Method for producing a composition containing a polypeptide with reduced coloration

Abstract

This method for producing a less colored composition containing a polypeptide comprises the steps of: a) culturing eukaryotic cells containing a nucleic acid encoding a polypeptide in a vitamin B12-free cell culture medium; and b) collecting a composition containing the polypeptide from the culture.

Description

[Technical field]

[0001] The present invention provides a method for producing a composition comprising a polypeptide that exhibits reduced discoloration. [Background technology]

[0002] Methods for producing polypeptides in vitro using recombinant cell cultures are well known and widely used for industrial-scale production. When a composition containing a polypeptide, such as an antibody, is formulated and provided as a pharmaceutical preparation, one quality attribute is required to keep the color of the preparation to an acceptable level, e.g., a level that satisfies regulatory requirements for marketing the product.

[0003] In particular, in preparations containing antibodies at high concentrations (eg, 150 mg / mL or more), coloring intensifies with concentration, so it is important to keep the color of the preparation at an acceptable level.

[0004] The color of antibody-containing preparations may be due to the incorporation of vitamin B 12 The adsorption of VB12 (hereinafter referred to as "VB12") has been reported (Non-Patent Documents 1-3, Patent Document 1). One molecule of VB12 is coordinated with one molecule of cobalt. VB12 exists in the cyanocobalamin form and the hydroxycobalamin form, and it has been reported that the hydroxycobalamin form binds to an antibody, causing the molecule to turn pink or red (Non-Patent Documents 1-3).

[0005] Furthermore, a culture method using a medium containing specific amounts of vitamins B2, B6, B9, B12, and cystine has been reported as a culture method aimed at preventing discoloration of antibodies (Patent Document 2). Also, a method has been reported that prevents discoloration by preventing the conversion of cyanocobalamin in the medium to hydroxycobalamin and the reduction of disulfide bonds in antibodies (Patent Document 1).

[0006] On the other hand, it has been reported that VB12 is an essential component of cell culture media for eukaryotic cells (Non-Patent Document 1), and that VB12 is an essential component for the culture of mammalian cells (Patent Document 1). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2018 / 208743 [Patent Document 2] Special table 2015-515281 [Non-patent literature]

[0008] [Non-Patent Document 1] MAbs. 2013 Nov 1; 5(6): 974-981 [Non-Patent Document 2] MAbs. 2014 May 1; 6(3): 679-688 [Non-Patent Document 3] Biotechnology and Bioengineering.2018;115:900-909 Summary of the Invention [Problem to be solved by the invention]

[0009] The objective of the present invention is to provide a method for solving the problem of discoloration of a polypeptide that is prone to discoloration, by relatively simply modifying the production method while maintaining the production amount and physical properties of the polypeptide within acceptable ranges. [Means for solving the problem]

[0010] As a result of intensive research to achieve the above-mentioned object, the inventors discovered that by using a medium that does not contain VB12, discoloration can be suppressed while maintaining the production amount of polypeptide, and thus completed the present invention.

[0011] Therefore, the present invention can be expressed as follows (1) to (17). (1) A method for producing a composition comprising a polypeptide with reduced discoloration, comprising the steps of: (a) culturing a eukaryotic cell comprising a nucleic acid encoding the polypeptide in a cell culture medium that does not contain vitamin B12; and (b) recovering a composition comprising the polypeptide from the culture. (2) The method for producing the composition described in (1), wherein the molar concentration ratio of vitamin B12 to the polypeptide in the composition is less than 0.26%. (3) A method for producing the composition according to (1) or (2), comprising carrying out fed-batch culture using an initial medium not containing vitamin B12 and a feed medium not containing vitamin B12. (4) The VCD (viable cell density) in the cell culture medium on the 7th day after the start of fed-batch culture was 80 × 10 5 The method for producing the composition according to (3), wherein the concentration of each of the compositions is 100 to 150 cells / mL or more. (5) The method for producing the composition according to any one of (1) to (4), wherein the eukaryotic cell is a CHO cell. (6) The method for producing the composition according to any one of (1) to (5), wherein the polypeptide is an Fc-containing polypeptide. (7) The method for producing the composition described in (6), wherein the Fc-containing polypeptide is an antibody. (8) A method for producing a composition described in (6) or (7), wherein the polypeptide includes a modification of at least one amino acid residue selected from the amino acid residues at positions 214, 234, 238, 250, 264, 307, 311, 330, 343, 428, 434, 436, 438, and 440 according to EU numbering in the Fc region. (9) A method for producing a composition according to any one of (6) to (8), wherein the polypeptide comprises at least one modification selected from 214R, 234Y, 238D, 250V, 264I, 307P, 311R, 330K, 343R, 428L, 434A, 436T, 438R, and 440E according to EU numbering in the Fc region. (10) A method for producing a composition according to any one of (6) to (9), wherein the polypeptide is an antibody comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 2. (11) A method for producing the composition described in (10), wherein the antibody is an IgG1 humanized antibody that binds to latent myostatin. (12) A method for producing an antibody-containing composition with suppressed coloration, comprising the following steps: a) The antibody of interest comprises a modification of at least one amino acid residue selected from the amino acid residues at positions 214, 234, 238, 250, 264, 307, 311, 330, 343, 428, 434, 436, 438, and 440 (EU numbering) in the Fc region. A process of confirming; b) selecting a cell culture medium that does not contain vitamin B12 for antibodies that contain the amino acid residue modification; c) culturing a eukaryotic cell comprising a nucleic acid encoding an antibody comprising the amino acid residue modification in the cell culture medium selected in step b); and d) recovering the antibody-containing composition from the culture. (13) The method according to (12), wherein step b) is a step of selecting an initial medium containing no vitamin B12 and a feed medium containing no vitamin B12. (14) The method according to (12) or (13), wherein the eukaryotic cell is a CHO cell. (15) A method for producing an antibody-containing formulation, comprising the steps of: (a) culturing a eukaryotic cell containing an antibody-encoding nucleic acid in a cell culture medium that does not contain vitamin B12; (b) recovering a composition comprising the antibody from the culture; and (c) formulating the resulting composition into a pharmaceutical formulation. (16) A method for suppressing antibody discoloration in the production of an antibody using a recombinant cell culture, comprising the steps of: (a) culturing eukaryotic cells containing nucleic acid encoding the antibody in a cell culture medium that does not contain vitamin B12; and (b) recovering a composition containing the antibody from the culture. (17) A method for producing a composition according to (7), a method for producing an antibody-containing preparation according to (15), or a method for suppressing discoloration of an antibody according to (16), wherein the antibody is an anti-IL-8 antibody or an anti-myostatin antibody. Effect of the Invention

[0012] The present invention provides a method for producing a composition containing a polypeptide that exhibits reduced discoloration. [Brief description of the drawings]

[0013] [Figure 1] In culturing cells producing a polypeptide (antibody A: anti-myostatin antibody), media containing VB12 in the initial medium and feed medium were designated VB+, and media not containing VB12 in the initial medium and feed medium were designated VB-. The number of viable cells was measured each day over the 14 days of culture using an automatic cell counting device, the Vi-CELL system (Beckman Courter, model Vi-CELL XR). [Diagram 2] In the culture of cells producing antibody A, a medium containing VB12 in the initial medium and the feed medium is designated as VB+, and a medium not containing VB12 in the initial medium and the feed medium is designated as VB-. The cell viability was measured each day over the 14 days of culture using an automatic cell measuring device, Vi-CELL System (Beckman Courter, model Vi-CELL XR). [Diagram 3] In the culture of cells producing antibody A, a medium containing VB12 in the initial medium and the feed medium is designated as VB+, and a medium not containing VB12 in the initial medium and the feed medium is designated as VB-. The cumulative cell number was calculated from the viable cell count measured using an automatic cell counting device, Vi-CELL System (Beckman Courter, model Vi-CELL XR) for each day over the 14 days of culture. [Figure 4]In the culture of cells producing antibody A, medium containing VB12 was designated as VB+, and medium not containing VB12 was designated as VB-. Subculture was carried out for up to 100 days (28 passages) at 3- or 4-day cycles, and the cell proliferation behavior for each day was plotted using the cell doubling time as an index. (Measurements were made using an automatic cell measurement device, the Vi-CELL system (manufactured by Beckman Courter), and the cell doubling time was calculated.) [Diagram 5] In the culture of cells producing a different polypeptide (antibody B: anti-IL-8 antibody) from that shown in Figure 1, a medium containing VB12 in the initial medium and the feed medium is designated VB+, and a medium not containing VB12 in the initial medium and the feed medium is designated VB-. The number of viable cells was measured each day over the 14 days of culture using an automatic cell counting device, the Vi-CELL system (Beckman Courter, model Vi-CELL XR). [Figure 6] In the culture of cells producing a polypeptide different from that shown in Figures 1 and 5 (antibody C: anti-FIXa / FX bispecific antibody), the medium containing VB12 in the initial medium and feed medium was designated VB+, and the medium not containing VB12 in the initial medium and feed medium was designated VB-. The number of viable cells was measured each day over the 14 days of culture using an automatic cell measuring device, the Vi-CELL system (Beckman Courter, model Vi-CELL XR). [Figure 7] In culturing cells producing antibody B, a medium containing VB12 in the initial medium and feed medium is designated as VB+, and a medium containing no VB12 in the initial medium but containing VB12 in the feed medium is designated as VB-. The number of viable cells was measured each day over the 14 days of culture using an automatic cell counting device, the Vi-CELL system (Beckman Courter, model Vi-CELL XR). [Figure 8] In culturing cells producing antibody C, a medium containing VB12 in the initial medium and the feed medium is designated as VB+, and a medium containing no VB12 in the initial medium but containing VB12 in the feed medium is designated as VB-. The number of viable cells was measured each day over the 14 days of culture using an automatic cell counting device, the Vi-CELL system (Beckman Courter, model Vi-CELL XR). [Figure 9]In the culture of cells producing antibody B, a medium containing VB12 in the initial medium and the feed medium is designated as VB+, and a medium not containing VB12 in the initial medium and the feed medium is designated as VB-. The cell viability was measured each day over the 14 days of culture using an automatic cell measuring device, Vi-CELL System (Beckman Courter, model Vi-CELL XR). [Figure 10] In the culture of cells producing antibody C, a medium containing VB12 in the initial medium and the feed medium is designated as VB+, and a medium not containing VB12 in the initial medium and the feed medium is designated as VB-. The cell viability was measured each day over the 14 days of culture using an automatic cell measuring device, Vi-CELL System (Beckman Courter, model Vi-CELL XR). [Figure 11] In culturing cells producing antibody B, a medium containing VB12 in the initial medium and feed medium is designated as VB+, and a medium containing no VB12 in the initial medium but containing VB12 in the feed medium is designated as VB-. The cell viability was measured each day over the 14 days of culture using an automatic cell measuring device, the Vi-CELL system (Beckman Courter, model Vi-CELL XR). [Figure 12] In culturing cells producing antibody C, a medium containing VB12 in the initial medium and feed medium is designated as VB+, and a medium containing no VB12 in the initial medium but containing VB12 in the feed medium is designated as VB-. The cell viability was measured each day over the 14 days of culture using an automatic cell measuring device, the Vi-CELL system (Beckman Courter, model Vi-CELL XR). [Figure 13] In the culture of cells producing antibody B, a medium containing VB12 in the initial medium and the feed medium is designated as VB+, and a medium not containing VB12 in the initial medium and the feed medium is designated as VB-. The cumulative cell number was calculated from the viable cell count measured using an automatic cell counting device, Vi-CELL System (Beckman Courter, model Vi-CELL XR) for each day over the 14 days of culture. [Figure 14]In the culture of cells producing antibody C, a medium containing VB12 in the initial medium and the feed medium was designated VB+, and a medium not containing VB12 in the initial medium and the feed medium was designated VB-. The cumulative cell number was calculated from the viable cell count measured using an automatic cell counting device, Vi-CELL System (Beckman Courter, model Vi-CELL XR) for each day over the 14 days of culture. [Figure 15] In culturing cells producing antibody B, a medium containing VB12 in the initial medium and the feed medium is designated as VB+, and a medium containing no VB12 in the initial medium but containing VB12 in the feed medium is designated as VB-. The cumulative cell number was calculated from the viable cell count measured using an automatic cell counting device, Vi-CELL System (Beckman Courter, model Vi-CELL XR), for each day over the 14 days of culture, and is shown here. [Figure 16] In culturing cells producing antibody C, a medium containing VB12 in the initial medium and the feed medium is designated as VB+, and a medium containing no VB12 in the initial medium but containing VB12 in the feed medium is designated as VB-. The cumulative cell number was calculated from the viable cell count measured using an automatic cell counting device, Vi-CELL System (Beckman Courter, model Vi-CELL XR) for each day over the 14 days of culture, and is shown here. [Figure 17] In the culture of cells producing antibody B, medium containing VB12 was designated as VB+, and medium not containing VB12 was designated as VB-. Subculture was performed for up to 136 days (39 passages) in 3- or 4-day cycles, and the cell proliferation behavior for each day was plotted using the cell doubling time as an index. (Measurements were performed using an automatic cell measurement device, the Vi-CELL system (manufactured by Beckman Courter), and the cell doubling time was calculated.) [Figure 18] In the culture of cells producing antibody C, medium containing VB12 was designated as VB+, and medium not containing VB12 was designated as VB-. Subculture was performed for up to 24 days (7 passages) at 3- or 4-day cycles, and the cell proliferation behavior for each day was plotted using the cell doubling time as an index. (Measurements were performed using an automatic cell measurement device, Vi-CELL System (manufactured by Beckman Courter), and the cell doubling time was calculated.) [Figure 19] The antibody-containing composition treated with potassium cyanide and the antibody-containing composition not treated with potassium cyanide were removed using a 5 KD cutoff filter, and the collected samples were analyzed using reverse phase chromatography (Column: Acclaim™ Polar Advantage II LC). The sample not treated with potassium cyanide is represented as KCN-, the sample treated with potassium cyanide is represented as KCN+, and the VB12 peak is represented as STD. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Hereinafter, an embodiment of the present invention will be described in detail.

[0015] The present invention relates to a method for producing a composition comprising a polypeptide that exhibits reduced discoloration.

[0016] VB12 VB12 refers to cyanocobalamin in the narrow sense, and vitamin B 12 Cobalamin is a general term for the VB12 medium component, which is cyanocobalamin, and when the medium components are in aqueous solution, they reach an equilibrium state of cyanocobalamin, hydroxycobalamin, aquacobalamin, adenosylcobalamin, and methylcobalamin.

[0017] One cobalt molecule is bound to each VB12 molecule. Therefore, in the examples of the present application, the relative concentration of VB12 to the antibody was quantified by quantifying the relative concentration of cobalt molecules to the antibody. In the present invention, a VB12-free initial medium and feed medium can be used for culturing eukaryotic cells. In the present invention, the "VB12-free" medium includes not only a medium in which the concentration of VB12 in the medium is 0 mg / L, but also a medium in which VB12 is mixed to an extent that does not meet the substantial content as a medium additive. Specifically, when the medium is used to perform culture under the same conditions as in Example 1 of the present invention and the obtained antibody-containing composition (antibody concentration of about 30 mg / mL) is measured by ICP-MS, the concentration of cobalt molecules contained in VB12 is less than the quantification limit of 20 ppb, and the medium corresponds to "not containing VB12".

[0018] eukaryotic cell Eukaryotic cells refer to cells having a nucleus surrounded by a nuclear membrane. In the present invention, a "eukaryotic cell containing a nucleic acid encoding a polypeptide" is a host cell that can be used as a production system for manufacturing a desired polypeptide. Such cells may be natural cells capable of producing the desired polypeptide, or may be cells into which a nucleic acid encoding (or expressing) the desired polypeptide has been artificially introduced, but transformed cells into which a nucleic acid encoding the desired polypeptide has been introduced are preferred. An example of such a transformed cell is a polypeptide-producing strain obtained by introducing foreign DNA encoding the desired polypeptide into a eukaryotic cell using recombinant gene technology. Therefore, the phrase "containing a nucleic acid encoding a polypeptide" can also be read as "a nucleic acid encoding a polypeptide has been artificially introduced" or "a foreign nucleic acid encoding a polypeptide has been introduced."

[0019] Typical examples of eukaryotic cells in the present invention are cells suitable as hosts for the production of recombinant proteins, and can be selected from cells of insect, fish, amphibian, reptile, or mammalian origin. Preferred examples of eukaryotic cells in the present invention are mammalian cells. Mammalian cells include CHO cells, NS0 cells, Sp2 / 0 cells, COS cells, HEK cells, BHK cells, PER.C6 cells, and the like. (商標登録) The cell is selected from among human cells, hybridoma cells, and the like, and more preferably is a CHO cell.

[0020] Cell culture media In the present invention, the cell culture medium refers to a medium used for culturing cells containing a nucleic acid encoding a polypeptide, and may also be simply referred to as a medium. As the cell culture medium, a commercially available medium or a known medium can be appropriately used. Except for the VB12-free medium used in the present invention, normal media contain VB12 as an essential component required for the transcription activity and nucleic acid synthesis of cells. In the examples of the present invention, a medium was used that does not contain, has removed, or has reduced the content of only the VB12 component while maintaining the composition of an existing medium.

[0021] Those skilled in the art can appropriately select the type of culture medium suitable for a particular cell line. For example, well-known or commercially available culture media for animal cell culture can be used, such as IMDM (Iscove's Modified Dulbecco's Medium), DMEM (Dulbecco's Modified Eagle Medium), Ham's F12 medium, D-MEM / F-12 1:1 Mixture (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12), RPMI1640, CHO-S-SFM II (Invitrogen), CHO-SF (Sigma-Aldrich), EX-CELL 301 (JRH biosciences), CD-CHO (Invitrogen), IS CHO-V (Irvine Scientific), and PF-ACF-CHO (Sigma-Aldrich), but it is understood by those skilled in the art that they are not limited thereto. Numerous media optimized for cell growth and enhanced yield of antibody or recombinant protein production are well known to those of skill in the art.

[0022] For example, examples of media that can be used for culturing the CHO cell line that produces a humanized antibody (IgG1 antibody) that binds to latent myostatin used in the examples include IMDM, DMEM, or Ham's F12 medium, or combinations thereof.

[0023] cell culture Generally, cell culture methods are classified into batch culture, continuous culture, and fed-batch culture.

[0024] The batch method is a culture method in which a small amount of seed culture liquid is added to a medium and cells are grown without adding new medium to the medium or discharging the culture liquid.

[0025] The continuous culture method is a culture method in which the medium is continuously added and continuously discharged during the culture. Note that the continuous culture method also includes perfusion culture.

[0026] Since fed-batch culture lies between the batch and continuous culture methods, it is also called semi-batch culture. During cultivation, medium is added continuously or incrementally, but unlike the continuous cultivation method, the medium is not continuously discharged. The medium added during fed-batch culture (hereinafter referred to as the fed-batch medium) does not have to be the same medium as the medium already used in cultivation (hereinafter referred to as the initial medium); a different medium may be added, or only specific components may be added. Alternatively, the fed-batch medium may be a medium with the same composition as the initial medium.

[0027] In the present invention, any of the culture methods including the batch method, the continuous method, and the fed-batch method may be used, but the fed-batch method is preferably used.

[0028] Generally, in order to produce a desired polypeptide by culturing cells, a certain amount of a seed medium containing the cells is added to an initial medium, and the cells are cultured. In order to increase the production amount of the desired polypeptide, a feed medium is further added during the culture.

[0029] The seed medium refers to a medium for expanding cells (working cell bank) that produce a desired polypeptide to obtain the number of cells required for transferring them to a medium (initial medium) for finally producing the desired polypeptide. The initial medium is usually a medium for culturing cells to produce a desired polypeptide, and is used in the initial stage of culturing the cells. The feed medium is usually a medium added to the medium during the initial culture. The feed medium may be added in multiple batches. The feed medium may be added continuously or intermittently.

[0030] In the present invention, the cells are subcultured in a seed medium, and then a certain amount of the seed medium containing the cells is added to the initial medium, and the cells are cultured in the initial medium to produce the desired polypeptide. In addition, a feed medium is optionally added to the medium during the culture one or more times.

[0031] Fed-batch culture is further classified according to the method of feeding. The constant-rate fed-batch method is a culture method in which a constant amount of feed medium is continuously added to the starting medium.

[0032] Polypeptides, compositions containing polypeptides, and recovery of compositions containing polypeptides The polypeptide used in the present invention is preferably an Fc-containing polypeptide that contains a region equivalent to the Fc of an antibody, and more preferably an antibody.

[0033] A composition comprising a polypeptide (polypeptide-containing composition) refers to a composition comprising a polypeptide and other components. In an embodiment of the present invention, a composition comprising a polypeptide can be recovered from a culture obtained by a cell culture process for producing a polypeptide. The term "recover" as used herein refers to recovering a supernatant (culture supernatant) containing the polypeptide from the culture or culture liquid obtained by the cell culture process, or recovering a filtrate containing the polypeptide using a filter.

[0034] The solution thus recovered can be subjected to a purification step such as affinity column chromatography and a concentration step to prepare a composition containing the polypeptide at an appropriate concentration.

[0035] When the polypeptide is an Fc-containing polypeptide or an antibody, a composition comprising the polypeptide (polypeptide-containing composition) can be read as a composition comprising an Fc-containing polypeptide (Fc-containing polypeptide-containing composition) or a composition comprising an antibody (antibody-containing composition).

[0036] Fc area (Fc) The term Fc region (or simply Fc) includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, it refers to a region in an antibody molecule consisting of the hinge region or part thereof, CH2 and CH3 domains. The Fc region is not limited to an antibody (IgA, IgD, IgE, IgG, IgM), particularly an IgG Fc region, but is preferably an Fc region of human IgG (IgG1, IgG2, IgG3, IgG4), and more preferably an Fc region of human IgG1.

[0037] Suppression of coloring The composition containing a polypeptide with suppressed coloring in the present invention refers to a composition containing a polypeptide with reduced coloring, specifically red or pink, caused by adsorption of VB12. In the examples of the present invention, a polypeptide (antibody)-containing composition in which the concentration of the polypeptide (antibody) is adjusted to about 30 mg / mL was evaluated as colorless by visual evaluation, and it was confirmed that coloring was suppressed. Furthermore, the composition containing a polypeptide (antibody) with suppressed coloring has a molar concentration ratio [%] of VB12 to the polypeptide quantified according to the conditions of Example 1 of less than 0.26, preferably less than 0.2, more preferably less than 0.1, and even more preferably less than 0.05.

[0038] Confirmation of antibodies and selection of cell culture media In one embodiment of the present invention, an antibody that is prone to staining is identified in advance, and a culture step suitable for suppressing staining that is different from a normal culture step is selected for the antibody. In this embodiment, the method first includes a step of confirming that the target polypeptide is a polypeptide containing a modification in at least one amino acid residue selected from the amino acid residues at positions 214, 234, 238, 250, 264, 307, 311, 330, 343, 428, 434, 436, 438, and 440 in the EU numbering system in the Fc region. "Confirming" does not necessarily mean identifying the specific site of the modification in the polypeptide by analytical techniques, but also includes recognizing in advance that the target polypeptide contains the modification.

[0039] Next, for the antibody confirmed to contain the amino acid residue modification, a process different from the normal culture process is selected. That is, in the normal culture process, a cell culture medium containing VB12 as an essential component (normal cell culture medium) is used, but in the culture process of the antibody, a cell culture medium not containing VB12 is used instead of the normal cell culture medium. Here, the VB12-free cell culture medium is made by removing only VB12 from the composition of the normal cell culture medium, and the other compositions can be the same.

[0040] A composition containing the polypeptide is produced by culturing cells containing a nucleic acid encoding the polypeptide using the cell culture medium selected in this manner and recovering the composition containing the polypeptide from the culture. EXAMPLES

[0041] The present invention will be specifically described below with reference to examples. Note that these examples are for the purpose of explaining the present invention and are not intended to limit the scope of the present invention. The examples were carried out using the following antibodies, cells and media.

[0042] antibody: Antibody A was an anti-myostatin antibody that is an IgG1 humanized antibody comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 2, and that binds to latent myostatin. This antibody contains the following modifications in the Fc region: 214R, 234Y, 238D, 250V, 264I, 307P, 311R, 330K, 343R, 428L, 434A, 436T, 438R and 440E according to EU numbering. As antibody B, an anti-IL-8 antibody that binds to IL-8 described in WO / 2016 / 125495 or WO / 2017 / 046994 was used. As antibody C, an anti-FIX(a) / FX bispecific antibody that binds to both FIX(a) and FX, as described in WO2019065795, was used.

[0043] cell: The CHO cells used were derived from CHO-DXB11.

[0044] Media: The initial medium and the feed medium were made by Thermo Fisher Scientific and Fujifilm Wako Pure Chemical Industries, Ltd. In this example, the initial medium containing VB12 (initial medium with a relative VB12 concentration of 100%), the initial medium from which VB12 was completely removed and the composition other than VB12 was the same (initial medium with a relative VB12 concentration of 0%), and the initial medium in which these two media were mixed at a ratio of 1:1 (initial medium with a relative VB12 concentration of 50%) were prepared as the initial medium. In addition, the feed medium containing VB12 (feed medium with a relative VB12 concentration of 100%) and the feed medium from which VB12 was completely removed and the composition other than VB12 was the same (feed medium with a relative VB12 concentration of 0%) were prepared as the feed medium.

[0045] Example 1. Examination of the effect of VB12 on antibody coloration Using cells producing antibody A, samples 1 to 9 were cultured under the same conditions by the constant-rate feeding method in 1 L to 25 L culture apparatuses, each of which was prepared by combining the initial medium and the feed medium as shown in Table 1. The culture was carried out for 14 days at a pH range of 6.7 to 7.2 and at a temperature range of 34°C to 38°C, and the feed medium was added on the third day after the start of culture.

[0046] After culturing for 14 days, a composition containing an antibody was collected from the culture medium and purified by Protein A affinity column chromatography, and then the eluate was concentrated and adjusted to an antibody concentration of about 30 mg / mL (26.7 mg / mL to 31.4 mg / mL) for Samples 1 to 8. Sample 9 was further concentrated to an antibody concentration of 200 mg / mL or more.

[0047] The antibody-containing compositions thus obtained were evaluated for coloration and VB12 content. Coloration was evaluated visually. Slight coloration (weak red) was observed in Samples 1, 5, and 7. Coloration (red) was observed in Sample 9. On the other hand, no coloration was observed in Samples 2 to 4 and 6 to 8 (colorless).

[0048] Since one molecule of cobalt is bound to each VB12 molecule in the antibody-containing composition, the concentration of cobalt in the antibody-containing composition was measured by ICP-MS (Inductively Coupled Plasma-Mass spectrometry) and the molar concentration ratio of cobalt to antibody (cobalt / antibody [%]) was calculated to estimate the VB12 content (molar concentration ratio of VB12 to antibody (VB12 / antibody [%])). These results are shown in Table 1.

[0049] [Table 1]

[0050] The experimental results demonstrated that VB12 contained in the antibody-containing composition was the cause of the coloration. It was also confirmed that VB12 contained in the antibody-containing composition after culture could be suppressed by suppressing VB12 contained in the initial medium and the feed medium.

[0051] Example 2. Examination of the effect of VB12 in the medium on cell culture The effects of the presence or absence of VB12 in the medium on the number of live cells, cell viability, and cumulative cell number were observed by carrying out cell culture under the same conditions as in Example 1. As a result, it was confirmed that the number of live cells, cell viability, and cumulative cell number on each day were almost equivalent in culture using a medium that did not contain VB12 in the initial medium and feed medium (VB-) compared to culture using a medium that contained VB12 in the initial medium and feed medium (VB+) (Figures 1, 2, and 3).

[0052] Cells producing antibody B or cells producing antibody C were used, and culture was performed under the same conditions by constant-rate feeding using a 1L culture apparatus with the initial medium and feed medium as Sample 10 (VB+) and Sample 11 (VB-) in Table 1-1. Culture was performed for 14 days at pH 6.7-7.2 and 34°C-38°C, and the feed medium was added on the 3rd day after the start of culture.

[0053] We observed the effect of the presence or absence of VB12 in the medium on the number of live cells, cell viability, and cumulative cell number in cell culture under these conditions. As a result, we confirmed that the number of live cells, cell viability, and cumulative cell number on each day were almost the same in culture using a medium that did not contain VB12 in the initial medium and feed medium (VB-) compared to culture using a medium that contained VB12 in the initial medium and feed medium (VB+) (Figures 5, 6, 9, 10, 13, 14).

[0054] Cells producing antibody B or cells producing antibody C were cultured under the same conditions as sample 10 (VB+) and sample 12 (VB-) in Table 1-1 using the initial medium and the feed medium in a 1L culture apparatus by the constant-rate feed method. Culture was performed for 14 days at pH 6.7-7.2 and 34°C-38°C, and the feed medium was added on the 3rd day after the start of culture.

[0055] We observed the effect of the presence or absence of VB12 in the medium on the number of live cells, cell viability, and cumulative cell number in cell culture under these conditions. As a result, we confirmed that the number of live cells, cell viability, and cumulative cell number on each day were almost equivalent in culture using a medium that did not contain VB12 in the initial medium but contained VB12 in the feed medium (VB-) compared to culture using a medium that contained VB12 in both the initial medium and the feed medium (VB+) (Figures 7, 8, 11, 12, 15, 16).

[0056] [Table 1-1]

[0057] Example 3. Examination of the effect of VB12 in the medium on the amount of antibody production The effect of the presence or absence of VB12 in the medium on the amount of antibody produced was observed by carrying out cell culture under the same conditions as in Example 1. As a result, it was confirmed that the amount of antibody produced after 14 days of culture was almost the same in culture medium (VB-) that did not contain VB12 in the initial medium and feed medium, compared to culture medium (VB+) that contained VB12 in the initial medium and feed medium (Table 2: the amount of antibody produced in VB+ control culture is shown as 100%).

[0058] Cells producing antibody B were also cultured under the conditions in Table 1-1, and the effect of the presence or absence of VB12 in the medium on the amount of antibody produced was observed. As a result, it was confirmed that the amount of antibody produced after 14 days of culture was almost the same in the culture medium (VB-) in which the initial medium and the feed medium did not contain VB12, which is the condition of sample 11, and the culture medium (VB-) in which the initial medium did not contain VB12 but the feed medium contained VB12, which is the condition of sample 12, compared to the culture medium (VB+) in which the initial medium and the feed medium contained VB12, which is the condition of sample 10 (Tables 2-1 and 2-2: the amount of antibody produced when VB+ was used as the control culture is shown as 100%).

[0059] [Table 2]

[0060] [Table 2-1]

[0061] [Table 2-2]

[0062] Cells producing antibody C were also cultured under the conditions in Table 1-1, and the effect of the presence or absence of VB12 in the medium on the amount of antibody produced was observed. As a result, it was confirmed that the amount of antibody produced after 14 days of culture was almost the same in the culture medium (VB-) in which the initial medium and the feed medium did not contain VB12, which is the condition of sample 11, and the culture medium (VB-) in which the initial medium did not contain VB12 but the feed medium contained VB12, which is the condition of sample 12, compared to the culture medium (VB+) in which the initial medium and the feed medium contained VB12, which is the condition of sample 10 (Tables 2-3 and 2-4: the amount of antibody produced when VB+ was used as the control culture is shown as 100%).

[0063] [Table 2-3]

[0064] [Table 2-4]

[0065] Example 4. Examination of the effect of VB12 in the medium on antibody properties Cell culture, affinity column chromatography, and concentration were performed under the same conditions as in Example 1, and the antibody in the obtained composition was analyzed to observe the effect of the presence or absence of VB12 in the medium on the physical properties of the antibody. As a result, it was confirmed that the antibody obtained by culture in a medium (VB-) that did not contain VB12 in the initial medium and feed medium was almost equivalent in glycosylation (afcosyl, fucosyl, galactosyl, high mannose, hybrid), cell-derived protein (HCP), cell-derived DNA (DNA), proportion of charge variants (acidic, basic), and proportion of polymers (HMWs) compared to the antibody obtained by culture in a medium (VB+) that contained VB12 in the initial medium and feed medium (Table 3: each physical property of the antibody obtained by culture with VB+ as the control is shown as 100%).

[0066] [Table 3]

[0067] Example 5. Examination of the effect of VB12 in the medium on cell passage The effect of removing VB12 from the cell subculture medium (Thermo Fisher Scientific) was observed. As a result, subculture in medium without VB12 (VB-) showed almost the same growth behavior as subculture in medium containing VB12 (VB+). It was confirmed that subculture of cells producing antibody A could be performed for 100 days (28 subcultures) (Figure 4), subculture of cells producing antibody B for 136 days (39 subcultures) (Figure 17), and subculture of cells producing antibody C for 24 days (7 subcultures) (Figure 18).

[0068] Example 6. Examination of the possibility of removing VB12 by cation exchange chromatography An initial medium and a feed medium containing VB12 were used to culture the cells under the same conditions as in Example 1, and affinity column chromatography was performed. The antibody-containing composition thus obtained was examined for its ability to remove VB12 by a cation exchange chromatography step, which is the binding / elute method. As in Example 1, the cobalt concentration was measured by ICP-MS and used as an index of the VB12 concentration.

[0069] As a result, no change was observed in the cobalt / antibody [%] ratio before and after passing through a CEX (cation exchange) column (Table 4). This confirmed that VB12 contained in the antibody-containing composition cannot be removed even if it is subjected to an additional chromatography step.

[0070] [Table 4]

[0071] Example 7. Further investigation of the effect of VB12 on antibody coloration Media containing VB12 were used as the initial medium and the feed medium, and the culture, affinity column chromatography treatment, polishing step, and concentration were carried out under the same conditions as in Example 1. An antibody-containing composition (antibody concentration approximately 30 mg / ml, colored (weak red)) was obtained, and further investigation was carried out to confirm that VB12 was adsorbed to the antibody.

[0072] Potassium cyanide was added to the antibody-containing composition to a concentration of 0.1% (a condition in which no potassium cyanide was added was also carried out as a control), and the composition was incubated at 37°C for 45 minutes. These samples were passed through a 5 KD cutoff filter, and the collected samples from which the antibodies had been removed were analyzed by reverse phase chromatography. As a result, no peak of VB12 (cyanocobalamin) was detected in the sample not treated with potassium cyanide (KCN-), whereas a peak indicating VB12 (cyanocobalamin) was observed in the sample treated with potassium cyanide (KCN+) (Figure 19, antibody A). From these results, it was confirmed that VB12 was released from the antibody by the treatment with potassium cyanide, and that VB12 affects the coloring of the antibody. [Industrial Applicability]

[0073] The present invention can be used in the production of pharmaceuticals containing the polypeptide.

Claims

1. A method for producing an antibody-containing composition with reduced discoloration, comprising the steps of: a) culturing mammalian cells comprising a nucleic acid encoding an antibody polypeptide in a cell culture medium lacking vitamin B12; b) recovering a composition comprising the antibody polypeptide from the culture; Culturing in a cell culture medium free of vitamin B12 includes feeding culture with a vitamin B12-free starting medium and a vitamin B12-free feed medium; A method for producing a composition, wherein the antibody polypeptide comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:1 and a light chain constant region comprising the amino acid sequence of SEQ ID NO:

2.

2. The method for producing the composition according to claim 1, wherein the molar concentration ratio of vitamin B12 to the polypeptide in the composition is less than 0.26%.

3. The VCD (viable cell density) in the cell culture medium on the 7th day after the start of fed-batch culture was 80 x 10 5 A method for producing the composition according to claim 1 or 2, wherein the concentration of said composition is 100% or more of cells / mL.

4. The method for producing the composition according to any one of claims 1 to 3, wherein the mammalian cells are CHO cells.

5. A method for producing a composition described in any one of claims 1 to 4, wherein the antibody is an IgG1 humanized antibody that binds to latent myostatin.

6. A method for producing an antibody-containing formulation, comprising the steps of: producing an antibody-containing composition by a method according to any one of claims 1 to 5; and formulating the resulting composition into a pharmaceutical formulation.

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