WO2006109781A1 - Process for production of glycoprotein composition - Google Patents

Abstract

A process for producing a composition of a glycoprotein having a complex glycoside-linked sugar chain from an animal cell, which is characterized in that the content of a fucose-free complex glycoside-linked sugar chain in the total amount of a complex glycoside-linked sugar chain contained in the composition is varied by culturing the animal cell in a culture medium under controlling the osmotic pressure of the culture medium.

Description

 Specification

 Method for producing glycoprotein composition

 Technical field

 [0001] The present invention relates to a method for producing a glycoprotein composition. Specifically, a glycoprotein composition having glycoside-linked sugar chains produced from animal cells, characterized by controlling the osmotic pressure in a medium in which animal cells are cultured, is contained in the composition. The present invention relates to a method for producing a glycoprotein composition in which the ratio of sugar chains in which fucose is not bound to sugar chains is changed with respect to all the glycoside-bonded sugar chains. The present invention also includes a glycoprotein composition having a glycoside-linked sugar chain produced from an animal cell, characterized in that the osmotic pressure in a medium in which the animal cell is cultured is controlled. The present invention relates to a method for changing the ratio of sugar chains in which fucose is not bonded to sugar chains to the total glycoside-linked sugar chains. Background art

 [0002] The osmotic pressure of the medium for producing useful substances using animal cells is 210_500mOsmZkg, which is widely used for the purpose of production research and industrialization of useful substances using animal cells. Yes (Non-Patent Document 1). In addition, in animal cells such as Chineseno, Muster ovarian tissue-derived cells (hereinafter referred to as CHO cells), and hyperidoma, which are often used in production, productivity is improved when the osmotic pressure is increased to 450 _600 mOsm / kg. Are known (Patent Documents 1 and 2).

 [0003] As a method of controlling the composition of the sugar chain bound to the glycoprotein, a method of changing the type of sugar chain bound to the glycoprotein by changing the sugar composition or sugar concentration in the medium (patent) Document 3), a method of modifying the sugar chain structure by changing the specific consumption rate of sugar in the medium (Patent Document 4), etc. are known.

In addition, as a method for controlling the composition of the sugar chain bound to the glycoprotein, the N-glycoside-linked sugar chain that binds to the immune function molecule as the sugar chain that binds to the glycoprotein is N- A method of regulating the activity of an immune function molecule by the presence or absence of fucose binding to acetylyldarcosamine (Patent Document 5), a composition comprising an antibody molecule having an N-glycoside-linked complex type sugar chain in the Fc region, All N- binding to Fc region contained in the composition Encodes an antibody molecule that produces an antibody composition in which the percentage of sugar chains that do not have fucose bound to N-acetylyldarcosamine at the sugar chain reducing end of glycoside-linked complex sugar chains is 20% or more. For example, a method for producing an antibody using CHo cells derived from Chinese or muster ovary tissues into which a gene to be transferred has been known (Patent Document 6) is known. In addition, a method for producing an antibody having high ADCC activity using YB2Z3.0Ag30 cells, which are rat myeloma cell lines, is known (Patent Document 7).

 Patent Literature l: W096 / 39488

 Patent Document 2: US4724206

 Patent Document 3: JP-A-62-292592

 Patent Document 4: WO02 / 02793

 Patent Document 5: WO00 / 61739

 Patent Document 6: WO02 / 31140

 Patent Document 7: WO01 / 29246

 Non-patent literature l: Biotechnol and Bioeng, 62, 1 120-123 (1999)

 Disclosure of the invention

 Problems to be solved by the invention

[0004] An object of the present invention is to provide a glycoprotein composition having a glycoside-linked sugar chain produced from an animal cell, characterized by controlling the osmotic pressure in a medium for culturing the animal cell. An object of the present invention is to provide a method for producing a glycoprotein composition in which fucose is bound to a sugar chain with respect to all glycoside-linked sugar chains contained in the composition, and the ratio of sugar chain and sugar chain is changed. .

 Means for solving the problem

That is, the present invention relates to the following (1) to (: 17).

 (1) In a glycoprotein composition having glycoside-linked sugar chains produced from animal cells by controlling the osmotic pressure in a medium for culturing animal cells, fucose is added to the glycoside-linked sugar chains of the glycoprotein. A method for producing a glycoprotein composition, wherein the ratio of sugar chains to which no is bonded is changed.

(2) Animal cells are cultured by controlling the osmotic pressure in a medium for culturing animal cells. A glycoprotein composition having a glycoside-linked sugar chain produced more, wherein the ratio of the sugar chain in which fucose is not bound to the glycoside-linked sugar chain of the glycoprotein is increased. Method.

 (3) The method according to (1) or (2) above, wherein the osmotic pressure in the medium is 200 to 400 mOsm / kg.

 (4) The method according to any one of (1) to (3) above, wherein the animal cell is a cell selected from rat cells, mouse cells, and hamster cells.

 (5) The method according to (4) above, wherein the rat cell is a myeloma cell or a hybrid cell of a myeloma cell line.

 (6) The method according to (4) above, which is a rat cell force SYB2 / 3HL.P2.G11.16Ag.20 (ATCC CRL 1662, ECACC No: 85110 501) cell.

 (7) The method according to (4) above, wherein the mouse cell is a cell selected from NS0 cells or Sp2 / 0 cells.

 (8) The method according to any one of (1) to (7) above, wherein the animal cell is a cell into which a recombinant DNA containing a DNA encoding a glycoprotein has been introduced.

 (9) The method according to any one of (1) to (8) above, wherein the glycoprotein is an antibody.

(10) In a glycoprotein composition having a glycoside-linked sugar chain produced from animal cells by controlling the osmotic pressure in a medium for culturing animal cells, the glycoside-linked sugar chain of the glycoprotein is produced. To change the proportion of sugar chains that do not have fucose bonded to them

(11) The method according to (10) above, wherein the osmotic pressure in the medium is 200 to 400 mOsmZkg.

(12) The method according to (10) or (11) above, wherein the animal cell is a cell selected from rat cells, mouse cells and hamster cells.

 (13) The method according to (12) above, wherein the rat cell is a myeloma cell or a hybrid cell of a myeloma cell line.

 (14) The method according to (12) above, which is a rat cell force YB2 / 3HL.P2.G11.16Ag.20 (ATCC CRL 1662, ECACC No: 8511 0501) cell.

(15) The above (11), wherein the mouse cell is a cell selected from NS0 cells or Sp2 / 0 cells. The method described.

 (16) The method according to any one of (10) to (15) above, wherein the animal cell is a cell into which a recombinant DNA containing a DNA encoding a glycoprotein has been introduced.

 (17) The method according to any one of (10) to (: 16) above, wherein the glycoprotein is an antibody.

The invention's effect

 [0006] A glycoprotein composition having a glycoside-linked sugar chain produced from an animal cell, characterized in that the osmotic pressure in a medium for culturing the animal cell is controlled and contained in the composition Provided is a method for producing a glycoprotein composition in which fucose is bonded to a sugar chain and the ratio of sugar chain and sugar chain is changed with respect to all glycoside-linked sugar chains.

 Brief Description of Drawings

 [0007] [FIG. 1] Each of anti-00 chimeric antibody producing 82/0 cell line 61_330 / ^ £ 1 ^ 1 BP— 7325)

 Three

 Cultivation in various osmotic pressure media prepared with a seed osmotic pressure regulator, and reduction of N-glycoside-linked complex sugar chains that bind to the Fc region of the antibody in the antibody composition obtained from the culture medium FIG. 5 is a graph showing the results of calculating the proportion of sugar chains in which fucose is not bound to the terminal N-acetylyldarcosamine. The vertical axis shows the ratio of the sugar chain in which the fucose is not bound to the N-acetylidanolosecosamine at the reducing end of the N-glycoside-bonded complex sugar chain that binds to the Fc region, contained in the antibody composition. The horizontal axis shows the value of the osmotic pressure cultured.

 [Fig.2] Production of anti-GD chimeric antibody YB2 / 0 cell line 61—33γ (FERM BP—7325)

 Three

 Fucose binds to N-acetylcyldarcosamine at the reducing end of the N-glycoside-linked complex-type glycan that binds to the Fc region, which is contained in the antibody composition obtained from the culture medium after culturing Fuedbachig in an osmotic medium. FIG. 5 is a diagram showing the results of calculating the proportion of sugar chains that are not used. The vertical axis shows the percentage of sugar chains in the antibody composition in which fucose is not bound to N-acetylyldarcosamine at the reducing end of the N-glycoside-bound complex sugar chain that binds to the Fc region of the antibody. The axis shows the value of the osmotic pressure cultured.

 [Fig.3] Anti-GD chimera antibody-producing cell line 61—33γ (FERM BP—7355)

 Three

Perform perfusion culture on the ground, in the antibody composition obtained from the culture medium. The ratio of the sugar chain in which fucose is not bound to N-acetyl darcosamine at the reducing end of the N-glycoside-linked complex sugar chain that binds to the Fc region of the antibody was calculated. The vertical axis shows the ratio of the sugar chain in which the fucose is not bound to the N-acetylyldarcosamine at the reducing end of the N-glycoside-bonded complex sugar chain that binds to the Fc region. The axis shows the osmotic pressure values cultured.

 [Fig. 4] Anti-CCR4 chimeric antibody production YB2 / 0 strain (FERM BP-7054) was cultured in each osmotic medium in an Erlenmeyer flask and bound to the Fc region of the antibody in the antibody composition obtained from the culture medium. The ratio of the sugar chain in which fucose is not bound to the N-acetylyldarcosamine at the reducing end of the N-glycoside-bonded sugar chain was calculated. The vertical axis represents the ratio of the sugar chain in which fucose is not bound to N-acetyldylcosamine at the reducing end of the N-glycoside-bonded complex type sugar chain that binds to the Fc region. Indicates the value of the osmotic pressure cultured.

 [Fig. 5] Anti-CCR4 antibody producing strain YB2 / 0 (FERM BP-7054) was cultured in each osmotic medium for 1 L, and in the antibody composition obtained from the culture medium, the antibody was in the Fc region. The ratio of the sugar chain in which fucose is not bound to N-acetylyldarcosamine at the reducing end of the N-glycoside-bonded complex sugar chain to be bound was calculated. The vertical axis represents the proportion of sugar chains in which fucose is not bound to N-acetylcylcosamine at the reducing end of the N-glycoside-bonded complex sugar chain that binds to the Fc region. Indicates the value of the osmotic pressure cultured.

 [Fig. 6] Transformed SP2 / 0 cell clone KM—871 (FERM

 Three

 BP-3512) was cultivated in batch in each osmotic medium, and in the antibody composition obtained from the culture medium, the reducing end of the N-glycoside-linked complex sugar chain that binds to the Fc region of the antibody. The proportion of sugar chains in which fucose is not bound to N-acetylyldarcosamine was calculated. The vertical axis shows the ratio of fucose bound to N-acetylcylcosamine at the reducing end of the N-glycoside-bonded complex-type sugar chain that binds to the Fc region contained in the antibody composition. The horizontal axis shows the osmotic pressure values cultured.

[Fig. 7] Batch production of anti-CCR4 antibody producing NS0 strain (FERM BP-7964) in each osmotic medium, and binding to the Fc region of the antibody in the antibody composition obtained from the culture medium The ratio of the sugar chain in which fucose is not bound to the N-acetylidanorecosamine at the reducing end of the N-glycoside-linked complex sugar chain was calculated. The vertical axis shows the ratio of the sugar chain that is not bound to fucose to N-acetylcylcosamine at the reducing end of the N-glycoside-bonded complex-type sugar chain that binds to the Fc region in the antibody composition. The axis shows the value of the osmotic pressure cultured.

 BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, the osmotic pressure (Osm) is

 (Formula 1)

 ○ sm = RTiC

 (The number of moles of solute in a unit volume: C, R: gas constant, T: absolute temperature, i: Fant's Hoff coefficient).

 [0009] The method for controlling the osmotic pressure in the medium in the present invention includes a method of changing the concentration of a substance that adjusts the osmotic pressure in the medium (hereinafter referred to as an osmotic pressure regulator), and an osmotic pressure different from that of the medium. To control the osmotic pressure by changing the molarity or electrolysis of the medium depending on the substance produced in the medium. Methods.

 [0010] As a method for changing the concentration of the osmotic pressure adjusting agent in the medium, the osmotic pressure adjusting agent is added to the medium, or the osmotic pressure adjusting agent in the medium is removed, thereby reducing the osmotic pressure in the medium. There are ways to change it.

 The osmotic pressure adjusting agent may be any substance that is involved in the osmotic pressure of the medium, that is, a substance that changes the molar concentration or the electrolysis degree. Specific examples include sodium chloride (NaCl), potassium chloride. (KC1), salts such as lithium chloride (LiCl), glucose, galactose, mannose, fucose, funolectose, sucrose, mannitol, sugars such as xylose, trehalose, sorbitol, glycerol, various amino acids, vitamins, soybeans, wheat, Hydrolyzate from plants such as rice and corn, hydrolyzate from animals such as ushi protein and milk protein hydrolyzate, yeast extract such as yeast ixstrata, sodium hydroxide (NaOH), sodium carbonate (Na CO 2), sodium bicarbonate (NaHCO 3)

2 3 3 Alkaline, Coenzyme Q, Creatine, Ficoll, Dairy Power capable of adding a medium additive such as an acid Preferably sodium chloride is used.

 [0011] Examples of the method of adding a solution having an osmotic pressure different from that of the medium to the medium include a method of adding a solution having a higher osmotic pressure, a solution or a lower osmotic pressure, and a solution.

 Solutions with high osmotic pressure include solutions that contain one or more salts such as sodium chloride in a medium that can be used for animal cells, solutions composed of high-concentration amino acids or vitamins, and animal cells. Examples include solutions in which salt, amino acids, vitamins, etc. are added to the medium.

 [0012] The solution having a low osmotic pressure includes a solution obtained by removing one or more salts such as sodium chloride and sodium from a medium usable for animal cells, a solution composed of minimum medium components such as amino acids and vitamins, Examples of media that can be used for animal cells include RPMI1640 medium, IMDM medium, and DMEM medium.

 [0013] Substances produced in the medium include substances produced by animal cells, substances produced by decomposition of medium components, and the like.

 Examples of substances produced by animal cells include lactic acid and ammonia. In addition, examples of the substance produced by the decomposition of the medium components include ammonia.

 In the present invention, the time for controlling the osmotic pressure may be any time as long as animal cells are producing the glycoprotein composition.

 [0014] Specific examples of the method for culturing while controlling the osmotic pressure in the medium include a method of appropriately adding the aforementioned osmotic pressure adjusting agent or a solution containing the osmotic pressure adjusting agent to the culture. As the addition method, the osmotic pressure of the medium is monitored, and an osmotic pressure adjusting agent or a solution containing the osmotic pressure adjusting agent is added as needed so that the osmotic pressure of the medium becomes constant. Examples thereof include a method of adding a regulator or a solution containing an osmotic pressure regulator, and a method of adding an osmotic pressure regulator or a solution containing an osmotic pressure regulator before seeding cells. When using a culture method with small fluctuations in osmotic pressure during the culture period, a method of adjusting the osmotic pressure only before seeding cells may be used.

[0015] The osmotic pressure in the medium can be measured as follows. In the present invention, when the osmotic pressure fluctuation during the culture period is small and the culture method is used, it may be measured by the osmotic pressure in the medium on the first culture day or the last culture day. When a culture method in which a solution containing a pressure regulator or an osmotic pressure regulator is added to the medium is used, the osmotic pressure fluctuates during the culture. It is preferable to measure the osmotic pressure in the section). Since the increase / decrease amount of the glycoprotein composition produced per unit time correlates with the increase / decrease value of the osmotic pressure in the medium, the osmotic pressure can be calculated by the following formula 2.

(Formula 2)

Osmotic pressure = ∑ (osmotic pressure in the unit interval) X (glycoprotein / total production of glycoprotein in the unit interval)

 In the present invention, in the glycoprotein composition having a glycoside-linked sugar chain produced from an animal cell, to increase the proportion of glycoprotein having a sugar chain in which fucose is not bound to a glycoside-linked complex type sugar chain, The osmotic pressure value is controlled to 200 to 400 mOsm / kg, preferably 240 to 360 mOsm / kg, more preferably 240 to 280 mOsm / kg.

 The animal cell in the present invention may be any animal cell, but animal cells such as rat cells, mouse cells, human cells, monkey cells, inu cells, hamster cells, or hybrid cells of these animal cells. System.

Specific examples of animal cells include Chinese nomstar ovarian tissue-derived cells (CH cells), rat myeloma cell line YB2Z0 cell, mouse myeloma cell line NS0 cell, mouse myeloma cell line SP2 / 0_Agl4 cell, Syrian hamster kidney tissue Derived from BHK cells (ATCC CCL 10), MDCK (ATCC CCL 34), PER_C6 ™, hybridoma cells, human leukemia cell line Namalva cells, embryonic stem cells, fertilized egg cells, etc. Preferably, rat cells, mouse cells Can be given. Examples of rat cells include Y 3Agl.2.3. (ATCC CRL— 1631), YO (ECACC No: 85110501) and YB2 / 0 (AT CC CRL 1662). Examples of mouse cells include NSO (ATCC CRL— 1827). , Sp 2/0 (ATCC CRL-1581) and the like, and myeloma cells or myeloma cell line hybrid cells. In addition, these cells obtained by subjecting these cells to mutation treatment or cell fusion with B cells obtained by immunizing a non-human mammal with an antigen Cells having equivalent properties are also included in the animal cells in the present invention.

[0017] In the present invention, the glycoprotein is preferably a eukaryotic cell-derived glycoprotein, more preferably a mammalian cell-derived glycoprotein. Further, it may be an artificially modified glycoprotein such as a fused glycoprotein or a partial fragment thereof.

 Glycoproteins include antibodies, erythropoietin (EPO) [J. Biol. Chem., 252, 5558 (1977)], thrombopoietin (TPO) [Nature, 369, 533 (1994)] tissue type plasminogen activator. , Prolokinase, thrombomodulin, antithrombin III, protein, blood coagulation factor VII, blood coagulation factor VIII, blood coagulation factor IX, blood coagulation factor X, blood coagulation factor XI, blood coagulation factor XII, prothrombin complex, fibrinogen, Albumin, gonadotropin, thyroid stimulating hormone, epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor, activin, osteogenic factor, granulocyte colony stimulating factor (G—CSF) [J. Biol Chem., 258, 9017 (1983)], stem cell factor (SCF) such as macrophage colony stimulating factor (M—CSF) [J. Exp. Med., 173, 269 (1992)], granulocyte-macrophage colony -Stimulating factor (GM-CSF) [J.Biol. Chem., 252, 1998 (1977)], granulocyte-macrophage colony stimulating factor (GM-CSF) [J. Biol. Chem., 252,1998 (1 977) )], Interferon α, Interferon β, Interferon γ, Interleukin — 2 (IL-2) [Science, 193, 1007 (1976)], Interleukin 6, Interleukin 10, Interleukin 11, Interleukin 1-12 ( IL—12) [J. Leuc. Biol., 55, 280 (1994)], soluble interleukin 4 receptor, tumor necrosis factor, Dnasel, galactosidase, glycosidase, darcocerebrosidase, hemoglobin, transferrin, etc. And partial fragments of these glycoproteins.

[0018] The sugar chain that binds to the glycoprotein is composed of a sugar chain that binds to asparagine (N-glycoside-linked sugar chain) and a sugar chain that binds to serine, threonine, etc. (0-glycoside-linked sugar). It is roughly divided into two types. These are collectively referred to as glycoside-linked sugar chains.

N-glycoside-linked sugar chains have various structures [Biochemical Experimental Method 23-Glucose Protein Glycan Research Method (Academic Publishing Center) Atsuko Takahashi (1989)], In some cases, a common core structure represented by the following structural formula (I) is included. [0019] [Chemical 1]

4GlcNAc

Structural formula (I)

 [0020] In Structural Formula (I), the end of the sugar chain that binds to asparagine is at the reducing end, and the opposite side is at the non-end.

 The N-glycoside-linked sugar chain is a high mannose-type sugar chain in which only mannose is bonded to the non-reducing end of the core structure, and galactose—N-acetylidanolecosamine (hereinafter referred to as Gato GlcNAc) on the non-reducing end of the core structure. )) In parallel with one or more branches, and Gal-GlcNAc

 A complex type (also called complex type) sugar chain with a structure such as sialic acid or bisecting N-acetylidanorecosamine on the non-reducing end side, and hymannose type and complex type on the non-reducing end side of the core structure Hybrid sugar chains with both branches

[0021] As the 0-glycoside-linked sugar chain, the reducing end of N-acetylgalatatosamine is α-bonded to the hydroxyl group of serine or threonine, and further galactose, Ν-acetyldarcosamine, Ν-acetylgalatato Examples include sugar chains to which samine, fucose, or sialic acid is bonded, sugar chains in which xylose is β-bonded to a hydroxyl group of selenium, and sugar chains in which galactose is β-bonded to a hydroxyl group of hydroxylysine.

 [0022] In sugar chains in which xylose is β-bonded to the hydroxyl group of serine, usually a plurality of sugars are bonded to the 4-position of the xylose, and a linear polysaccharide consisting of disaccharides is bonded to the end of the bonded sugar. ing. Examples of the substance having such a sugar chain structure include cartilage proteodalycan. Examples of the substance having a sugar chain structure in which galactose is β-bonded to the hydroxyl group of hydroxylysine include collagen.

[0023] The glycoprotein composition refers to a composition comprising glycoprotein molecules having Ν-glycoside-linked sugar chains or 0-glycoside-linked sugar chains. There are many sugar chains that bind to glycoproteins Since the sugar chain structure is diverse, there are many combinations of sugar chains in the sugar chains of glycoproteins. Accordingly, the glycoprotein composition in the present invention includes a composition composed of glycoprotein molecules bound to a single sugar chain structure, and a glycoprotein molecule bound to a plurality of different sugar chain structures. Such as a composition.

 [0024] The composition of glycoprotein molecules to which glycoside-linked sugar chains are bound is contained in a composition comprising fucose bound to a glycoside-linked sugar chain. Using known methods such as degradation and enzymatic digestion [Biochemical Experimental Method 23-Glycoprotein Glycan Research Method (Academic Publishing Center) Takahashi Eiko (1989)], the sugar chain is released and the released sugar chain is fluorescent. It can be determined by labeling or isotope labeling and separating the labeled sugar chain by a chromatographic method. Moreover, the released sugar chain can be analyzed and determined by the HPAED-PAD method [Journal 'Ob' Liquid 'Chromatography (J. Liq. Chromatogr.), 6, 1577 (1983)].

 [0025] In the present invention, as a sugar chain in which fucose is not bonded to a glycoside-linked sugar chain, for example, the 1-position of fucose is α-bonded to the 6-position of N-acetylcylcosamine of the N-glycoside-linked sugar chain. Ν-glycoside-linked sugar chains are not included.

 Among the glycoproteins listed above, the antibody composition refers to a composition comprising antibody molecules having a Ν-glycoside-linked sugar chain in the Fc region.

 [0026] An antibody is a tetramer in which two molecules of two types of polypeptide chains, a heavy chain and a light chain, are associated with each other. About one-quarter of the N-terminal side of the heavy chain and about one-half of the N-terminal side of the light chain (each about 100 amino acids) are called variable regions and are diverse and directly involved in antigen binding To do. Most of the parts other than the variable region are called constant regions. Antibody molecules are classified into IgG, IgM, IgA, IgD, and IgE classes based on the homology of the constant region.

 [0027] The IgG class is further classified into IgGl to IgG4 subclasses based on the homology of the constant region.

The heavy chain is divided into four immunoglobulin domains, VH, CH1, CH2, and CH3, from the N-terminal side. Between CH1 and CH2, there is a highly flexible peptide region called a hinge region, and CH1 and CH2 are separated. It is done. The structural unit consisting of CH2 and CH3 after the hinge region is called the Fc region. N-glycoside-linked sugar chains are attached. This region also binds Fc receptors, complements, etc. (Immunology Illustrated Original 5th Edition, published February 10, 2000, Nanedo Edition, Introduction to Antibody Engineering, January 1994 25 First edition, Jinjinshokan).

 [0028] Since the Fc region of an antibody molecule has a region where N-glycoside-linked sugar chains are bound one by one, two sugar chains are bound per antibody molecule. N-glycoside-linked sugar chains that bind to antibody molecules have various structures, and the sugar structures of the sugar chains include those of the core structure represented by the structural formula (I). There are many combinations of sugar chains in the two N-Dalcoside-linked sugar chains that bind to the antibody. Therefore, as an antibody composition in the present invention, as long as the effects of the present invention can be obtained, a composition composed of an antibody molecule having a single sugar chain structure, an antibody molecule having a plurality of different sugar chain structures And the like, and the like.

 [0029] As the sugar chain in which fucose is not bonded to N-acetylyldarcosamine, the 1-position of the fucose is α-bonded to the 6-position of N-glycidyl-linked sugar chain reducing terminal N-acetylyldarcosamine. Ν-glycoside-linked sugar chain, preferably 1-position of the fucose is not α-bonded to the 6-position of ァ -glycylside glycosamine at the reducing end of Ν-glycoside-bonded sugar chain Examples include sugar chains.

 [0030] ADCC activity refers to the fact that an antibody bound to a cell surface antigen such as a tumor cell in a living body is bound to an Fc region by binding an antibody Fc region to the Fc receptor present on the effector cell surface. Refers to the activity of activating the tumor cells and damaging tumor cells etc. [Monoclonal Antibodies: Principles and Applications, Wiley—Liss, In, Capter 2.1 (1995)]. Effector cells include killer cells, natural killer cells, activated macrophages, etc.

[0031] The antibody may be an antibody having such an antigen-binding property, but it binds to an antibody that binds to a tumor-related antigen, an antibody that binds to an antigen related to allergy or inflammation, or an antigen that relates to cardiovascular disease. The antibody class is preferably an antibody that binds to an antigen associated with an autoimmune disease, or an antibody that binds to an antigen associated with a virus or bacterial infection, and the antibody class is preferably IgG. [0032] Antibodies that bind to tumor-associated antigens include anti-GD2 antibody (Anticancer Res., 13, 331-33 6, 1993), anti-GD3 antibody (Cancer Immunol. Immunother., 36,260-266, 1993), anti-GM 2 antibodies (Cancer Res., 54, 1511-1516, 1994), anti-HER2 antibodies (Proc. Natl. Acad. Sci. USA, 89, 4285-4289, 1992), anti-CD52 antibodies (Nature, 332, 323- 327, 1988), anti-MAGE antibody (British J. Cancer, 83, 493-497, 2000), anti-HMl. 24 antibody (MolecularlmmunoL, 36, 387-395, 1999), anti-parathyroid hormone-related protein (PTHrP) Antibody (Cancer, 88, 2909-2911, 2000), anti-FGF8 antibody (Proc. Natl. Acad. Sci. USA, 86, 9911-9915, 1989) Anti-basic fibroblast growth factor antibody, anti-FGF8 receptor Antibody G. Biol. Chem., 265, 16455-16 463, 1990), anti-basic fibroblast growth factor receptor antibody, anti-insulin-like growth factor antibody (J. Neurosci. Res., 40, 647-659) , 1995), anti-insulin-like growth factor receptor antibody (J. Neurosci. Res., 40, 647-659, 1995), anti-PMSA antibody (J. U rology, 160,2396-2401, 199 8), anti-vascular endothelial growth factor antibody (Cancer Res., 57, 4593-4599, 1997), anti-vascular endothelial cell growth factor receptor antibody (Oncogene, 19, 2138- 2146, 2000), anti-CA125 antibody, anti-17-1A antibody, anti-integrin α ν3 antibody, anti-CD33 antibody, anti-CD22 antibody, anti-HLA antibody, anti-HLA-DR antibody, anti-CD20 antibody, anti-CD19 antibody, anti-EGF Examples include receptor antibodies (Immunology Today, 21 (8), 403-410 (2000)), anti-CD10 antibodies (American Journal of Clinical Pathology, 113, 374-382, 2000) and the like.

[0033] Examples of antibodies that bind to antigens related to allergy or inflammation include anti-interleukin 6 antibody (Immunol. Rev., 127, 5-24, 1992), anti-interleukin 6 receptor antibody (Mole cular Immunol. , 31, 371-381, 1994), anti-interleukin 5 antibody (Immunol. Rev., 127,5 -24, 1992), anti-interleukin 5 receptor antibody, anti-interleukin 4 antibody (Cytokine, 3, 562- 567, 1991), anti-interleukin 4 receptor antibody (J. Immunol. Meth., 217, 41-50, 1 998), anti-tumor necrosis factor antibody (Hybridoma, 13, 183-190, 1994), anti-tumor necrosis Factor receptor antibody (MolecularPharmacol., 58, 237-245, 2000), anti-CCR4 antibody (Nature, 400,77 6-780, 1999), anti-chemokine antibody (J. Immunol. Meth., 174, 249-257, 1994), anti-chemoin receptor receptor antibody (J. Exp. Med., 186, 1373-1381, 1997), anti-IgE antibody, anti-CD23 antibody, anti-CDlla antibody (Immunology Today, 21 (8), 403-410 (2000)), anti-CRTH2 antibody (JI mm unol "162, 1278-1286 (1999)), anti-CCR8 antibody (W099 / 25734), anti-CCR3 antibody (US620 7155).

[0034] Examples of antibodies that bind to an antigen associated with cardiovascular disease include anti-GpIIb / IIIa antibody (J. Immuno 1 "152, 2968-2976, 1994), anti-platelet-derived growth factor antibody (Science, 253, 1129- 1132, 1991), anti-platelet-derived growth factor receptor antibody (J. Biol. Chem., 272, 17400-17404, 199 7) or anticoagulant factor antibody (Circulation, 101, 1158-1164, 2000) It is possible.

 Antibodies that bind to antigens associated with autoimmune diseases (such as psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, multiple sclerosis) include anti-self DNA antibodies (Immunol. Letters, 72 , 61-68, 2000), anti-CDlla antibody, anti-ICAM3 antibody, anti-CD80 antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-integrin α4 7 antibody, anti-CD40L antibody, anti-IL-2 receptor And antibodies (Immunology Today, 21 (8), 403-410 (2000)).

 [0035] As an antibody that binds to an antigen associated with virus or bacterial infection, anti-gpl20 antibody

 (Structure, 8, 385-395, 2000), anti-CD4 antibody (J. Rheumatology, 25, 2065-2076, 1998), anti-CCR4 antibody, anti-verotoxin antibody (J. Clin. Microbiol., 37, 396-399) , 1999).

 [0036] An antibody is a protein produced in a living body by an immune reaction as a result of stimulation with a foreign antigen, and has an activity that specifically binds to an antigen. If it is a molecule containing the Fc region of an antibody, , Such molecules are also included.

 Specifically, in addition to antibodies, antibody fragments, fusion proteins containing an Fc region, and the like can be mentioned.

 [0037] As an antibody, in addition to an antibody secreted by a hybridoma cell produced from a spleen cell of an immunized animal, an antibody produced by gene recombination technology, that is, an antibody gene is inserted. Examples thereof include an antibody obtained by introducing the antibody expression vector into a host cell. Specifically, antibodies produced by Hypridoma, humanized antibodies, human antibodies and the like can be mentioned.

[0038] In the present invention, the hyperidoma is obtained by fusing a B cell obtained by immunizing a mammal other than a human with a myeloma cell derived from a rat or mouse. Means a cell producing a monoclonal antibody having a desired antigen specificity. Examples of humanized antibodies include human chimeric antibodies, human type homology determining regions (hereinafter referred to as CDR) -grafted antibodies, and the like.

 [0039] A human chimeric antibody is a non-human animal antibody heavy chain variable region (hereinafter, the heavy chain is also referred to as H chain, the variable region is also referred to as HV or VH) and an antibody light chain variable region (hereinafter referred to as "H chain"). The light chain is also referred to as LV or VL as the L chain) and the heavy chain constant region of the human antibody (hereinafter the constant region is also referred to as CH as the C region) and the light chain constant region of the human antibody (hereinafter also referred to as CL). Is an antibody. As animals other than humans, mice, rats, mice, musters, rabbits, etc. can be used as long as they can produce mice and hybridomas.

 [0040] For a human chimeric antibody, cDNAs encoding VH and VL are obtained from a hybridoma producing a monoclonal antibody, and inserted into expression vectors for host cells having genes encoding human antibody CH and human antibody CL, respectively. Thus, it is possible to construct a human chimeric antibody expression vector, introduce it into a host cell, express it and produce it.

 The CH of the human chimeric antibody may be any of those belonging to human immunoglobulin (hereinafter referred to as hlg), but is preferably of the hlgG class, and more preferably hlgG1, hIgG2, hIgG3, Any of the subclasses such as hIgG4 can be used. As the CL of the human chimeric antibody, any κ class or λ class can be used as long as it belongs to hlg.

 [0041] The human CDR-grafted antibody means an antibody obtained by grafting the VH and VL CDR amino acid sequences of a non-human animal antibody to appropriate positions of the human antibody VH and VL.

 The human CDR-grafted antibody constructs a cDNA encoding the V region obtained by grafting the VH and VL CDR sequences of non-human animal antibodies to the VH and VL CDR sequences of any human antibody. A human CDR-grafted antibody is constructed by constructing a human CDR-grafted antibody expression vector by inserting it into an expression vector for a host cell having a gene encoding the CL of a human antibody, and introducing the expression vector into the host cell. Can be expressed and produced.

[0042] The CH of the human CDR-grafted antibody may be any of those that belong to hlg, but those of the hlgG class are preferred, and those of hlgG, hIgG2, hIgG3, hIgG4 belonging to the hlgG class are preferred. Any of the subclasses can be used. As the CL of the human CDR-grafted antibody, any KL class or I class can be used as long as it belongs to hlg.

 [0043] The animal cell used in the present invention can be used as it is as long as it produces a glycoprotein composition. An animal into which a recombinant vector containing a DNA encoding a glycoprotein has been introduced. Cells can also be used.

Examples of expression vectors used for preparing a recombinant vector containing a DNA encoding a glycoprotein include pcDNAI, pcDM8 (Funakoshi), pAGE 107 (Japanese Patent Laid-Open No. 3-22979, 7) (: 1111010 _§ 3, 3,133 (1990)), pAS3-3 (Japanese Patent Laid-Open No. 2-227075), pCDM8 (Nature, 329, 840 (1987)), pcDNAl / Amp (Invitrogen), p REP4 (Invitrogen) PAGE103 [J. Biochem., 101, 1307 (1987)], pAGE210 and the like.

 [0044] As a promoter for preparing a recombinant vector, any promoter that functions in animal cells can be used. For example, the promoter of cytomegalovirus (CMV) IE (immediateearly) gene And SV40 early promoter, retrovirus promoter, metamouthonein promoter, heat shock promoter, SRa promoter, and the like. In addition, an enhancer of the IE gene of human CMV may be used together with a promoter.

 [0045] The host cell into which the recombinant vector is introduced may be any animal cell as described above, and preferably a rat cell or a mouse cell, such as Y3A gl.2.3., Y0, ΥΒ2 / 0, NS0, Sp2 / 0, etc., myeloma cells or myeloma cell line hybrid cells. In addition, these cells can be obtained by subjecting these cells to mutation treatment, immunizing a non-human mammal with an antigen and cell fusion with a B cell obtained, and the like. .

[0046] As a method for introducing a recombinant vector into a host cell, any method can be used as long as it is a method for introducing DNA into the cell. For example, an electoresis method [Cytote chnology, 3, 133 ( 1990)), calcium phosphate method (JP-A-2-227075), ribofunction method (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987), Virology, 52,456 (1973)), etc. The ability to boil S.

 [0047] A glycoprotein composition can be produced in a cell or in a culture supernatant by culturing animal cells into which a recombinant vector has been introduced by the above method in an appropriate medium. Examples of animal cells that produce glycoprotein compositions include transformed cells that produce anti-GD human chimeric antibodies 7-9-151 (FERM BP-6691), anti-CC

Three

 Transformed cells producing R4 chimeric antibody KM2760 (FERM BP-7054), transformed cells producing anti-CCR4 humanized antibody KM8759 (FERM BP— 8129) j; KM87 60 (FERM BP— 8130), anti-IL 5 receptor Transformed cells producing α-chain chimeric antibody KM7399 (FERM BP—5649), transformed cells producing anti-IL 5 receptor α-chain human CDR-grafted antibody KM8399 (FERM BP—5648) and KM9399 (FERM BP—5647), transformed cells producing anti-GM2 human CDR-grafted antibodies KM8966 (FER M BP—5105), KM8967 (FERM BP—5106), KM8969 (FERM BP—55 27) and KM8970 (FERM BP— 5528 ) Etc.

[0048] In the present invention, as a method for culturing animal cells, in a glycoprotein composition having a glycoside-linked sugar chain, fucose is bound to a glycoside-linked sugar chain that binds to the glycoprotein. Any of the commonly used animal cell culture methods can be used as long as it is a culture method capable of changing the chain ratio and efficiently producing glycoprotein. For example, batch culture, repeat batch culture, fed-batch culture, perfusion culture, etc. In order to increase the productivity of glycoprotein, it is preferable to use feedbatch culture or perfusion culture.

[0049] Fuedbatch culture is a culture method in which physiologically active substances, nutrient factors, and the like are additionally supplied in small amounts continuously or intermittently. Fuedbachi culture can prevent a decrease in the cell density of the cultured cells due to accumulation of waste products in the culture medium where the metabolic efficiency of the cells is high. In addition, since the desired glycoprotein in the collected culture medium has a higher concentration than that obtained in batch culture, the glycoprotein can be easily separated and purified, and compared with batch culture, The production amount of the glycoprotein can be increased. Furthermore, the osmotic pressure can be controlled using the added solution, and is easier to control than the notch culture. [0050] Perfusion culture is efficiently separated by a device that separates the culture solution and cells, the concentrated cells are returned to the original culture tank, and the reduced amount of fresh medium is newly supplied to the culture tank. Is the method. This method is relatively easy to control because the culture environment in the culture tank is always kept good. Further, since the osmotic pressure in the tank can be controlled by supplying a fresh medium, it is preferable for controlling the osmotic pressure in the medium.

 [0051] As a medium used in the method of the present invention, any basal medium used for normal animal cell culture can be used. If necessary, those containing each of the physiologically active substances and nutrient factors and containing a carbon source, a nitrogen source, etc. that can be assimilated by animal cells are used.

 Specifically, RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 ( 1959), 199 medium (Proceeding of the Society for the Biological Medicine, 73, 1 (1950)), F12 medium (Proc. Natl. Acad. Sci. USA, 53,288 (1965)), IMDM medium J. Experimental Medicine , 147, 923 (1978)], etc., preferably DMEM medium, F12 medium, IMDM medium, Hybridoma Serum Free medium (Invitrogen), Chenically Defined Hybridoma Serum Free medium (Invitrogen) It is done.

 [0052] The medium is supplemented with nutrient factors, physiologically active substances, and the like necessary for the growth of animal cells as necessary. These additives are appropriately added to the culture medium during the cultivation as necessary, depending on the force previously contained in the medium before the cultivation, or if necessary. The supply method may be in the form of one solution or two or more solutions. Moreover, the addition method does not ask | require continuous or intermittent. Nutritional factors include sugars, amino acids, vitamins, hydrolysates, lipids and the like. Examples of physiologically active substances include insulin, IGF_1, transferrin, anolebumin, coenzyme Q and the like.

 Ten

 Used as needed.

 [0053] Examples of the sugar include glucose, mannose, fructose, and the like, and they are used alone or in combination of two or more.

Amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamic acid, L-glutamine, glycine, L-histidine, L- Isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-purelin, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-noline, etc. Used in combination of one or more.

[0054] As vitamins, d_piotine, D-pantothenic acid, choline, folic acid, myo_inositol

, Niacinamide, pyridoxanol, riboflavin, thiamine, cyanocobalamin, DL-a

-Tocopherol, etc. are used, and one or more are used in combination.

 Examples of the hydrolyzate include soybeans, wheat, rice, peas, cottonseed, yeast extract, etc., which are used as necessary.

[0055] Examples of lipids include cholesterol, linoleic acid, linolenic acid, and the like, which are used as necessary.

 Various cultures are usually carried out under conditions such as pH 6-8 and 30-40 ° C for 3-12 days, and perfusion cultures for 3-40 days. In addition, antibiotics such as streptomycin and penicillin may be added to the medium as needed during the culture. In addition, dissolved oxygen concentration control, pH control, temperature control, stirring, etc. can be performed according to the method used for normal animal cell culture.

[0056] As described above, saccharides obtained by culturing animal cells and changing the proportion of sugar chains in which fucose is not bound to glycoside-bound complex type sugar chains in the glycoprotein composition having glycoside-bound complex type sugar chains are changed. A glycoprotein composition can be produced by accumulating a protein composition and collecting the glycoprotein composition from the culture.

 The production method of the glycoprotein composition of the present invention includes a direct expression method in which the glycoprotein composition is produced in the host cell, a method in which the glycoprotein composition is secreted and produced outside the host cell (Molecular 'Cloning 2nd Edition). ) Etc.

[0057] The glycoprotein composition was prepared by the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], the method of Lou et al. [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989). Genes Develop., 4, 1288 (1990)], or by applying the method described in JP-A-5-336963, WO94Z23021, etc., it can be actively secreted outside the host cell. That is, the glycoprotein composition can be actively secreted out of the host cell by expressing it with a signal peptide added using a genetic recombination technique. [0058] Further, according to the method described in JP-A-2-227075, the production amount of the glycoprotein composition may be increased by using a gene amplification system using a dihydrofolate reductase gene or the like. it can.

 The glycoprotein produced by the method of the present invention can be isolated and purified using a normal glycoprotein isolation and purification method.

 [0059] When the glycoprotein composition produced by the method of the present invention is expressed in a dissolved state in the cells, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer, Disrupt the cells with a sonic breaker, French press, Manton Gaurin homogenizer, dynomill, etc. to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, an ordinary enzyme isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion-exchange chromatographic method using resin such as Jetylaminoethyl (DEAE) —Sepharose, DIAION HPA—75 (Mitsubishi Chemical), and positive using resin such as S-Sepharose FF (Falmasia) Ion exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography using protein A, chromatofocusing, A purified preparation can be obtained by using electrophoresis methods such as isoelectric focusing alone or in combination.

[0060] When the glycoprotein composition produced by the method of the present invention is secreted extracellularly, the glycoprotein composition can be recovered in the culture supernatant. That is, a culture supernatant is obtained from the culture by a technique such as centrifugation as described above, and a purified preparation is obtained from the culture supernatant by using the same isolation and purification method as described above. Can do.

 Among the glycoprotein compositions produced by the method of the present invention, the antibody composition contains an N-glycoside-linked N-glycoside-linked complex-type sugar chain that binds to the Fc region of the antibody. An antibody composition with an increased proportion of sugar chains in which fucose is not bound to acetylyldarcosamine is used for the treatment of diseases such as tumors, inflammation, allergies, congenital diseases, etc. with high antibody-dependent cytotoxicity (ADCC activity). Useful.

[0061] The present invention will be described more specifically with reference to the following examples. However, the examples are merely illustrative of the present invention and do not limit the scope of the present invention. Example 1

 [0062] Production of anti-GD antibody in batch culture using YB2 / 0 cell line

 Three

 Transformed rat-rat fusion cells producing anti-GD antibody YB2 / 0 strain 61—33 γ (FE

Three

 The following batch culture was performed using RM BP-7325).

 As a culture medium for batch culture, CD-Hybridoma medium (Invitrogene) was used. The osmotic pressure in the initial medium was 320 mOsm / kg.

[0063] CD-Hybridoma medium was diluted with sterile water to prepare a low osmotic pressure medium of about 250 mOsm / kg. Next, 10 mg / mL recombinant human insulin (hereinafter referred to as INS) (manufactured by Invitrogen), lmmol / L Methotrexate (hereinafter referred to as MT X) (manufactured by Sigma), 200 mmol / LL-glutamine (Invitrogen) was added.

 [0064] As an osmotic pressure regulator, sodium chloride, potassium salt (manufactured by Wako Pure Chemical Industries, Ltd.), Creatine (manufactured by Sigma), Fucose, Fructose (manufactured by Nacalai), and Mannitol were added to the medium. The medium was adjusted so as to have an osmotic pressure of 60 to 390 mOsm / kg and used for the following expansion culture.

CD-Hybridoma medium was used for expansion culture. After a sufficient number of cells was obtained, the cells were seeded in 3 × 10 ⁶ cells ZmL in the various osmotic pressure media prepared in the 25 OmL conical flasks (manufactured by Cojung). Thereafter, the cells were cultured at 37 ° C for 11 days.

 [0065] The antibody composition produced from the cells was collected in a culture medium, and the sugar composition in the antibody composition was analyzed. The results are shown in Fig. 1. As shown in Figure 1, regardless of the type of osmotic pressure regulator, when the osmotic pressure of the medium increases, the N-glycoside-binding complex that binds to the Fc region of the antibody in the antibody composition produced by animal cells. The proportion of sugar chains in which fucose is not bound to N-acetylidanolosecosamine at the reducing end of the sugar chain decreased.

 Example 2

 [0066] Production of anti-GD antibody in Fuedbatch culture using YB2 / 0 cell line

 Three

 Transformed rat producing rat anti-GD antibody—rat fusion cell line YB2 / 0 61— 33 γ (FE

Three

The following food batch culture was performed using RM BP-7325). As the medium, CD_Hybridoma medium (Invitrogen) having an initial osmotic pressure of 325 mOsmZkg was used. As the low osmotic pressure medium, CD_Hybridoma medium was diluted with sterile water to prepare low osmotic pressure media of 285 and 300 mOsmZkg, respectively. As a hyperosmotic medium, NaCl was added to CD_Hybridoma medium to prepare a 340 mOsmZkg medium. Subsequently, lOmgZmL INS (manufactured by Invitrogen), 1 mmol / L MTX (manufactured by Sigma), and 200 mmol / LL-glutamine (manufactured by Invitrogen) were added to these media.

[0067] Expansion culture was performed on CD-Hybridoma medium, and when a sufficient number of cells were obtained, a 5 L jar containing media adjusted to osmotic pressures of 285, 300, 325, and 345 mOsm / kg was added. Cells were seeded at 3 × 10 ⁵ cells / mL in the fermenter. Then 37. C, pH 7.1 and DO 50% were cultured for 11 days.

 From the beginning of the culture to the end of the culture, the culture broth was sampled once daily from each fermenter, and the viable cell density (cells / mL) and antibody concentration (mg / L) were measured. The sugar chain of the antibody composition was analyzed. The viable cell density was measured by a dye exclusion method using 0.4% trypan blue solution (Invitrogen), and the antibody concentration was measured by high performance liquid chromatography (hereinafter referred to as HPLC) (Shimadzu Corporation). did. The result is shown in Figure 2. Figure 2 [As shown, N-glycoside-linked complex-type glycans that bind to the Fc region of an antibody against an antibody composition produced in 285, 300, 325, 345 mOsm / kg osmotic pressure adjustment medium The percentages of sugar chains in which fucose was not bound to N-acetylidanorecosamine at the reducing end were 70, 58, 45, and 40%, respectively.

[0068] In addition, the cumulative number of cells was shown as the sum of products of viable cell density and elapsed time. In this example, since the viable cell density was measured once a day, the cumulative number of cells was simply shown as a value obtained by adding the viable cell density measured each time (cell ZmL x day). The specific antibody production rate was calculated from the following formula.

 (Formula 3)

 Specific antibody production rate (ng / cell / day) = antibody concentration (mg / L) ÷ cumulative viable cell count (cells / mL x day)

As a result, the specific antibody production rate at the end of the culture greatly affects the initial osmotic pressure. I did not receive it. Similarly, no significant change was observed in the cumulative number of viable cells.

 Example 3

 [0069] Production of anti-GD antibody in perfusion culture using YB2 / 0 cell line

 Three

 Transformed rat-rat fusion cells producing anti-GD antibody YB2 / 0 strain 61—33 γ (FE

Three

 The following perfusion culture was performed using RM BP-7325).

 As the medium, CD-Hybridoma medium (Invitrogen) having an initial osmotic pressure of 350 mOsm / kg was used. As the low osmotic pressure medium, CD-Hybridoma medium was diluted with sterile water, and 245, 260, 280, 300, 330 mOsm / kg low osmotic pressure medium was prepared. Next, 10 mg / mL INS (manufactured by Invitrogen), 1 mmol / L MTX (manufactured by Sigma), and 200 mmol / LL-glutamine (manufactured by Invitrogen) were added to the medium after adjusting the osmotic pressure.

[0070] Expansion culture was performed with CD-Hybridoma medium, and after obtaining a sufficient number of cells, the cells were seeded in a 1 L bioreactor. After the start of culture, perfusion culture using a centrifuge (SORVAL LABII) was started on the third day. The medium exchange rate was lwd. Each of the 280, 260, 245, 300, and 330 mOsm / kg osmotic pressure media prepared in advance was changed every 5 days, and the cell density was maintained to be about 5 × 10 ⁶ cells ZmL. The culture was performed at 35 ° C, pH 7.1, DO50% for 35 days.

 [0071] From the start of culture to the end of culture, the culture solution was sampled once a day, and the viable cell density (cell ZmL) and antibody concentration (mgZU were measured. In addition, sugar chains that bind to the antibody produced in the medium The viable cell density was measured by a dye exclusion method using a 0.4% trypan blue solution (manufactured by Invitrogen), and the antibody concentration was measured by HPLC (manufactured by Shimadzu Corporation). This is shown in Fig. 3. As shown in Fig. 3, N-glycosidic conjugates that bind to the Fc region of antibodies in antibody compositions produced in 260, 280, 300, 330 mOs m / kg osmotic pressure adjusted medium. The proportions of sugar chains in which fucose was not bound to N-acetylidanorecosamine at the reducing end of the type sugar chain were 85, 81, 76 to 64, and 47%, respectively.

[0072] It was considered that 245 mOsm / kg was a force S that showed a culture failure, and the cause was that alkali was added and the measured infiltration pressure increased to 278 mOsm / kg. The N-glycoside-linked complex type sugar chain that binds to the Fc region contained in the antibody composition produced in the osmotic pressure adjustment medium at this time The ratio of the sugar chain in which fucose was not bound to N-acetylyldarcosamine at the reducing end was 76%.

 [0073] From the above, when the osmotic pressure of the medium is high, in the antibody composition, fucose is present in the reducing terminal N-acetylidanorecosamine of the N-glycoside-bonded complex sugar chain that binds to the Fc region of the antibody. The proportion of unbound sugar chains decreases, and when the osmotic pressure of the medium is low, N-acetylyldarcosamine at the reducing end of the N-glycoside-linked complex sugar chain that binds to the Fc region of the antibody in the antibody composition In addition, the proportion of sugar chains without fucose was increased. Example 4

 [0074] Production of anti-CCR4 antibody by Fuedbatch culture using YB2 / 0 strain

 (1) Food batch culture in Erlenmeyer flask

 Using the transformed rat-rat fusion cell strain YB2 / 0 (FERM BP — 7054) that produces anti-CCR4 antibody, the following Fuedbac culture was performed.

 CD-Hybridoma medium (Invitrogen) was used as the medium. By adding sodium chloride (manufactured by Wako Pure Chemical Industries, Ltd.) to the CD-Hybridoma medium, the osmotic pressure of the medium was adjusted to 226, 242, 253, 284, 304, 313, 337, 359, and 375 mOsmZkg, respectively. Next, lOmg / mLINS (manufactured by Invitrogene), 1 mmol / L MTX (manufactured by Sigma), and 200 mmol / L L-glutamine (manufactured by Invitrogene) were added to the various media after osmotic pressure adjustment, followed by culturing.

[0075] Expansion culture was performed in a medium with an osmotic pressure of 313 mOsm / kg, and when a sufficient number of cells were obtained, each of 226, 242, 253, 284, 226, 242, 253, 284, It was seeded in a medium of 304, 337, 359, 375 mOsm / kg osmotic pressure to give 3 × 10 ⁵ Itodatsuki sachet / mL. Each Erlenmeyer flask was injected with 7.5% carbon dioxide gas at a flow rate of 2 L / min for 1 minute, and then cultured at 35 ° C. and lOOrpm for 8 days.

[0076] From the start of the culture to the end of the culture, the culture solution was collected once every day, and the viable cell density (cells / mL) and antibody concentration (mg / L) were measured. In addition, a sample for analyzing a sugar chain bound to the antibody produced in the culture solution at the end of the culture was obtained. The viable cell density was measured by a dye exclusion method using a 0.4% trypan blue solution (manufactured by Invitrogen), and the antibody concentration was measured by HPLC (manufactured by Shimadzu Corporation). The results are shown in Fig. 4. Figure 4 As shown, the results show that N_glycosidic linkages that bind to the Fc region of antibodies in antibody compositions produced in 226, 242, 253, 284, 304, 337, 359, 375 mOsm / kg permeation pressure media. Harm of leucose chain without fucose binding to N-acetylidanorecosamine at the reducing end of complex type sugar chain | J-joint was 90, 92, 92, 92, 89, 84, 80, 77% respectively It was. Therefore, in the conical flask fed batch culture of the anti-CCR4 antibody-producing YB2Z0 strain, the lower the osmotic pressure of the medium, the lower the osmotic pressure of the medium, the N-glycoside-conjugated complex sugar chain that binds to the antibody Fc region. It was shown that the proportion of sugar chains in which fucose is not bound to the N-acetylyldarcosamine at the reducing end of the protein increased.

[0077] In addition, the cumulative number of viable cells and the specific antibody production rate were calculated by the same method as described in Example 2. As a result, the specific antibody production rate at the end of the culture increased as the medium osmotic pressure decreased. The cumulative number of viable cells did not change significantly due to the osmotic pressure of the medium.

 (2) Culture in 1L reactor

 Example 4 Using the same cells as in (1), the following Fuedbatch culture was performed.

 [0078] As the medium, CD-Hybridoma medium was used. A medium with osmotic pressures of 227, 313, 333 and 406 mOsm / kg was prepared by adding sodium chloride to the CD-Hybridoma medium. Subsequently, 10 mg / mL INS, 1 mmol / L MTX, 200 mmol / L L-gnoretamine was added to the medium after adjusting the osmotic pressure, and used for the culture.

Enlargement culture was performed in a medium with an osmotic pressure of 313 mOsm / kg. When a sufficient number of cells were obtained, each medium with 227, 333, 406 mOsm / kg osmotic pressure prepared in a 1 L bioreactor (manufactured by ABLE) was used. 3. Seeded at 5 × 10 ⁵ cells / mL. Culturing was performed for 11 days under conditions of 35 ° C, pH 7.1, and DO50%.

[0079] From the start to the end of the culture, the culture broth was collected once a day, and the viable cell density (cell ZmL) and antibody concentration (mgZmL) were measured. In addition, the production antibody was purified from the culture solution at the end of the culture, and a sample for analyzing the sugar chain bound to the antibody was obtained. The viable cell count was measured by a dye exclusion method using 0.4% trypan blue solution, and the production antibody concentration was measured by HPLC. As a result of analyzing the sugar composition that binds to the obtained antibody, the antibody Fc region was produced in the antibody composition produced in the osmotic pressure adjustment medium 227, 333, 406 mOsm / kg. The proportions of sugar chains in which fucose was not bound to N-acetylyldarcosamine at the reducing end of the N-glycoside-bonded complex sugar chain bound to N was 94, 79 and 61%, respectively. Therefore, in the 1L reactor culture of the anti-CCR4 antibody-producing YB2Z0 cell line, the lower the osmotic pressure of the medium, the lower the osmotic pressure of the culture medium, It was shown that the proportion of sugar chains in which fucose is not bound to acetylyldarcosamine increases.

[0080] Further, the cumulative number of viable cells and the specific antibody production rate were calculated by the same method as described in Example 2. As a result, the specific antibody production rate at the end of the culture was 6.9, 4.4, 4.6 mg / 10 ⁶ cells / day in the order of 227, 333, 406 11103111 / 1¾ osmotic medium, and the cumulative number of cells was the specific antibody production rate. The lower it was, the more it increased.

 Example 5

 [0081] Production of anti-GD antibody in batch culture using Sp2Z0 strain

 Three

 Transformed mouse myeloma strain Sp2 / 0 that produces anti-GD monoclonal antibodies (FER

Three

 The following batch culture was performed using M BP — 3512).

 As the medium, RPMI1640 medium (manufactured by Invitrogene) was used. The hypoosmotic medium was prepared by diluting RPMI1640 medium with sterile water. Similarly, the hyperosmotic medium was adjusted by adding sodium chloride to RPMI1640 medium. Next, 10% Daigo GF21 (manufactured by Nippon Pharmaceutical Co., Ltd.), lmmol / L MTX, and 50 mg / mL G418 (manufactured by Nacalai Testa) were added to the medium after adjusting the osmotic pressure. The following culture was carried out using the medium of osmotic pressure 210, 238, 277, 312, 353, 407 mOsm / kg obtained by the above method.

[0082] Expansion culture was performed in RPMI1640 medium supplemented with 10% urine fetal serum JRH), 1 mmol / L MT X, 50 mg / mL G418. It seed | inoculated so that it might become 2.5 * 10 < ⁵ > cell / mL in the culture medium of each 210,238,277,312,353,407mOsm / kg osmotic pressure prepared for CELL BAG (made by Medtronic). The culture was performed in a 37 ° C, 5% carbon dioxide incubator for 7 days.

[0083] The produced antibody was purified from the culture medium after completion of the culture, and a sample for analyzing the sugar chain bound to the antibody was obtained. As a result of analyzing the sugar composition that binds to the obtained antibody, the antibody composition produced in 210, 23, 38, 277, 312, 353, 407 mOsm / kg osmotic pressure adjusted medium The percentage of sugar chains in which fucose is not bound to N-acetylyldarcosamine at the reducing end of the N-glycoside-bonded complex sugar chain that binds to the Fc region of the antibody is 25, 20, They were 20, 16, 16, and 15% (Fig. 6).

 Example 6

 [0084] Production of anti-CCR4 antibody in batch culture using NS0 strain

 The following batch culture was performed using a transformed NS0 strain (FERM BP-7964) that produces an anti-CCR4 antibody.

 As the medium, RPMI1640 medium (manufactured by Invitrogene) was used. The hypotonic medium was prepared by diluting RPMI1640 medium with sterile water. To adjust the hyperosmotic medium, sodium chloride was added to RPMI medium. Subsequently, 10% Daigo GF21 (manufactured by Nippon Pharmaceutical Co., Ltd.) and 500 nmol / L MTX were added to the medium after adjusting the osmotic pressure. Culturing was carried out using a medium having an osmotic pressure of 217, 241, 276, 305, 349, 399 mOsm / kg obtained by the above method.

[0085] Enlarged cultures in RPMI 1640 medium10. /. 217, 241, 276, 305 prepared in a T225cm ² flask (manufactured by Iwaki Glass Co., Ltd.) when a sufficient number of cells was obtained. , 349, 399 mOsmZkg Osmotic medium was seeded at 3 × 10 ⁵ cells ZmL. Cultivation was carried out in a 37 ° C, 5% carbon dioxide incubator for 5 days.

 [0086] A sample for analyzing a sugar chain bound to the antibody was obtained from the culture medium after completion of the culture. As a result of analyzing the sugar composition that binds to the obtained antibody, it binds to the Fc region of the antibody in the antibody composition produced in the conditioned medium of 217, 241, 276, 305, 349, 399mOsm / kg osmotic pressure. N-glycidyl-linked glycan N-acetylyldarcosamine at the reducing end of fucose force S-bonded to the damage of low-grade glycans, 70, 73, 73, 69, 56, 40%, respectively (Fig. 7).

 Industrial applicability

[0087] By culturing while controlling the osmotic pressure in a medium for culturing animal cells, the glycoprotein composition having glycoside-linked complex-type sugar chains produced from animal cells is reduced to a glycoprotein. There is provided a method for producing a glycoprotein composition, characterized in that the ratio of the sugar chain in which fucose is not bound to the glycoside-linked sugar chain is changed.

Claims

 The scope of the claims

 [I] In a glycoprotein composition having a glycoside-linked sugar chain produced from animal cells by controlling the osmotic pressure in a medium for culturing animal cells, fucose is added to the glycoside-linked sugar chain of the glycoprotein. The method for producing a glycoprotein composition is characterized in that the ratio of les, nares and sugar chains is changed by binding.

 [2] In a glycoprotein composition having glycoside-linked sugar chains produced from animal cells by controlling the osmotic pressure in a medium for culturing animal cells, fucose is added to the glycoside-linked sugar chains of the glycoprotein. The method for producing a glycoprotein composition is characterized in that the ratio of les, nares and sugar chains is increased by binding.

 [3] The method according to claim 1 or 2, wherein the osmotic pressure in the medium is 200 to 400 mOsm / kg.

 [4] The method according to any one of claims 1 to 3, wherein the animal cell is a cell selected from a rat cell, a mouse cell and a hamster cell.

 5. The method according to claim 4, wherein the rat cell is a myeloma cell or a hybrid cell of a myeloma cell line.

 [6] The method according to claim 4, wherein the rat cell force is SYB2 / 3H P2.G11.16Ag.20 (ATCC CRL 1662, ECACC No: 85110501) cells.

[7] The method according to claim 4, wherein the mouse cell is a cell selected from NS0 cells or Sp2 / 0 cells.

 [8] The method according to any one of [7] to [7] above, wherein the animal cell is a cell into which a recombinant DNA containing a DNA encoding a glycoprotein has been introduced.

[9] The method according to any one of claims 1 to 8, wherein the glycoprotein is an antibody.

[10] In a glycoprotein composition having glycoside-linked sugar chains produced from animal cells by controlling the osmotic pressure in a medium for culturing animal cells, fucose is added to the glycoside-linked sugar chains of the glycoprotein. Is a method of changing the proportion of sugar chains.

 [I I] The method according to claim 10, wherein the osmotic pressure in the medium is 200 to 400 mOsm / kg.

 12. The method according to claim 10 or 11, wherein the animal cell is a cell selected from a rat cell, a mouse cell and a hamster cell.

[13] The rat cell according to claim 12, wherein the rat cell is a myeloma cell or a hybrid cell of a myeloma cell line. The method of publication.

 [14] The method according to claim 12, wherein the rat cell force is YB2 / 3H P2.G11.16Ag.20 (ATCC CRL 1662, ECACC No: 85110501) cells.

15. The method according to claim 11, wherein the mouse cell is a cell selected from NS0 cells or Sp2 / 0 cells.

 [16] The method according to any one of [10] to [15], wherein the animal cell is a cell into which a recombinant DNA containing a DNA encoding a glycoprotein has been introduced.

[17] The method according to any one of [10] to [16], wherein the glycoprotein is an antibody.