary to maintain and / or support the in vitro growth of cells and a culture medium supplement or feed. In a preferred embodiment, the cell culture medium is a full culture medium, a perfusion culture medium or a feed medium. The full culture medium is also called a basal culture medium, and its pH is generally between 6.8 and 7.8. Preferably, the pH of the feed medium is lower than 8.5, preferably between 6.5 and 8.5.

[0045] The cell culture medium can be a liquid culture medium or a dry powder culture medium.

[0046] Typically, the cell culture medium according to the present invention is used to maintain and / or support the growth of cells in a bioreactor.

[0047] Feed or feed medium is such cell culture medium, and it is not the basal medium of initial growth and production in supporting cell culture, but the culture medium added in later stage, to prevent the exhaustion of nutrients and to maintain the production stage.Compared with basal medium, feed medium can have some components of higher concentration.For example, some components (for example comprising the nutrient of amino acid or carbohydrate) can be present in feed medium with the concentration of about 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100X, 200X, 400X, 600X, 800X or even about 1000X in basal medium.

[0048] Mammalian cell culture medium is a mixture of components that maintain and / or support the in vitro growth of mammalian cells. Examples of mammalian cells are human or animal cells, preferably CHO cells, COS cells, IVERO cells, BHK cells, AK-1 cells, SP2 / 0 cells, L5.1 cells, hybridoma cells or human cells.

[0049] Chemically defined cell culture media are cell culture media that do not contain any chemically undefined substances. This means that the chemical composition of all chemicals used in the culture 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 might add poorly chemically defined proteins to the culture medium. Chemically undefined or poorly defined chemical components are those whose chemical composition and structure are unknown, exist in varying compositions, or can only be defined with tremendous experimental effort—comparable to evaluating the chemical composition and structure of proteins such as albumin or casein.

[0050] Powdered cell culture medium or dry powder culture medium is a cell culture medium that is typically obtained by a grinding process, a freeze-drying process, or a dry or wet granulation process. This means that the powdered cell culture medium is a granular, particulate culture medium rather than a liquid culture medium. The term "dry powder" can be used interchangeably with the term "powder"; however, as used herein, "dry powder" refers only to the overall appearance of the granular material and is not intended to mean that the material is completely free of complexing or agglomerated solvents, unless otherwise specified. Dry powder culture medium obtained by grinding or freeze-drying processes typically has a particle size of less than 0.5 mm, for example, between 0.05 and 0.5 mm.

[0051] The dry powder culture medium obtained by a dry or wet granulation process (e.g., by spray drying, wet granulation, or dry compaction) typically has a particle size greater than 0.5 mm, for example, between 0.5 and 5 mm. Dry compaction is typically performed in a roller press. US 6,383,810 B2 discloses a method for producing an agglomerated eukaryotic cell culture medium powder. The method comprises wetting the dry powder cell culture medium with a solvent and then re-drying the wetted medium to obtain a dry, agglomerated cell culture medium.

[0052] In one embodiment, the dry powder culture medium according to the invention is produced by dry compaction.

[0053] The cells to be cultured with the culture medium according to the present invention can 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 normal cells, immortalized cells, diseased cells, transformed cells, mutant cells, somatic cells, germ cells, stem cells, precursor cells or embryonic cells, any of which can be established or transformed cell lines or obtained from natural sources. Host cells are cells that have been incorporated with a gene for expressing an antibody having an Fc domain or another Fc-containing protein.

[0054] The particle size refers to the average diameter of the particles. If 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 given particle size range. The particle size is determined by laser scattering.

[0055] An inert atmosphere is created by filling each container or device with an inert gas. Suitable inert gases are rare gases, such as argon or preferably nitrogen. These inert gases are non-reactive and prevent undesirable chemical reactions from occurring. In the method according to the invention, creating an inert atmosphere means, for example, reducing the concentration of oxygen to below 10% (v / v) absolute value by introducing liquid nitrogen or nitrogen.

[0056] Different types of mills are known to those skilled in the art.

[0057] Pin mills (also called centrifugal impact mills) pulverize solids where protruding pins on a high-speed rotating disk provide the breaking energy. Pin mills are sold, for example, by Munson Machinery (USA), Premium Pulman (India) or Sturtevant (USA).

[0058] Jet mills use compressed gas to accelerate particles, causing them to impact each other in a process chamber.Jet mills are sold, for example, by Sturtevant (USA) or PMT (Austria).

[0059] The fitz mill commercialized by Fitzpatrick (USA) uses a rotor with blades for grinding.

[0060] A continuously operated process is one that is not batch-wise. If the milling process is run continuously, this means that the culture components are fed to the mill permanently and steadily over time.

[0061] The cell culture medium according to the present invention (especially a complete culture medium) generally 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.

[0062] Substratum can also comprise sodium pyruvate, insulin, vegetable protein, lipid acid and / or fatty acid derivative and / or pluronic acid and / or surface active component (as chemically prepared nonionic surfactant).An example of suitable nonionic surfactant is the difunctional segmented block copolymer surfactant that is terminated in primary hydroxyl group, also referred to as poloxamer, for example can be with trade name pluronic From BASF, Germany obtains.

[0063] The sugar components are all monosaccharides or disaccharides, such as glucose, galactose, ribose or fructose (examples of monosaccharides) or sucrose, lactose or maltose (examples of disaccharides).

[0064] 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, and non-proteinogenic amino acids such as D-amino acids, preferably L-amino acids.

[0065] Tyrosine refers to L- or D-tyrosine, preferably L-tyrosine.

[0066] Cysteine ​​refers to L- or D-cysteine, preferably L-cysteine.

[0067] 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 (tocopherols, tocotrienols), and vitamin K (phylloquinone, menaquinone). Also includes vitamin precursors.

[0068] Examples of salts are compositions 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), ferrous sulfate (II), anhydrous sodium dihydrogen phosphate (NaH2PO4), anhydrous magnesium sulfate (MgSO4), anhydrous disodium hydrogen phosphate (Na2HPO4), magnesium chloride hexahydrate (MgCl2 . 6H2O), zinc sulfate heptahydrate.

[0069] Examples of buffers are CO2 / HCO3 (carbonate), phosphate, HEPES, PIPES, ACES, BES, TES, MOPS and TRIS.

[0070] Examples of cofactors are thiamine derivatives, biotin, vitamin C, NAD / NADP, cobalamin, flavin mononucleotides and derivatives, glutathione, heme nucleotide phosphates and derivatives.

[0071] According to the invention, nucleic acid components 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, adenosine diphosphate or adenosine triphosphate.

[0072] Feed media can have a different composition than the complete medium. They usually contain amino acids, trace elements, and vitamins. They may also contain sugar components, but sometimes, for production reasons, sugar components are added as a separate feed.

[0073] Suitable feed media may, for example, contain one or more of the following compounds:

[0074] L-Asparagine Monohydrate

[0075] L-Isoleucine

[0076] L-Phenylalanine

[0077] Sodium L-glutamate monohydrate

[0078] L-Leucine

[0079] L-Threonine

[0080] L-Lysine monohydrochloride

[0081] L-Proline

[0082] L-Serine

[0083] L-Arginine monohydrochloride

[0084] L-histidine monohydrochloride monohydrate

[0085] L-Methionine

[0086] L-Valine

[0087] Monosodium L-aspartate monohydrate

[0088] L-Tryptophan

[0089] Choline chloride

[0090] Inositol

[0091] Niacinamide

[0092] D(+) Calcium Pantothenate

[0093] Pyridoxine hydrochloride

[0094] Thiamine chloride hydrochloride

[0095] Micronized vitamin B12 (cyanocobalamin)

[0096] Biotin

[0097] folic acid

[0098] Riboflavin

[0099] Anhydrous magnesium sulfate

[0100] Copper(II) sulfate pentahydrate

[0101] Zinc sulfate heptahydrate

[0102] 1,4-Diaminobutane dihydrochloride

[0103] Ammonium heptamolybdate tetrahydrate

[0104] Cadmium sulfate hydrate

[0105] Magnesium(II) chloride tetrahydrate

[0106] Nickel(II) chloride hexahydrate

[0107] Sodium metasilicate

[0108] Sodium metavanadate

[0109] Tin(II) chloride dihydrate

[0110] Sodium selenite (approximately 45% Se)

[0111] Sodium dihydrogen phosphate monohydrate

[0112] Ammonium iron(III) citrate (approximately 18% Fe)

[0113] Freezing according to the present invention refers to cooling to a temperature below 0°C.

[0114] The term "antibody" refers to (a) immunoglobulin polypeptides and immunologically active portions of immunoglobulin polypeptides, i.e., polypeptides of the immunoglobulin family or fragments thereof, which contain an antigen binding site that immunospecifically binds a particular antigen (e.g., CD70) and an Fc domain comprising one or more complex N-glycoside-linked sugar chains, or (b) conservatively substituted derivatives of such immunoglobulin polypeptides or fragments that immunospecifically bind an antigen (e.g., CD70). Antibodies are generally described in, for example, Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988).

[0115] An Fc-containing protein is any protein that comprises an Fc domain or region that contains complex N-glycoside-linked sugar chains.

[0116] 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 not the method by which it is produced. Thus, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology.

[0117] The term "Fc region" refers to the constant region of an antibody, such as C H 1-Hinge-C H 2-C H 3 domains, optionally with C H 4 domains, or conservatively substituted derivatives of such Fc regions.

[0118] The term "Fc domain" refers to the constant region domain of an antibody, such as C H 1. Hinge, C H 2. C H 3 or C H 4 domains, or conservatively substituted derivatives of such Fc domains.

[0119] The term "Fab region" refers to the variable region of an antibody that binds to an antigen.

[0120] An "antigen" is a molecule to which an antibody, usually an antibody Fab domain, specifically binds.

[0121] The terms "specific binding" and "specifically bind" mean that an antibody or antibody derivative will bind to its corresponding target antigen in a highly selective manner and not to a large number of other antigens. Typically, an antibody or other Fc-containing protein will bind to a specific antigen with a specific binding affinity of at least about 1×10 -7 M, and preferably 10 -8 M to 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 The antibody binds to the predetermined antigen with an affinity that is at least twice as strong as that of M and binds to a nonspecific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen.

[0122] The term "inhibit" or "inhibition of" refers to reducing by a measurable amount, or completely preventing, particularly reducing by a measurable amount, or completely preventing, the activity of a particular enzyme that catalyzes a reaction.

[0123] The term "afucosylation" or "afucosylated" refers to a glycan structure that lacks fucose at the internal core structure.

[0124] As used herein, "thiofucosylation" or "thiofucosylated" refers to a glycan structure comprising at least one thiofucose. Typically in a "thiofucosylated" glycan structure, fucose is replaced by thiofucose.

[0125] As used herein, "5-thio-L-fucose" or "ThioFuc" refers to a fucose derivative in which the oxygen in the sugar ring is replaced by a sulfur atom. "5-thio-L-fucose" refers to the α and / or β anomer.

[0126]

[0127] As used herein, "acetylated 5-thio-L-fucose" or "AcThioFuc" refers to all forms of acetyl groups on thiofucose, which refers to 5-thio-L-fucose carrying 1-4 acetyl groups. Figure 1 For example, fully acetylated 5-thio-L-fucose carrying 4 acetyl groups is shown.

[0128] The derivatives of 5-thio-L-fucose are non-acetylated, partially acetylated 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-fucosephosphonate.

[0129] The non-acetylated, partially acetylated or fully acetylated forms of 2-F-5-thio-L-fucose are molecules according to formula I:

[0130] I

[0131] wherein R is independently H or acetyl.

[0132] The non-acetylated, partially acetylated or fully acetylated forms of 5-alkynyl-5-thio-L-fucose are molecules according to formula II:

[0133] II

[0134] wherein R is independently H or acetyl.

[0135] The non-acetylated, partially acetylated or fully acetylated forms of 6,6,6-trifluoro-5-thio-L-fucose are molecules according to formula III:

[0136] III

[0137] wherein R is independently H or acetyl.

[0138] The non-acetylated, partially acetylated or fully acetylated forms of 5-thio-L-fucosephosphonate are molecules according to formula IV:

[0139] IV

[0140] wherein R is independently H or acetyl.

[0141] The following picture shows the non-acetylated form. In the acetylated form, one or more OH groups are acetylated.

[0142]

[0143] As used herein, "core fucosylation" or "fucosylation" refers to the addition of fucose to N-acetylglucosamine ("GlcNAc") at the reducing terminus of an N-linked glycan.

[0144] As used herein, "N-glycoside-linked sugar chains" or "N-glycoside-linked glycans" are typically bound to asparagine 297 (according to Kabat's numbering), although complex N-glycoside-linked sugar chains can also be attached to other asparagine residues. As used herein, complex N-glycoside-linked sugar chains have biantennary complex sugar chains, primarily having the following structure:

[0145]

[0146] Wherein + / - indicates that sugar molecules may be present or absent, and the numbers indicate the connection position between sugar molecules. In the above structure, the end of the sugar chain bound to asparagine is called the reducing end (right side), and the opposite side is called the non-reducing end. Fucose is usually combined with the N-acetylglucosamine ("GlcNAc") of the reducing end, usually through α 1,6 bonds (the 6-position of GlcNAc is connected to the 1-position of fucose). "Gal" refers to galactose, and "Man" refers to mannose.

[0147] "Complex N-glycoside-linked sugar chains" do not include high-mannose type sugar chains in which mannose is incorporated only into the non-reducing termini of the core structure, but include

[0148] 1) a complex type, wherein the non-reducing terminal side of the core structure has one or more galactose-N-acetylglucosamine (also known as "gal-GlcNAc") branches, and the non-reducing terminal side of Gal-GlcNAc optionally has sialic acid, bisecting N-acetylglucosamine, etc.; or

[0149] 2) A hybrid type in which the non-reducing terminal side of the core structure has two branches of a high-mannose N-glycoside-linked sugar chain and a complex N-glycoside-linked sugar chain.

[0150] In some embodiments, a "complex N-glycoside-linked sugar chain" includes a complex type in which the non-reducing terminal side of the core structure has zero, one or more galactose-N-acetylglucosamine (also known as "gal-GlcNAc") branches, and the non-reducing terminal side of the Gal-GlcNAc optionally further has a structure (e.g., sialic acid, bisecting N-acetylglucosamine, etc.).

[0151] The gist of the present invention is to provide reagents and methods for reducing core fucosylation (i.e., fucosylation of N-glycoside-linked sugar chains of Fc-containing proteins or antibodies). In one embodiment, fucosylation is inhibited and thereby reduced or completely suppressed. In another embodiment, the N-glycoside-linked sugar chains of Fc-containing proteins or antibodies are instead thiofucosylated. Both effects can also occur in parallel, in different antibodies or in a single antibody carrying two different glycan structures.

[0152] Also provided is the antibody produced by such method.In other aspects, provide the culture medium comprising effective dose of 5-thio-L-fucose and / or 5-thio-L-fucose derivative, described derivative is selected from partially or completely 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 5-thio-L-fucophosphonate non-acetylated, partially acetylated or completely acetylated form.Hereinafter, derivative is not specified at every turn.Obviously, if mentioning 5-thio-L-fucose, it also includes the derivative specified above.However, preferred 5-thio-L-fucose and / or partially or completely acetylated 5-thio-L-fucose.

[0153] In some embodiments, fucosylation of complex N-glycoside-linked sugar chains that bind to the Fc region (or domain) is completely inhibited or reduced compared to antibodies or other Fc-containing proteins from the same host cells cultured under otherwise identical culture conditions but in the absence of 5-thio-L-fucose. "Under otherwise identical culture conditions but in the absence of 5-thio-L-fucose" generally means that the cell culture contains the same components and is performed under the same conditions, but with the exception that 5-thio-L-fucose is absent.

[0154] To inhibit or reduce core fucosylation, the host cells are cultured in a cell culture medium comprising 5-thio-L-fucose and / or its derivatives.

[0155] In a preferred embodiment, the cell culture medium in which the host cells are cultured does not contain L-fucose.

[0156] The amount of 5-thio-L-fucose contained in the liquid cell culture medium in which the host cells are cultured is an amount effective to reduce fucose incorporation. In this context, an "effective amount" refers to an amount of 5-thio-L-fucose sufficient to reduce fucose incorporation into complex N-glycoside-linked sugar chains of an antibody or another Fc-containing protein by at least 10%, at least 20%, at least 30%, at least 40%, or at least 60%.

[0157] Typically, this amount is between 0.1 µM and 1 mM.

[0158] If feed medium is added to cell cultures, they may contain higher amounts of 5-thio-L-fucose.

[0159] The amount of effective 5-thio-L-fucose can be determined by standard cell culture methods. For example, cell culture assays can be used to help identify the optimal dosage range. The exact amount to be used also depends on the time of administration, host cell line, cell density, etc.

[0160] The fucosylation and / or thiofucosylation pattern of an antibody can be modulated by varying the concentration of 5-thio-L-fucose in the culture medium and / or the duration of exposure to 5-thio-L-fucose.

[0161] The powdered cell culture medium of the present invention is preferably produced by mixing all components and grinding them. Mixing the components is known to those skilled in the art of producing dry powdered cell culture media by grinding. Preferably, all components are thoroughly mixed so that all parts of the mixture have approximately the same composition. The higher the homogeneity of the composition, the better the quality of the resulting culture medium in terms of uniform cell growth.

[0162] Grinding can be carried out using any type of mill suitable for producing powdered cell culture medium. Typical examples are ball mills, pin mills, fitz mills or jet mills. Pin mills, fitz mills or jet mills are preferred, with pin mills being very preferred.

[0163] A person skilled in the art knows how to operate such a mill.

[0164] For example, in the case of a pin mill, large-scale equipment mills having a disc diameter of about 40 cm are usually operated at 1 to 6500 revolutions per minute, preferably 1 to 3000 revolutions per minute.

[0165] Milling can be carried out under standard milling conditions to give a powder with a particle size between 10-300 μm, most preferably between 25-100 μm.

[0166] Preferably, all components of the mixture subjected to milling are dry. This means that, if they contain water, they contain only water of crystallization, but not more than 10% by weight, preferably not more than 5% by weight, most preferably not more than 2% by weight of unbound or uncoordinated water molecules.

[0167] In a preferred embodiment, the milling is carried out in an inert atmosphere. The preferred inert protective gas is nitrogen.

[0168] In another preferred embodiment, all components of the mixture are frozen prior to grinding. Freezing the components prior to grinding can be performed by any means that ensures the components are cooled to a temperature below 0°C and most preferably below -20°C. In a preferred embodiment, freezing is performed with liquid nitrogen. This means that the components are treated with liquid nitrogen prior to introduction into the mill, for example by pouring liquid nitrogen into the container in which the components are stored. In a preferred embodiment, the container is a feeder. If the container is a feeder, the liquid nitrogen is preferably introduced on or near the side of the feeder where the components are introduced.

[0169] Typically, the components were treated with liquid nitrogen for 2-20 seconds.

[0170] Preferably, cooling of the ingredients is performed in such a way that the temperature of all ingredients entering the mill is below 0°C, most preferably below -20°C.

[0171] In a preferred embodiment, all ingredients are placed in a container and the mixture is transferred from the container to a feeder, most preferably a metering screw feeder. In the feeder, the ingredients are sometimes further mixed (depending on the type of feeder) and further cooled. The frozen mixture is then transferred from the feeder to the mill, so that the mixture ground in the mill is still preferably below 0°C, more preferably below -20°C.

[0172] Typically, the blending time (ie, the residence time of the mixture of ingredients in the feeder) is greater than one minute, preferably between 15 and 60 minutes.

[0173] The metering screw feeder (also called metering worm wheel) usually runs at a speed of 10-200 revolutions per minute, preferably it runs at 40-60 revolutions per minute.

[0174] Typically, the temperature of the mill is maintained between -50°C and +30°C. In a preferred embodiment, the temperature is maintained at about 10°C.

[0175] Oxygen levels during milling are preferably below 10% (v / v).

[0176] The process can be carried out, for example, batchwise or continuously. In a preferred embodiment, the process according to the invention is carried out continuously by permanently filling the mixture of the components into a feeder for cooling over a certain period of time and permanently filling the cooled mixture from the feeder into the mill.

[0177] After milling, the resulting dry powder culture medium can be further compacted, for example by dry compacting in a roller compactor, to enlarge the size of the particles.

[0178] To use a dry powdered culture medium, e.g. produced by grinding or wet or dry compaction, a solvent, preferably water (most particularly distilled and / or deionized water or purified water or water for injection) or an aqueous buffer, is added to the culture medium and the components are mixed until the culture medium is completely dissolved in the solvent and a ready-to-use liquid culture medium is produced.

[0179] The solvent may also contain saline, soluble acid or base ions to provide a suitable pH range (typically within the range of pH 1.0 to pH 10.0), stabilizers, surfactants, preservatives, and alcohols or other polar organic solvents.

[0180] Other substances may also be added to the mixture of cell culture medium and solvent, such as buffer substances for adjusting pH, fetal bovine serum, sugars, etc. The resulting liquid cell culture medium is then contacted with the cells to be grown or maintained.

[0181] The dry powder or granular culture medium according to the present invention typically contains 5-thio-L-fucose in an amount such that, after dissolution, the resulting liquid culture 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 culture medium preferably does not contain any L-fucose.

[0182] The present invention further relates to a method for culturing cells, said method being carried out as follows:

[0183] a) providing a bioreactor,

[0184] b) mixing the cells to be cultured with a cell culture medium produced by dissolving the dry powder culture medium according to the present invention,

[0185] c) Incubating the mixture from step b).

[0186] In one embodiment, the bioreactor is a perfusion bioreactor.

[0187] A bioreactor is any container, flask, or tank in which cells can be cultured. Incubation is typically performed under suitable conditions (e.g., a suitable temperature, etc.). Those skilled in the art will appreciate suitable incubation conditions for supporting or maintaining cell growth / culture, such as suitable temperature, pH, osmotic pressure, aeration, agitation, etc., as well as bioreactors that limit or ideally avoid contamination by exogenous microorganisms from the environment.

[0188] A perfusion bioreactor is a bioreactor in which perfusion cell culture can be performed. It includes a bioreactor container that is usually closed during cell culture, an agitator in the container, a pipeline for introducing fresh culture medium, a harvesting line for removing a harvesting stream containing cells, liquid culture medium and target product from the bioreactor, and a cell retention device in the harvesting line, wherein the cell retention device retains the cells while the liquid portion of the harvest can be collected. A review of perfusion cell culture that provides details of advantageous settings 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.

[0189] The present invention also relates to a method for culturing cells in a bioreactor, said method being carried out as follows:

[0190] - filling the bioreactor with cells and aqueous cell culture medium,

[0191] - incubating cells in bioreactors,

[0192] - adding the cell culture medium to the bioreactor continuously during the entire incubation time of the cells in the bioreactor or once or several times during said incubation time,

[0193] The culture medium preferably has a pH of less than pH 8.5 and comprises at least 5-thio-L-fucose and / or its derivatives as defined above.

[0194] In a preferred embodiment, the culture medium is a feed medium.

[0195] Typically the feed medium contains between 50-300 g / l of solid components dissolved in a solvent.

[0196] In another embodiment, the culture medium is a perfusion medium.

[0197] In one embodiment, in the method of the invention, the feed medium added to the bioreactor continuously during the incubation period or once or several times within said time has a different composition.

[0198] In another generally preferred embodiment, in the process according to the invention, the feed medium which is added to the bioreactor during the incubation period, either continuously or once or several times within the time, always has the same composition.

[0199] Antibodies and antibody derivatives produced by the methods of the invention can be isolated from cell cultures and purified using, for example, gel electrophoresis, filtration, dialysis, and chromatography (such as, for example, affinity chromatography and / or ion exchange chromatography).

[0200] In some embodiments, the antibodies or antibody derivatives produced by the methods of the present invention have higher effector function (e.g., ADCC activity) than antibodies or antibody derivatives produced in the absence of 5-thio-L-fucose. ADCC activity can be measured using assays known in the art, and in exemplary embodiments, ADCC activity 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 a parent antibody with core fucosylation.

[0201] 5-Sulfo-L-fucose and / or its derivatives are particularly suitable for modifying core fucosylation because they generally have no significant effect on cell performance (VCD and titer). The glycosylation pattern of antibodies produced by the method of the present invention shows that sometimes core fucosylation is reduced, whereby less overall core fucosylation occurs, and sometimes core fucosylation is reduced due to its replacement by sulfofucosylation. Sometimes both effects can be observed simultaneously. Generally, the level of fucosylation can be reduced by using increasing amounts of 5-Sulfo-L-fucose, and in parallel, the incorporation of 5-Sulfo-L-fucose can be increased by using increasing amounts of 5-Sulfo-L-fucose, whereby the effects are not completely antiparallel, such that afucosylation and sulfofucosylation can be modified by carefully selecting the concentration of 5-Sulfo-L-fucose added to the cell culture.

[0202] Compared to the known inhibitor 2F-peracetylfucose, 5-thio-L-fucose showed higher efficiency. As shown in Example 4, the production of antibodies treated with 5-thio-L-fucose showed a 64%, 68%, and 67% reduction in fucosylation at days 7, 10, and 12, respectively, compared to the control. In contrast, the addition of the same concentration of 2F-peracetylfucose (a known fucose analog) was only able to achieve a reduction of 11%, 50%, and 58% at days 7, 10, and 12, respectively.

[0203] It could further be shown that antibodies produced using the method of the present invention using a culture medium comprising 5-thio-L-fucose exhibited higher binding affinity to their receptors.

[0204] The present invention is further illustrated by the following figures and examples, however, the present invention is not limited thereto.

[0205] The entire disclosure of all applications, patents and publications cited above and below, and the corresponding EP 19218172.5 filed on December 19, 2019, are incorporated herein by reference. Example

[0206] The following examples represent practical applications of the present invention.

[0207] Example 1: Expression of recombinant protein in the presence of 5-thio-L-fucose.

[0208] A common fed-batch method was used to determine the 5-thio-L-fucose ( Figure 1 ) on the glycosylation profile of recombinant proteins. Four different Chinese hamster ovary (CHO) cell clones expressing three different monoclonal antibodies (mAbs) and fusion proteins were tested. Cells were cultured in rotating tubes at 37°C, 5% CO2, and 80% humidity for between 14 and 20 days. As a starting cell density, 3 × 10 cells were grown in 30 mL of Cellvento® 4 CHO medium (pH 7.0 ± 0.1). 5Cells were grown at 320 rpm agitation for clones 1 and 2 (Cellvento® Platform), producing mAb1 and mAb2, respectively. Cells were cultured with 3% (v / v) Cellvento® 4Feed on days 3, 5, 10, 12, and 14, and fed with 6% (v / v) feed on day 7. Clones 3 and 4, producing mAb3 and fusion proteins, were seeded at the same cell density in 30 mL of Ex-Cell® Advanced Media (pH 7.2 ± 0.1) with agitation set at 230 rpm. Clones 3 and 4 were fed with 5% (v / v) Ex-Cell® Advanced Feed on days 3, 5, 7, 10, 12, 14, and 17. Feeds were supplemented with 400 μM 5-thio-L-fucose, 400 μM acetylated 5-thio-L-fucose, 400 μM acetylated 2F-fucose as a positive control, or with the corresponding amount of DMSO (1.17%) as a solvent for the two acetylated fucose analogs. The pH of all Cellvento® feeds was adjusted to 7.0 ± 0.1, and the pH of Ex-Cell® Feed was adjusted to 8.5 ± 0.1.

[0209] Glucose levels were maintained above 4 g / L by adding specific amounts of 400 g / L glucose stock solution as needed during the week to a maximum of 6 g / L and to a maximum of 13 g / L over the weekend. Glucose and titer in the supernatant were determined spectrophotometrically and turbidimetrically using a bioprocess analyzer, CEDEX Bio HT (Roche, Mannheim, Germany). Viable cell density (VCD) and viability were assessed using a Vi-CELL™ XR 2.04 cell counter (Beckman Coulter, Fullerton, CA, USA).

[0210] Typically, 5-thio-L-fucose treatments were compared to control conditions without any supplementation. Acetylated 5-thio-L-fucose was compared to a known positive control, 2F-peracetylfucose, and a DMSO control used to solubilize both acetylated fucose analogs.

[0211] As in Figure 2-5As shown in, in four biological replicates, when compared with two control conditions and the positive control, the cell performance (VCD and titer) of the CHO cell lines studied with or without acetylated 5-sulfo-L-fucose was not significantly affected. Slight changes were detected in the two platforms for mAb2 and mAb3. Compared with the control that was not supplemented, the production of mAb2 caused a slight reduction in titer after the 12th day under all conditions. On the 17th day, the titer of 2.18 g / L in the control was reduced to 2.02 g / L and 1.99 g / L with 5-sulfo-L-fucose and DMSO control, respectively. Detected with acetylated 5-sulfo-L-fucose, it was further reduced to 1.82 g / L, to a level similar to the positive control 2F-full acetylfucose (1.78 g / L). Because two fully acetylated fucose analogs are all dissolved in DMSO, the titer portion that reduces is due to the solvent.

[0212] For mAb3 production, approximately 13% and 24% reduced VCD were detected for 5-thio-L-fucose and acetylated 5-thio-L-fucose by calculating the area under the curve (compared to the control) up to day 17. The DMSO control showed a similar 13% reduction, indicating that the solvent for acetylated 5-thio-L-fucose most likely resulted in a further reduction in VCD. Since the reduced VCD did not affect the titer (2.25 g / L to 2.33 g / L), it was considered within the biological variability of the experiment.

[0213] Example 2: Incorporation of 5-thio-L-fucose into glycan structures

[0214] The glycosylation profiles of recombinant proteins produced in a control fed-batch were compared to the profiles 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).

[0215] Glycosylation patterns were analyzed by ultra-performance liquid chromatography coupled to a mass spectrometer (UPLC-MS). Purified antibodies were denatured, and glycans were released from the antibodies by digestion with peptide N-glycosidase F (PNGase F). 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 × 150 mm) was used, which separates glycans based on their hydrophobicity. Chromatography was performed at 45°C using a 50 mM ammonium formate (A) (pH 4.4) solution and an acetonitrile (B) gradient. Specifically, the A:B ratio was 20:80 at the beginning (0 minutes) and, at a flow rate of 0.5 ml / min, changed to 27:73 at 3 minutes and 37:63 at 35 minutes. From 36.5 to 39.5 minutes, A alone 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 minutes until the process was completed at 55 minutes. Detection was performed using a fluorescence detector with an excitation wavelength of 265 nm and an emission wavelength of 425 nm. The identification of glycan structures was performed by MS using positive electrospray ionization (ESI) and calibration with standards every 30 s.

[0216] Glycans containing non-natural 5-thio-L-fucose were identified by retention time shift, mass shift, and fragmentation pattern. The mass shift of approximately +16 Da is caused by the exchange of the epoxy group in fucose (164.0684 g / mol) with the sulfur in the thio-L-fucose derivative, resulting in a theoretical mass of 180.0455 g / mol for the 5-thio-L-fucose derivative. This mass is similar to that of galactose (180.0633 g / mol) but can be distinguished by a higher-resolution mass spectrometer (mass difference 115 ppm).

[0217] In addition, fragment spectra were obtained using LC-MS / MS. The fragmentation pattern of glycans containing thiofucose is similar to that of the corresponding fucosylated glycans, but is different for each glycan fragment containing fucose. For example, the theoretical mass of the Rapifluor-GlcNAc-Fuc fragment is 679.3304 g / mol, while the theoretical mass of Rapifluor GlcNAc-ThioFuc is 695.3076 g / mol. This difference allows for the unambiguous identification of the sugar structure ( Figure 6 , A fucosylated G0F, B thiofucosylated G0ThioF and C G1 fragmentation patterns).

[0218] In addition, the retention time of sulfofucosylated glycans can be distinguished from fucosylated glycans. Figure 7Figure 2 shows an exemplary shift in the fluorescence peak retention time of a mAb treated with 800 µM 5-thio-L-fucose. The thiofucosylated G0F peak was detected at 17.19 minutes, while the G0 peak eluted at 17.38 minutes. Separation of the thiofucosylated G1F isoform peaks can be observed at 20.88 minutes and 21.52 minutes, compared to retention times of 22.81 minutes and 23.51 minutes for the two fucosylated G1F isoforms. Overall, this data confirms the replacement of core fucosylation by 5-thio-L-fucose in the glycan structure.

[0219] Figure 7 Shown are the fluorescent signals obtained after separation of released, labeled glycans using UPLC, showing the shifted retention time of the peak corresponding to sulfofucosylated glycans compared to fucosylated glycans.

[0220] Example 3: 5-Thio-L-fucose can reduce fucosylation by incorporating 5-thio-L-fucose.

[0221] A common fed-batch method was used to determine the effect of 5-thio-L-fucose on the glycosylation profile. Four different Chinese hamster ovary (CHO) cell clones expressing three different monoclonal antibodies (mAbs) and fusion proteins were tested. The cells were cultured in rotating tubes at 37°C, 5% CO2 and 80% humidity for between 14 and 20 days. As a starting cell density, 3×10 5Cells were grown at 320 rpm agitation for clones 1 and 2 (Cellvento® Platform), producing mAb1 and mAb2, respectively. Cells were cultured with 3% (v / v) Cellvento® 4Feed on days 3, 5, 10, 12, and 14, and fed with 6% (v / v) feed on day 7. Clones 3 and 4, producing mAb3 and fusion proteins, were seeded at the same cell density in 30 mL of Ex-Cell® Advanced Media (pH 7.2 ± 0.1) with agitation set to 230 rpm (Ex-Cell® Platform). Clones 3 and 4 were fed with 5% (v / v) Ex-Cell® Advanced Feed on days 3, 5, 7, 10, 12, 14, and 17. Feeds were supplemented with 400 μM 5-thio-L-fucose, 400 μM acetylated 5-thio-L-fucose, and 400 μM acetylated 2F-fucose as positive controls, or with the corresponding amount of DMSO (1.17%) 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 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 as needed during the week to a maximum of 6 g / L and to a maximum of 13 g / L over the weekend. Glucose and titer in the supernatant were measured using the CEDEX Bio HT bioprocess analyzer (Roche, Mannheim, Germany).

[0222] To investigate glycosylation profiles 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 using the GlycoWorks™ RapiFluor-MS™ N-Glycan Kit as described by ultra-performance liquid chromatography coupled to a mass spectrometer (UPLC-MS).

[0223] Typically, treatments with or without acetylated 5-thio-L-fucose were compared to control conditions without any supplementation, to a known positive control 2F-peracetylfucose, and to a DMSO control used to solubilize two acetylated fucose analogs. Since harvesting of recombinantly produced proteins is common on day 12, the glycosylation profiles on day 12 were visualized. Quantification of glycans was obtained using the relative peak areas of the fluorescent signals. Almost all peaks were attributed to glycan structures based on their mass. Unknown glycan structures and signals that were not mass-tested were summarized as unknown species (not shown).

[0224] Four different Chinese hamster ovary (CHO) cell clones expressing three different monoclonal antibodies (mAbs) and fusion proteins were tested. In all tested cell lines producing different recombinant proteins, core-fucosylation was effectively reduced by 5-thio-L-fucose treatment.

[0225] mAb1 produced in clone 1 achieved reduced fucosylation by 5-thio-L-fucose through high incorporation of the fucose analog. Addition of 400 μM 5-thio-L-fucose with or without acetylation resulted in a reduction in core-fucosylated glycans to approximately 26% on day 12, compared to 94% core-fucosylated glycans in the control ( Figure 8 Figure 4. A). Addition of the same concentration of acetylated 2F-fucose (a fucose analog known to reduce fucosylation) was less efficient at day 12, resulting in 36% core-fucosylation. Compared to acetylated 2F-fucose, native fucosylation was reduced by 5-thio-L-fucose, independently of acetylation, by high incorporation of 5-thio-L-fucose into approximately 70% of glycans. Compared to the control and 5-thio-L-fucose (5%), acetylated 2F-fucose only increased afucosylation to 60% at day 12.

[0226] Production of mAb2 resulted in 8% core-fucosylation ( 12 days after treatment with or without acetylated 5-thio-L-fucose ) compared to 77% fucosylation in the control. Figure 8 B). In contrast to mAb1, whose afucosylation level did not change in response to 5-thio-L-fucose treatment, mAb2 achieved reduced fucosylation with 32% incorporation of 5-thio-L-fucose and 60% afucosylation (compared to 20% in the control condition). By increasing afucosylation to 78%, the known positive control achieved a fucosylation level of 14% on day 12.

[0227] Compared to 2F-fucose, a known fucose analogue, acetylated, only the same amount of afucosylation was achieved by clone 3 expressing mAb3, resulting in approximately 34% afucosylation at day 12 ( Figure 8 C). Incorporation of 43% 5-thio-L-fucose resulted in a lower fucosylation level of 21% compared to 90% in the control and 65% in the positive control at day 12.

[0228] On day 12, a glycosylation pattern similar to that of mAb2 was observed in the fusion protein produced in clone 4 ( Figure 8D) Compared to the control, core-fucosylation was reduced by approximately 63% by treatment with or without acetylated 5-thio-L-fucose. For both, 5-thio-L-fucose incorporation was observed in 39% of glycans, and 52% were afucosylated. The positive control achieved 82% afucosylation, with no detectable incorporation of the fucose analog.

[0229] In summary, at day 12, increased amounts of incorporated 5-thio-L-fucose were detected at approximately 32%, 39%, 43%, and 70% for mAb2, fusion protein, mAb3, and mAbl, respectively.

[0230] For mAbl, mAb3, fusion protein and mAb2, increasing amounts of afucosylation were detected, approximately 5%, 34%, 52% and 60%.

[0231] Figure 8 Shown are the effects of 5-thio-L-fucose on fucosylation profiles using CHO cell lines producing (A) mAb1, (B) mAb2, (C) mAb3, and (D) fusion proteins. Data represent the mean ± SEM of two biological replicates at day 12.

[0232] Example 4: Addition of 5-thio-L-fucose to the process results in a faster reduction of fucosylation compared to addition of 2F-peracetylfucose.

[0233] In order to identify the time required for 5-thio-L-fucose supplementation to affect the glycosylation profile of recombinantly produced proteins, samples collected at different time points during the fed-batch phase were studied. Samples in which cell viability was higher 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 A PhyTips® (PhyNexus Inc, San Jose, CA). Glycosylation patterns were analyzed using the GlycoWorks™ RapiFluor-MS™ N-glycan kit as described by ultra-high performance liquid chromatography (UPLC-MS) coupled to a mass spectrometer.

[0234] Early studies of glycan profiles during fed-batch experiments indicated the high efficiency of 5-thio-L-fucose. Compared to the control, mAb1 produced with 5-thio-L-fucose treatment showed a 64%, 68% and 67% reduction in fucosylation at 7, 10 and 12 days, respectively. By contrast, adding the same concentrations of 2F-peracetylfucose (a known fucose analogue) was only able to achieve a reduction of 11%, 50% and 58% at 7, 10 and 12 days, respectively. This indicates that 5-thio-L-fucose is more efficient than the known inhibitor 2F-peracetylfucose.

[0235] To examine the impact of 5-thio-L-fucose earlier in the fed-batch phase, the glycosylation profile of mAb3 was additionally investigated on day 5. Treatment with 400 μM 5-thio-L-fucose on day 5 resulted in a low fucosylation level of approximately 48%, whereas 400 μM acetylated 2F-fucose on day 5 resulted in approximately 84% fucosylation, similar to the 87% fucosylation level observed 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 contrast, the known inhibitor, acetylated 2F-fucose, only showed a reduction in fucosylation to approximately 78%, 72%, 65%, and 57%, respectively, on days 7, 10, 12, and 14. Therefore, 5-thio-L-fucose may be advantageous for use with short culture times, such as batch or perfusion methods. In addition, reduced amounts or different feeding schedules can be used to reduce the amount of fucose derivatives required to be added by this method or to titrate the level of fucosylation.

[0236] Figure 9 Shown is the accelerating 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.

[0237] Example 5: Increasing the concentration of 5-thio-L-fucose results in higher incorporation.

[0238] The ability to achieve different fucosylation levels by addition of 5-thio-L-fucose during fed-batch was investigated using two platforms and two different CHO clones producing mAbl or mAb3.

[0239] The cells were cultured in rotating tubes at 37°C, 5% CO2 and 80% humidity for 14-20 days. As a starting cell density, 3×10 cells were grown in 30 mL of Cellvento®4 CHO medium (pH 7.0±0.1). 5Clone 1 (Cellvento® Platform) producing mAb 1 was grown at 320 rpm agitation and fed with 3% (v / v) Cellvento® 4Feed on days 3, 5, 10, 12, and 14, and 6% (v / v) fed on day 7. Clone 3, producing mAb 3, was seeded at the same cell density in 30 mL of Ex-Cell® Advanced Media (pH 7.2 ± 0.1) with agitation set at 230 rpm (Ex-Cell® Platform). Clone 3 was fed with 5% (v / v) Ex-Cell® Advanced Feed on days 3, 5, 7, 10, 12, 14, and 17.

[0240] Feeds were supplemented with increasing concentrations of 5-thio-L-fucose, acetylated 5-thio-L-fucose, or a corresponding amount of DMSO (1.17%) used as a solvent for acetylated 5-thio-L-fucose. The pH of all Cellvento® feeds was adjusted to 7.0 ± 0.1, and the pH of Ex-Cell® Feed was adjusted to 8.5 ± 0.1. Glucose levels were maintained above 4 g / L by adding a specified amount of 400 g / L glucose stock solution as needed during the week to a maximum of 6 g / L and to a maximum of 13 g / L over the weekend. Glucose and titer in the supernatant were measured using the bioprocess analyzer CEDEX Bio HT (Roche, Mannheim, Germany).

[0241] Dose response of clone 1 producing mAb1 was performed with 200, 400 and 800 μM acetylated 5-thio-L-fucose ( Figure 10 A). Glycosylation profiles were investigated on days 7, 10, and 12. Since only minor changes were detected over time, the glycan structures of the produced IgG are presented as averages for all three days. The data indicate that by adding 200 μM, 400 μM, and 800 μM acetylated 5-thio-L-fucose, respectively, the incorporation of the non-natural fucose analog increased from 50% to 81% and 87%. Thus, compared to 96% fucosylation in the two control conditions (including 1.17% DMSO as a solvent for acetylated 5-thio-L-fucose), the fucosylation levels were reduced to 46%, 16%, and 9%.

[0242] In addition, the effect of increasing concentrations of 5-thio-L-fucose on clone 3, producing mAb 3, was investigated. Concentrations of 5-thio-L-fucose ranging from 50 μM to 800 μM resulted in increasing amounts of incorporated 5-thio-L-fucose and increasing amounts of afucosylation. At day 12, the highest applied concentration of 800 μM 5-thio-L-fucose resulted in approximately 36% afucosylation, and approximately 48% incorporation of the fucose analog, resulting in approximately 15% remaining natively fucosylated glycans. Thus, when compared to the control, the addition of 800 μM 5-thio-L-fucose reduced fucosylation by approximately 75% at day 12. Gradually decreasing the concentration to 400 μM, 200 μM, and 50 μM 5-thio-L-fucose resulted in approximately 44%, 36%, and 11% reductions in fucose analog incorporation at day 12, respectively. This data indicates that by careful selection of concentration, a target amount of 5-thio-L-fucose incorporation can be specified.

[0243] Figure 10 Shown is the dose response of 5-thio-L-fucose on the glycosylation profile. (A) Effect of acetylated 5-thio-L-fucose on the CHO cell line producing mAb1. Data represent mean ± SEM at days 7, 10, and 12. (B) Effect of 5-thio-L-fucose on the CHO cell line producing mAb3. Data represent mean ± SEM of two biological replicates at day 12.

[0244] Example 6: Effect of 5-thio-L-fucose treatment on FcγRIIIa binding

[0245] The aim of this section was to investigate whether antibodies produced in the thiofucose-containing method would exhibit higher binding affinity to FcγRIIIa and thus potentially increased ADCC.

[0246] Indeed, increased binding of afucosylated antibodies to FcγRIIIa and thus enhanced ADCC activity have been described in the literature. Two isoforms of FcγRIIIa are known, with either a phenylalanine (F158) or a valine (V158) residue at position 158. The latter isoform is reported to have a higher affinity for IgG1 than the F isoform and was used in these studies.

[0247] The binding of samples after 5-ThioFuc treatment was studied by surface plasmon resonance using a Biacore T200 system (GE Healthcare, Uppsala, Sweden). The assay temperature was set at 25°C, and the sample compartment temperature was set at 15°C. A His capture kit from GE Healthcare was used, according to the manufacturer's instructions, in active and reference flow cells, with an S-series sensor chip CM 5 coupled to an anti-His antibody. For all experiments, the immobilization level was 12185 ± 436 resonance units (RU; 1 RU ≈ 1 pg / mm 2 ) range. Anti-His antibody was further 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 into the active flow cell at a flow rate of 10 μL / min 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 reference and active flow cells, ranging from 1.9, 5.6, 16.7, 50, 150, and 450 nM. Binding was recorded continuously while automatically subtracting nonspecific binding and injection artifacts. Single-cycle kinetics were used with a dissociation time of 600 s and a flow rate of 30 μL / min. Data collection was performed at a rate of 10 Hz. 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 using a 1:1 binding model. The overall dissociation constant, KD, was determined from the ratio of the dissociation kinetic rate constant (kd) and the association kinetic rate constant (ka). Data are presented in Table 1 as fold change compared to the control.

[0248] Cells treated with increasing concentrations of 5-thio-L-fucose produced antibodies with increased affinity for FcγRIIIa. Samples treated with 50 µM 5-thio-L-fucose or 400 µM 2F-PerAcFuc achieved 1.2- and 1.3-fold enhanced binding from day 5 compared to the control. Samples treated with 200, 400, and 800 μM 5-thio-L-fucose showed a 2.0-, 2.9-, and 2.6-fold increase in affinity, respectively, on day 5 compared to the control. At later time points, increased affinity for FcγRIIIa was observed for all treated samples. On day 12, treatment with 50, 200, 400, and 800 μM 5-thio-L-fucose detected an increased affinity of 2.0, 3.1, 3.8, and 4.8-fold, respectively. Sensorgrams obtained by injecting the same concentration series of control and treated antibodies ( Figure 11 ) illustrates the higher affinity of the antibody 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), resulting in a 3.6-fold increase in affinity. On day 14, due to similar glycosylation patterns, the treated samples showed similar KD values ​​compared to day 12. Enhanced binding of 1.7-, 3.6-, 4.1-, and 4.5-fold was detected by 5-thio-L-fucose treatment at 50, 200, 400, and 800 μM, respectively.

[0249] Table 1: Effect of 5-thio-L-fucose treatment on affinity of mAb3 to FcγRIIIa at day 5 (A), day 12 (B), and day 14 (C). Data are representative of two biological replicates.

[0250]

[0251]

[0252]

[0253] Figure 11 Overlays are shown of FcγRIIIa bound to a mAb3 control (black) or to 5-thio-L-fucose-treated mAb3 (grey) at day 12. Sensorgrams show binding levels to captured FcγRIIIa across a concentration range of 6-486 nM (n = 3). All sensorgrams were double-referenced.

Claims

1. A method for producing an antibody or other Fc-containing protein with reduced fucosylation, the method comprising: culturing a host cell in a medium comprising 5-thio-L-fucose and / or a 5-thio-L-fucose derivative selected from the group consisting of partially or fully acetylated 5-thio-L-fucose, 2-F-5-thio-L-fucose, and non-acetylated, partially acetylated, or fully acetylated forms of 6,6,6-trifluoro-5-thio-L-fucose, wherein the host cell expresses an antibody or other Fc-containing protein having an Fc domain having at least one N-glycoside-linked sugar chain bound to the Fc domain via an N-acetylglucosamine at the reducing terminus of the sugar chain, and The antibody or other Fc-containing protein is isolated from the cell, wherein the antibody or other Fc-containing protein has reduced fucosylation at the carbohydrate chain compared to the antibody or other Fc-containing protein from the same host cell cultured under otherwise identical culture conditions but in the absence of 5-thio-L-fucose and / or its derivatives.

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

3. The method according to claim 1, 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 method according to any one of claims 1 to 3, characterized in that The host cells were cultured as follows: - filling a bioreactor with said host cells and aqueous cell culture medium, - incubating said cells in said bioreactor, adding a cell culture medium to the bioreactor continuously during the entire incubation time of the cells in the bioreactor or once or several times during the incubation time, wherein the cell culture medium comprises 5-thio-L-fucose and / or a 5-thio-L-fucose derivative selected from the group consisting of partially or fully acetylated 5-thio-L-fucose, 2-F-5-thio-L-fucose and non-acetylated, partially acetylated or fully acetylated forms of 6,6,6-trifluoro-5-thio-L-fucose.

6. The method according to any one of claims 1 to 3, characterized in that The pH of the added culture medium is lower than pH 8.5 and contains 5-thio-L-fucose and / or its derivatives at a concentration between 10 nM and 100 mmol / l.

7. A method for preparing a thiofucosylated antibody or other Fc-containing protein, the method comprising: culturing a host cell in a medium comprising 5-thio-L-fucose and / or a 5-thio-L-fucose derivative selected from the group consisting of partially or fully acetylated 5-thio-L-fucose, 2-F-5-thio-L-fucose, and non-acetylated, partially acetylated, or fully acetylated forms of 6,6,6-trifluoro-5-thio-L-fucose, wherein the host cell expresses an antibody or other Fc-containing protein having an Fc domain having at least one N-glycoside-linked sugar chain bound to the Fc domain via an N-acetylglucosamine at the reducing terminus of the sugar chain, and The antibody or other Fc-containing protein is isolated 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 the group consisting of partially or fully acetylated 5-thio-L-fucose, 2-F-5-thio-L-fucose and non-acetylated, partially acetylated or fully acetylated forms of 6,6,6-trifluoro-5-thio-L-fucose.

9. The cell culture medium according to claim 8, characterized in that The cell culture medium is a feed medium.

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

11. The cell culture medium according to claim 8 or 9, 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 method for producing a cell culture medium according to any one of claims 8 to 11, the method being carried out as follows: a) mixing 5-thio-L-fucose and / or a 5-thio-L-fucose derivative selected from the group consisting of partially or fully acetylated 5-thio-L-fucose, 2-F-5-thio-L-fucose and non-acetylated, partially acetylated or fully acetylated forms of 6,6,6-trifluoro-5-thio-L-fucose with other components of the cell culture medium, b) subjecting said mixture of step a) to milling.

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

14. Method for producing a cell culture medium according to claim 12 or 13, characterized in that Prior to grinding, the mixture from step a) is cooled to a temperature below 0°C.

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