Method for preparing a composition containing a polypeptide that inhibits coloration

Abstract

A method for preparing a composition containing a polypeptide that inhibits coloration, comprising: a) culturing eukaryotic cells containing a nucleic acid encoding the polypeptide in a cell culture medium without vitamin B12; and b) recovering the composition containing the polypeptide from the culture.

Description

Technical Field

[0001] The present invention provides a method for preparing a composition containing a polypeptide that inhibits coloration. Background Art

[0002] Methods for preparing polypeptides in vitro using recombinant cell cultures are well known and widely used in industrial-scale production. When a composition containing a polypeptide, such as an antibody, is formulated and provided as a pharmaceutical preparation, it is necessary to suppress the color of the preparation to an acceptable level, such as a level that meets regulatory requirements for the marketing of the product, as a quality characteristic.

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

[0004] Vitamin B12 has been reported as a cause of coloration of antibody-containing preparations. 12 (hereinafter referred to as "VB12") adsorption to antibodies (Non-patent Documents 1 to 3, Patent Document 1). One molecule of cobalt is coordinated to one molecule of VB12. VB12 contains cyanocobalamin and hydroxocobalamin. It is reported that the molecule is colored pink or red by binding of the hydroxocobalamin to the antibody (Non-patent Documents 1 to 3).

[0005] In addition, as a culture method for preventing antibody coloration, a culture method using a culture medium containing specific amounts of vitamins B2, B6, B9, B12, and cystine has been reported (Patent Document 2). In addition, a method for preventing coloration by preventing cyanocobalamin in the culture medium from being converted into hydroxocobalamin and reduction of the disulfide bonds of the antibody has been reported (Patent Document 1).

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

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: WO2018 / 208743

[0010] Patent Document 2: Japanese Patent Application No. 2015-515281

[0011] Non-patent literature

[0012] Non-patent document 1: MAbs. 2013 Nov 1; 5(6): 974-981

[0013] Non-patent document 2: MAbs. 2014 May 1; 6(3): 679-688

[0014] Non-patent literature 3: Biotechnology and Bioengineering. 2018; 115: 900-909 SUMMARY OF THE INVENTION

[0016] Problems to be solved by the invention

[0017] The object of the present invention is to provide a method for solving the problem of coloration while maintaining the production amount and physical properties of a polypeptide that is prone to coloration within an acceptable range by changing a relatively simple preparation method.

[0018] Means for solving problems

[0019] The present inventors conducted intensive studies to achieve the above-mentioned objectives and found that the production of polypeptides can be maintained while coloration can be suppressed by using a culture medium that does not contain VB12, thereby completing the present invention.

[0020] Therefore, the present invention can be expressed as follows (1) to (17).

[0021] (1) A method for preparing a composition containing a polypeptide that inhibits coloration, comprising: (a) culturing eukaryotic cells containing a nucleic acid encoding the polypeptide in a cell culture medium without vitamin B12; and (b) recovering the composition containing the polypeptide from the culture.

[0022] (2) The method for preparing the composition according to (1), wherein the molar concentration ratio of vitamin B12 to the polypeptide in the composition is less than 0.26%.

[0023] (3) The method for producing the composition according to (1) or (2), wherein the fed-batch culture is carried out using an initial medium not containing vitamin B12 and a fed-batch medium not containing vitamin B12.

[0024] (4) The method for preparing the composition according to (3), wherein the VCD (viable cell density) in the cell culture medium on day 7 from the start of fed-batch culture is 80×10 5 More than cells / mL.

[0025] (5) The method for producing a composition according to any one of (1) to (4), wherein the eukaryotic cell is a CHO cell.

[0026] (6) The method for producing a composition according to any one of (1) to (5), wherein the polypeptide is an Fc-containing polypeptide.

[0027] (7) The method for producing a composition according to (6), wherein the Fc-containing polypeptide is an antibody.

[0028] (8) The method for preparing a composition described in (6) or (7), wherein the polypeptide comprises a change in at least one amino acid residue selected from the group consisting of amino acid residues 214, 234, 238, 250, 264, 307, 311, 330, 343, 428, 434, 436, 438 and 440 in the Fc region according to EU numbering.

[0029] (9) A method for preparing a composition according to any one of (6) to (8), wherein the polypeptide comprises at least one alteration selected from the group consisting of 214R, 234Y, 238D, 250V, 264I, 307P, 311R, 330K, 343R, 428L, 434A, 436T, 438R and 440E in the Fc region according to EU numbering.

[0030] (10) A method for preparing a composition described in (6) to (9), wherein the polypeptide is an antibody comprising a heavy chain constant region containing the amino acid sequence of sequence number: 1 and a light chain constant region containing the amino acid sequence of sequence number: 2.

[0031] (11) The method for producing a composition according to (10), wherein the antibody is an IgG1 humanized antibody that binds to latent myostatin.

[0032] (12) A method for preparing a composition containing an antibody that inhibits coloration, comprising:

[0033] a) confirming that the target antibody is an antibody comprising an alteration of at least one amino acid residue selected from the group consisting of amino acid residues 214, 234, 238, 250, 264, 307, 311, 330, 343, 428, 434, 436, 438 and 440 in the Fc region according to EU numbering;

[0034] b) a step of selecting a cell culture medium without vitamin B12 for the antibody comprising the change in the amino acid residue;

[0035] c) the step of culturing eukaryotic cells containing the nucleic acid encoding the antibody containing the change in the amino acid residue using the cell culture medium selected in step b); and

[0036] d) a step of recovering the antibody-containing composition from the culture.

[0037] (13) The production method described in (12), wherein step b) is a step of selecting an initial culture medium without vitamin B12 and a fed-batch culture medium without vitamin B12.

[0038] (14) The production method described in (12) or (13), wherein the eukaryotic cells are CHO cells.

[0039] (15) A method for preparing a preparation containing an antibody, comprising: (a) culturing eukaryotic cells containing a nucleic acid encoding the antibody in a cell culture medium without vitamin B12; (b) recovering a composition containing the antibody from the culture; and (c) preparing the obtained composition into a pharmaceutical preparation.

[0040] (16) A method for inhibiting antibody coloring, comprising: in the preparation of an antibody using a recombinant cell culture, (a) culturing eukaryotic cells containing a nucleic acid encoding the antibody in a cell culture medium without vitamin B12; and (b) recovering a composition containing the antibody from the culture.

[0041] (17) The method for preparing the composition described in (7), the method for preparing the preparation containing the antibody described in (15), or the method for inhibiting the coloring of the antibody described in (16), wherein the aforementioned antibody is an anti-IL-8 antibody or an anti-myostatin antibody.

[0042] Effects of the Invention

[0043] According to the present invention, a method for preparing a composition containing a polypeptide that inhibits coloration is provided. Sequence Listing <110> Chugai Pharmaceutical Co., Ltd. <120> Method for preparing a composition containing a polypeptide that inhibits coloration <130> FA0001-20314 <150> JP 2020-014739 <151> 2020-01-31 <160> 2 <210> 1 <211> 328 <212> PRT <213> Artificial sequence <220> <223> Artificially synthesized sequences <400> 1 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Ile Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu 180 185 190 His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ala His Thr Thr 305 310 315 320 Arg Lys Glu Leu Ser Leu Ser Pro 325 <210> 2 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Artificially synthesized sequences <400> 2 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 BRIEF DESCRIPTION OF THE DRAWINGS

[0045] [ Figure 1] shows that in the culture of cells producing polypeptide (antibody A: anti-myostatin antibody), the culture medium containing VB12 in the initial culture medium and the fed-batch culture medium is VB+, and the culture medium not containing VB12 in the initial culture medium and the fed-batch culture medium is VB-. For each day during the 14-day culture, the number of live cells was measured by the cell automatic counting device Vi-CELL system (manufactured by Beckman Courter, model Vi-CELL XR).

[0046] [ Figure 2 ] shows that in the culture of cells producing antibody A, the culture medium containing VB12 in the initial culture medium and the fed-batch culture medium is VB+, and the culture medium not containing VB12 in the initial culture medium and the fed-batch culture medium is VB-. For each day during the 14-day culture, the cell survival rate was measured by the cell automatic metering device Vi-CELL system (manufactured by Beckman Courter, model Vi-CELL XR).

[0047] [ Figure 3 ] shows that in the culture of cells producing antibody A, the culture medium containing VB12 in the initial culture medium and the fed-batch culture medium is VB+, and the culture medium not containing VB12 in the initial culture medium and the fed-batch culture medium is VB-. For each day during the 14-day culture, the cumulative cell number is calculated based on the number of live cells measured by the cell automatic counting device Vi-CELL system (manufactured by Beckman Courter, model Vi-CELL XR).

[0048] [ Figure 4 ] shows that in the culture of cells producing antibody A, the culture medium containing VB12 is VB+, the culture medium without VB12 is VB-, and the subculture is performed in a 3-day or 4-day cycle until 100 days (28 subcultures), and the cell doubling time is used as an indicator to plot the results of cell proliferation behavior on each day. (The cell doubling time is calculated by measuring the cell automatic metering device Vi-CELL system (manufactured by Beckman Courter))

[0049] [ Figure 5 ] is displayed in the generated Figure 1 In the culture of cells expressing different polypeptides (antibody B: anti-IL-8 antibody), the medium containing VB12 in the initial medium and the fed-batch medium was denoted as VB+, and the medium not containing VB12 in the initial medium and the fed-batch medium was denoted as VB-. The number of viable cells was measured on each day during the 14-day culture period using the cell automatic counting device Vi-CELL system (manufactured by Beckman Courter, model Vi-CELL XR).

[0050] [ Figure 6 ] is displayed in the generated Figure 1 , Figure 5 In the culture of cells expressing different polypeptides (antibody C: anti-FIXa / FX bispecific antibody), the medium containing VB12 in the initial culture medium and the fed-batch culture medium was denoted as VB+, and the medium not containing VB12 in the initial culture medium and the fed-batch culture medium was denoted as VB-. The number of viable cells was measured on each day during the 14-day culture period using the cell automatic counting device Vi-CELL system (manufactured by Beckman Courter, model Vi-CELL XR).

[0051] [ Figure 7 ] shows that in the culture of cells producing antibody B, the culture medium containing VB12 in the initial culture medium and the fed-batch culture medium is VB+, and the culture medium not containing VB12 in the initial culture medium but containing VB12 in the fed-batch culture medium is VB-. For each day during the 14-day culture, the number of live cells was measured by the cell automatic counting device Vi-CELL system (manufactured by BeckmanCourter, model Vi-CELL XR).

[0052] [ Figure 8 ] shows that in the culture of cells producing antibody C, the culture medium containing VB12 in the initial culture medium and the fed-batch culture medium is VB+, and the culture medium not containing VB12 in the initial culture medium but containing VB12 in the fed-batch culture medium is VB-. For each day during the 14-day culture, the number of live cells was measured by the cell automatic counting device Vi-CELL system (manufactured by BeckmanCourter, model Vi-CELL XR).

[0053] [ Fig. 9 ] shows that in the culture of cells producing antibody B, the culture medium containing VB12 in the initial culture medium and the fed-batch culture medium is VB+, and the culture medium not containing VB12 in the initial culture medium and the fed-batch culture medium is VB-. For each day during the 14-day culture, the cell survival rate was measured by the cell automatic metering device Vi-CELL system (manufactured by Beckman Courter, model Vi-CELL XR).

[0054] [ Fig.10] shows that in the culture of cells producing antibody C, the culture medium containing VB12 in the initial culture medium and the fed-batch culture medium is VB+, and the culture medium not containing VB12 in the initial culture medium and the fed-batch culture medium is VB-. For each day during the 14-day culture, the cell survival rate was measured by the cell automatic metering device Vi-CELL system (manufactured by Beckman Courter, model Vi-CELL XR).

[0055] [ Fig.11 ] shows that in the culture of cells producing antibody B, the culture medium containing VB12 in the initial culture medium and the fed-batch culture medium is VB+, and the culture medium not containing VB12 in the initial culture medium but containing VB12 in the fed-batch culture medium is VB-. For each day during the 14-day culture, the cell survival rate was measured by the cell automatic measuring device Vi-CELL system (manufactured by BeckmanCourter, model Vi-CELL XR).

[0056] [ Fig.12 ] shows that in the culture of cells producing antibody C, the culture medium containing VB12 in the initial culture medium and the fed-batch culture medium is VB+, and the culture medium not containing VB12 in the initial culture medium but containing VB12 in the fed-batch culture medium is VB-. For each day during the 14-day culture, the cell survival rate was measured by the cell automatic metering device Vi-CELL system (manufactured by BeckmanCourter, model Vi-CELL XR).

[0057] [ Fig.13 ] shows that in the culture of cells producing antibody B, the culture medium containing VB12 in the initial culture medium and the fed-batch culture medium is VB+, and the culture medium not containing VB12 in the initial culture medium and the fed-batch culture medium is VB-. For each day during the 14-day culture, the cumulative cell number is calculated based on the number of live cells measured by the cell automatic counting device Vi-CELL system (manufactured by Beckman Courter, model Vi-CELL XR).

[0058] [ Fig.14 ] shows that in the culture of cells producing antibody C, the culture medium containing VB12 in the initial culture medium and the fed-batch culture medium is VB+, and the culture medium not containing VB12 in the initial culture medium and the fed-batch culture medium is VB-. For each day during the 14-day culture, the cumulative cell number was calculated based on the number of live cells measured by the cell automatic counting device Vi-CELL system (manufactured by Beckman Courter, model Vi-CELL XR).

[0059] [ Fig.15] shows that in the culture of cells producing antibody B, the culture medium containing VB12 in the initial culture medium and the fed-batch culture medium is VB+, and the culture medium not containing VB12 in the initial culture medium but containing VB12 in the fed-batch culture medium is VB-. For each day during the 14-day culture, the cumulative cell number was calculated based on the number of live cells measured by the cell automatic counting device Vi-CELL system (manufactured by BeckmanCourter, model Vi-CELL XR).

[0060] [ Fig.16 ] shows that in the culture of cells producing antibody C, the culture medium containing VB12 in the initial culture medium and the fed-batch culture medium is VB+, and the culture medium not containing VB12 in the initial culture medium but containing VB12 in the fed-batch culture medium is VB-. For each day during the 14-day culture, the cumulative cell number was calculated based on the number of live cells measured by the cell automatic counting device Vi-CELL system (manufactured by BeckmanCourter, model Vi-CELL XR).

[0061] [ Fig.17 ] shows that in the culture of cells producing antibody B, the culture medium containing VB12 is VB+, the culture medium without VB12 is VB-, and the subculture is performed in a 3-day or 4-day cycle until 136 days (39 subcultures), and the cell doubling time is used as an indicator to plot the results of cell proliferation behavior on each day. (The cell doubling time is calculated by measuring the cell automatic metering device Vi-CELL system (manufactured by Beckman Courter))

[0062] [ Fig.18 ] shows that in the culture of cells producing antibody C, the culture medium containing VB12 is VB+, the culture medium without VB12 is VB-, and the subculture is performed in a 3-day or 4-day cycle until 24 days (7 subcultures), and the cell doubling time is used as an indicator to plot the results of cell proliferation behavior on each day. (The cell doubling time is calculated by measuring the cell automatic metering device Vi-CELL system (manufactured by Beckman Courter))

[0063] [ Fig.19 ] shows that the antibody-containing composition treated with potassium cyanide and the antibody-containing composition not treated with potassium cyanide were each removed by a 5KD cutoff filter and analyzed by reverse phase chromatography (Column: Acclaim TM The results of the analysis of the recovered samples were performed using PolarAdvantage II LC. The potassium cyanide-untreated samples were represented by KCN-, the potassium cyanide-treated samples were represented by KCN+, and the VB12 peak was represented by STD.

[0064] Modes for carrying out the invention

[0065] Hereinafter, embodiments of the present invention will be described in detail.

[0066] The present invention relates to a method for preparing a composition containing a polypeptide that inhibits coloration.

[0067] VB12

[0068] VB12 refers to cyanocobalamin in a narrow sense and vitamin B12 in a broad sense. 12 The general term for cobalamin. VB12 as a culture medium component is cyanocobalamin. When the culture medium components become an aqueous solution, they are in a state of equilibrium among cyanocobalamin, hydroxocobalamin, hydrocobalamin, adenosylcobalamin and methylcobalamin.

[0069] In VB12, each molecule binds to 1 molecule of cobalt molecule. Therefore, in the embodiments of the present application, the relative concentration of VB12 relative to the antibody is quantified by quantifying the relative concentration of cobalt molecules relative to the antibody. In the present invention, for the culture of eukaryotic cells, an initial culture medium and a fed-batch culture medium that do not contain VB12 can be used. The culture medium "without VB12" in the present invention not only includes the case where the concentration of VB12 in the culture medium is 0 mg / L, but also includes a culture medium mixed with VB12 that is lower than the actual content level of the culture medium additive. Specifically, in the case of culturing using the culture medium under the same conditions as in Example 1 of the present invention, in the case of an antibody-containing composition (antibody concentration of about 30 mg / mL) obtained by ICP-MS measurement, in the case where the concentration of cobalt molecules contained in VB12 is lower than the quantitative limit value of 20 ppb, it is equivalent to "without VB12".

[0070] Eukaryotic cells

[0071] Eukaryotic cells refer to cells having a nucleus surrounded by a nuclear membrane. The "eukaryotic cells containing nucleic acids encoding polypeptides" in the present invention are host cells that can be used as a production system for the preparation of desired polypeptides. Such cells can be natural cells that can produce the desired polypeptides, or they can be cells into which nucleic acids encoding (or expressing) the desired polypeptides are artificially introduced, preferably transformed cells into which nucleic acids encoding the desired polypeptides are introduced. An example of such transformed cells is a strain of polypeptides obtained by introducing exogenous DNA encoding the desired polypeptides into eukaryotic cells using genetic recombination technology. Therefore, the term "containing nucleic acids encoding polypeptides" can also be read alternatively as "artificial introduction of nucleic acids encoding polypeptides" or "introduction of exogenous nucleic acids encoding polypeptides".

[0072] 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 from insects, pyrophores, amphibians, reptiles, or mammals. Preferred examples of eukaryotic cells in the present invention are mammalian cells. Mammalian cells are selected from CHO cells, NS0 cells, Sp2 / 0 cells, COS cells, HEK cells, BHK cells, PER.C6 (注册商标) Cells, and hybridoma cells, etc., more preferably CHO cells.

[0073] Cell culture medium

[0074] In the present invention, the cell culture medium refers to a culture medium used for culturing cells containing a nucleic acid encoding a polypeptide, and may also be referred to as a culture medium. As a cell culture medium, a commercially available culture medium or a known culture medium may be used appropriately. In addition to the culture medium not containing VB12 used in the present invention, a conventional culture medium contains VB12 as an essential component necessary for the transcriptional activity and nucleic acid synthesis of the cell. In an embodiment of the present invention, a culture medium is used in which the composition of the existing culture medium is maintained while excluding, removing or reducing only the component of VB12.

[0075] Those skilled in the art can appropriately select the type of culture medium suitable for a specific cell line. For example, as a known culture medium or commercially available culture medium for animal cell culture, IMDM (Iscove's Modified Dulbecco's Medium), DMEM (Dulbecco's Modified Eagle Medium), Ham's F12 medium, D-MEM / F-121:1 Mixture (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12), RPMI1640, CHO-S-SFMII (Invitrogen), CHO-SF (Sigma-Aldrich), EX-CELL 301 (JRH biosciences), CD-CHO (Invitrogen), IS CHO-V (Irvine Scientific), PF-ACF-CHO (Sigma-Aldrich) and the like can be used, but the culture medium is not limited to these, as will be understood by those skilled in the art. A variety of culture media optimized for cell proliferation and increased yield of antibody and recombinant protein production are known to those skilled in the art.

[0076] For example, examples of a medium that can be used to culture a CHO cell line producing a humanized antibody (IgG1 antibody) binding to latent myostatin used in the Examples include IMDM, DMEM, or Ham's F12 medium, or a combination thereof.

[0077] Cell culture

[0078] Generally, cell culture methods are classified into batch culture, continuous culture, and fed-batch culture.

[0079] The batch method is a culture method in which a small amount of seed culture solution is added to the culture medium, and cells are proliferated without newly adding culture medium to the culture medium or draining the culture medium.

[0080] The continuous method is a culture method in which a culture medium is continuously added and discharged during the culture. In addition, the continuous method also includes perfusion culture.

[0081] Since the fed-batch culture method is between the batch method and the continuous method, it is also called semi-batch culture, which is a culture method in which culture medium is added continuously or sequentially during culture, but the continuous culture fluid is not discharged as in the continuous method. The culture medium added during the fed-batch culture (hereinafter referred to as the fed-batch culture medium) does not have to be the same culture medium as the culture medium already used in the culture (hereinafter referred to as the initial culture medium), and a different culture medium may be added, or only specific components may be added. Alternatively, the fed-batch culture medium may also be a culture medium with the same composition as the initial culture medium.

[0082] In the present invention, any culture method of a batch method, a continuous method, or a fed-batch culture method can be used, but the fed-batch culture method is preferably used.

[0083] Generally, when culturing cells to produce a desired polypeptide, a certain amount of a seed culture medium containing cells is added to an initial culture medium to culture the cells. Furthermore, in order to increase the production of the desired polypeptide, a fed-batch culture medium is added during the culture.

[0084] Seed culture medium refers to a culture medium for amplifying cells (working cell bank) producing the desired polypeptide, and obtaining the number of cells required for the final transfer to the culture medium (initial culture medium) for producing the desired polypeptide. In addition, the initial culture medium generally refers to the culture medium used for culturing cells to produce the desired polypeptide, i.e., the culture medium used in the initial stage of the culture of the cells. Fed-batch culture medium generally refers to the culture medium added to the culture medium in the initial culture. Fed-batch culture medium is sometimes added in several times. In addition, the fed-batch culture medium is sometimes added continuously or intermittently.

[0085] In the present invention, the cells are subcultured using a seed culture medium, and thereafter, the seed culture medium containing the cells is added to an initial culture medium in a certain amount, and culture is performed in the initial culture medium to produce the desired polypeptide. Further, depending on the situation, a fed-batch culture medium is added to the culture medium one or more times.

[0086] Fed-batch culture methods are further classified according to the feeding method. Isokinetic fed-batch method is a culture method in which a certain amount of fed-batch medium is continuously added to the initial medium.

[0087] Polypeptide, composition comprising polypeptide, recovery of composition comprising polypeptide

[0088] The polypeptide used in the present invention is preferably an Fc-containing polypeptide comprising a region corresponding to the Fc region of an antibody, and more preferably an antibody.

[0089] A composition comprising a polypeptide (composition containing a polypeptide) means a composition comprising a polypeptide and other ingredients. In an embodiment of the present invention, a composition comprising a polypeptide can be recovered from a culture obtained by a cell culture step for polypeptide production. "Recovery" as described herein refers to recovering a supernatant (culture supernatant) containing a polypeptide from a culture or culture fluid obtained by a cell culture step or recovering a filtrate containing a polypeptide using a filter.

[0090] The solution thus recovered can be adjusted to a composition containing an appropriate concentration of the polypeptide through purification and concentration steps such as affinity column chromatography.

[0091] In the case where the polypeptide is an Fc-containing polypeptide or an antibody, the composition comprising the polypeptide (polypeptide-containing composition) can be read alternatively as a composition comprising an Fc-containing polypeptide (Fc-containing polypeptide-containing composition) or a composition comprising an antibody (antibody-containing composition).

[0092] Fc region (Fc)

[0093] The term Fc region (or simply Fc) includes native sequence Fc regions and variant Fc regions. The boundaries of the Fc region of an immunoglobulin heavy chain can vary, and refers to a region consisting of a hinge portion or a portion thereof, CH2, and CH3 domains in an antibody molecule. The Fc region is not limited as long as it is an antibody (IgA, IgD, IgE, IgG, IgM), especially an IgG Fc region, preferably a human IgG (IgG1, IgG2, IgG3, IgG4) Fc region, and more preferably a human IgG1 Fc region.

[0094] Suppression of coloration

[0095] The composition containing a polypeptide that inhibits coloring in the present invention refers to a composition containing a polypeptide that reduces coloring caused by adsorption of VB12, specifically red or pink. In an embodiment of the present invention, for a composition containing a polypeptide (antibody) in which the concentration of the polypeptide (antibody) is adjusted to about 30 mg / mL, a colorless evaluation is performed by visual evaluation to confirm that coloring is inhibited. In addition, for a composition containing a polypeptide (antibody) that inhibits coloring, the molar concentration ratio [%] of VB12 to the polypeptide quantified under the conditions of Example 1 is less than 0.26, preferably less than 0.2, more preferably less than 0.1, and further preferably less than 0.05.

[0096] Antibody confirmation and cell culture medium selection

[0097] In one embodiment of the present invention, an antibody that is prone to coloration is confirmed in advance, and a culture step suitable for inhibiting coloration is selected for it, which is different from the usual culture step. In this embodiment, there is first a step of confirming that the target polypeptide is a polypeptide containing an alteration in at least one amino acid residue selected from the group consisting of amino acid residues 214, 234, 238, 250, 264, 307, 311, 330, 343, 428, 434, 436, 438 and 440 in the Fc region according to EU numbering. "Confirmation" does not necessarily mean specifying the specific altered position of the polypeptide by an analytical method, and includes pre-recognition that the target polypeptide contains the alteration.

[0098] Then, for the antibody confirmed to contain the modification of the amino acid residue, a step different from the usual culture step is selected. That is, in the usual culture step, a cell culture medium containing VB12 as an essential component (usual cell culture medium) is used, but in the culture step of the antibody, a cell culture medium not containing VB12 is used instead of the usual cell culture medium. Here, the cell culture medium not containing VB12 can be a cell culture medium that is different from the composition of the usual cell culture medium except that VB12 is not contained, and the other components are the same.

[0099] The composition containing the polypeptide is prepared through the steps of culturing cells containing the nucleic acid encoding the polypeptide in the cell culture medium thus selected, and recovering the composition containing the polypeptide from the culture. Example

[0100] The present invention will be specifically described below by way of examples. In addition, these examples are for illustrating the present invention and do not limit the scope of the present invention.

[0101] The following antibodies, cells and culture media were used in the examples.

[0102] Antibody:

[0103] As antibody A, an IgG1 humanized antibody comprising a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 1 and a light chain constant region containing the amino acid sequence of SEQ ID NO: 2, i.e., an anti-myostatin antibody that binds to latent myostatin, was used. The antibody comprises alterations in the Fc region according to EU numbering 214R, 234Y, 238D, 250V, 264I, 307P, 311R, 330K, 343R, 428L, 434A, 436T, 438R and 440E.

[0104] As antibody B, an anti-IL-8 antibody that binds to IL-8 described in WO / 2016 / 125495 or WO / 2017 / 046994 is used.

[0105] As antibody C, the anti-FIX(a) / FX bispecific antibody that binds to both FIX(a) and FX described in WO2019065795 was used.

[0106] cell:

[0107] CHO cells used were derived from CHO-DXB11.

[0108] Culture medium:

[0109] The initial culture medium and the fed-batch culture medium are prepared using culture media manufactured by Thermo Fisher Scientific, Fujifilm and Koh Pure Chemical Industries, Ltd. In this embodiment, as the initial culture medium, an initial culture medium containing VB12 (initial culture medium with a relative VB12 concentration of 100%), an initial culture medium in which VB12 is completely removed and the composition other than VB12 is the same (initial culture medium with a relative VB12 concentration of 0%), and an initial culture medium in which these two culture media are mixed at a ratio of 1:1 (initial culture medium with a relative VB12 concentration of 50%) are prepared. In addition, as the fed-batch culture medium, a fed-batch culture medium containing VB12 (a fed-batch culture medium with a relative VB12 concentration of 100%) and a fed-batch culture medium in which VB12 is completely removed and the composition other than VB12 is the same (a fed-batch culture medium with a relative VB12 concentration of 0%) are prepared.

[0110] Example 1. Study on the effect of VB12 on antibody coloring

[0111] Using cells producing antibody A, samples 1 to 9 in which the initial medium and the fed-batch medium were combined as shown in Table 1 were cultured under the same conditions by an isokinetic fed-batch method using 1 L to 25 L culture apparatuses, respectively. Culture was performed at a pH range of 6.7 to 7.2 and a temperature range of 34° C. to 38° C. for 14 days, and the fed-batch medium was added on the 3rd day after the start of culture.

[0112] The composition containing the antibody was recovered from the culture solution after culturing for 14 days, purified by affinity column chromatography using Protein A, and the eluate was concentrated to adjust the antibody concentration to about 30 mg / mL (26.7 mg / mL to 31.4 mg / mL) in samples 1 to 8. Sample 9 was further concentrated to an antibody concentration of 200 mg / mL or more.

[0113] The coloring of the antibody-containing composition thus obtained and the content of VB12 were evaluated. Coloring was performed by visual evaluation. Some coloring (light red) was observed in samples 1, 5, and 7. In addition, coloring (red) was observed in sample 9. On the other hand, no coloring (colorless) was observed for samples 2 to 4 and 6 to 8.

[0114] Since one molecule of VB12 in the composition containing the antibody binds to one molecule of cobalt, the molar concentration ratio of cobalt to antibody (cobalt / antibody [%]) was calculated by measuring the concentration of cobalt contained in the composition containing the antibody using ICP-MS (Inductively Coupled Plasma-Mass spectrometry), and the content of VB12 (the molar concentration ratio of VB12 to antibody (VB12 / antibody [%])) was estimated. These results are shown in Table 1.

[0115] [Table 1]

[0116]

[0117] ※ The cobalt concentration [ppb], cobalt / antibody [%] and VB12 / antibody [%] of samples 2-4, 6 and 8 are below the quantitative limit

[0118] The experimental results confirmed that VB12 contained in the antibody-containing composition is the cause of coloration. In addition, it was confirmed that VB12 contained in the antibody-containing composition after culture can be suppressed by suppressing VB12 contained in the initial culture medium and the fed-batch culture medium.

[0119] Example 2. Study on the effect of VB12 in culture medium on cell culture

[0120] By carrying out cell culture under the same conditions as in Example 1, the effect of the presence or absence of VB12 in the culture medium on the number of live cells, cell survival rate, and cumulative cell number was observed. As a result, it was confirmed that the number of live cells, cell survival rate, and cumulative cell number on each day were almost the same when cultured in a medium (VB-) that did not contain VB12 in the initial culture medium and the fed-batch culture medium and cultured in a medium (VB+) that contained VB12 in the initial culture medium and the fed-batch culture medium. ( Figure 1 , 2 、3)

[0121] Using cells producing antibody B or cells producing antibody C, the initial medium and the fed-batch medium were set to the conditions of sample 10 (VB+) and sample 11 (VB-) in Table 1-1, and culture was performed under the same conditions by an isokinetic fed-batch method using a 1 L culture apparatus. Culture was performed for 14 days in the range of pH 6.7 to 7.2 and 34° C. to 38° C., and the fed-batch medium was added on the 3rd day after the start of culture.

[0122] In the cell culture under this condition, the effect of the presence of VB12 in the culture medium on the number of live cells, cell survival rate, and cumulative cell count was observed. As a result, it was confirmed that the number of live cells, cell survival rate, and cumulative cell count on each day were almost the same when the culture medium (VB-) containing VB12 in the initial culture medium and the fed-batch culture medium was compared with the culture medium (VB+) containing VB12 in the initial culture medium and the fed-batch culture medium ( Figure 5 , 6 , 9, 10, 13, 14).

[0123] Using cells producing antibody B or cells producing antibody C, the initial medium and the fed-batch medium were set to the conditions of sample 10 (VB+) and sample 12 (VB-) in Table 1-1, and culture was performed under the same conditions by an isokinetic fed-batch method using a 1 L culture apparatus. Culture was performed for 14 days in the range of pH 6.7 to 7.2 and 34° C. to 38° C., and the fed-batch medium was added on the 3rd day after the start of culture.

[0124] In the cell culture under this condition, the effect of the presence of VB12 in the culture medium on the number of live cells, cell survival rate, and cumulative cell number was observed. As a result, it was confirmed that the number of live cells, cell survival rate, and cumulative cell number on each day were almost the same when the culture medium (VB-) containing VB12 in the initial culture medium and the fed-batch culture medium was cultured in the culture medium containing VB12 (VB+) without VB12 in the initial culture medium ( Figure 7 , 8 , 11, 12, 15, 16)

[0125] [Table 1-1]

[0126]

[0127] Example 3. Study on the effect of VB12 in the culture medium on the amount of antibody produced

[0128] The effect of the presence of VB12 in the culture medium on the amount of antibody produced was observed by culturing cells 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 when cultured in a medium (VB-) containing no VB12 in the initial culture medium and the fed-batch culture medium and in a medium (VB+) containing VB12 in the initial culture medium and the fed-batch culture medium (Table 2: the amount of antibody produced in the culture with VB+ as the control was expressed as 100%).

[0129] Cells producing antibody B were also cultured under the conditions of Table 1-1 to observe the effect of the presence of VB12 in the culture medium on the amount of antibody produced. As a result, it was confirmed that the amount of antibody produced after 14 days of culture was almost equal to that of culture in the medium containing VB12 (VB-) in the initial culture medium and the fed-batch culture medium as the conditions of sample 11 and the medium containing VB12 (VB-) in the initial culture medium and the fed-batch culture medium as the conditions of sample 12, compared with the culture in the medium containing VB12 (VB+) in the initial culture medium and the fed-batch culture medium as the conditions of sample 10) (Tables 2-1 and 2-2: The amount of antibody produced by the culture with VB+ as the control was expressed as 100%).

[0130] [Table 2]

[0131] Production volume (%) Standard error (%) VB+ 100 1.36 VB- 104.6 1.53

[0132] [Table 2-1]

[0133] Antibody B

[0134] VB+ is the condition for sample number 10, VB- is the condition for sample number 11

[0135] Production volume (%) VB+ 100 VB- 100.5

[0136] [Table 2-2]

[0137] Antibody B

[0138] VB+ is the condition for sample number 10, VB- is the condition for sample number 12

[0139] Production volume (%) Standard error (%) VB+ 100 1.709 VB- 100.7 1.861

[0140] Cells producing antibody C were also cultured under the conditions of Table 1-1 to observe the effect of the presence of VB12 in the medium on the amount of antibody produced. As a result, it was confirmed that the amount of antibody produced after 14 days of culture was almost equal to that of culture in the medium containing VB12 (VB-) in the initial medium and the fed-batch medium as the conditions of sample 11 and the medium containing VB12 (VB-) in the initial medium and the fed-batch medium as the conditions of sample 12, compared with the culture in the medium containing VB12 (VB+) in the initial medium and the fed-batch medium as the conditions of sample 10 (Tables 2-3 and 2-4: the amount of antibody produced by the culture with VB+ as the control was expressed as 100%).

[0141] [Table 2-3]

[0142] Antibody C

[0143] VB+ is the condition for sample number 10, VB- is the condition for sample number 11

[0144] Production volume (%) Standard error (%) VB+ 100 1.027 VB- 106.2 1.558

[0145] [Table 2-4]

[0146] Antibody C

[0147] VB+ is the condition for sample number 10, VB- is the condition for sample number 12

[0148] Production volume (%) Standard error (%) VB+ 100 1.027 VB- 107.4 1.623

[0149] Example 4. Study on the effect of VB12 in the culture medium on the physical properties of antibodies

[0150] Cell culture, affinity column chromatography and concentration were performed under the same conditions as in Example 1, and the antibodies in the obtained composition were analyzed to observe whether the presence of VB12 in the culture medium had an effect on the physical properties of the antibodies. As a result, it was confirmed that the antibodies obtained by culturing in a medium (VB-) without VB12 in the initial culture medium and the fed-batch culture medium were almost equal to the antibodies obtained by culturing in a medium (VB+) containing VB12 in the initial culture medium and the fed-batch culture medium in terms of sugar chain modification (Afcosyl, fucosyl, galactosyl, high mannose, hybrid), cell-derived protein (HCP), cell-derived DNA (DNA), the ratio of charge variants (acidic, basic), and the ratio of polymers (HMWs) (Table 3: each physical property of the antibody obtained by culturing with VB+ as the control was expressed as 100%).

[0151] [Table 3]

[0152] Afucosyl fucosyl Galactosyl High Mannose Heterozygote HCP DNA Acidic Alkaline HMWs VB+(%) 100 100 100 100 100 100 100 100 100 100 VB-(%) 93.5 100.4 84.0 94.5 88.3 93.8 113.8 91.4 106.8 113.9 VB + standard error (%) 8.2 0.3 3.1 2.3 14.6 4.9 31.4 1.6 2.5 21.8 VB-standard error (%) 5.4 0.2 1.7 2.3 6.6 8.9 26.2 1.0 1.3 14.0

[0153] Example 5. Study on the effect of VB12 in culture medium on cell passage

[0154] The effect of removing VB12 from the cell subculture medium (manufactured by Thermo Fisher Scientific) was observed. As a result, subculture in a medium without VB12 (VB-) showed almost the same proliferation behavior as subculture in a medium containing VB12 (VB+). It was confirmed that subculture of cells producing antibody A could be carried out for 100 days (28 subcultures) ( Figure 4 ), the subculture of cells producing antibody B can be carried out for 136 days (39 subcultures) ( Fig.17 ), and the subculture of cells producing antibody C can be carried out for 24 days (7 subcultures) ( Fig.18 ).

[0155] Example 6. Study on the possibility of removing VB12 by cation exchange chromatography

[0156] The initial medium and the fed-batch medium containing VB12 were cultured under the same conditions as in Example 1, and affinity column chromatography was performed. For the obtained antibody-containing composition, it was studied whether VB12 could be removed by a cation exchange chromatography step as a binding / elution method. As in Example 1, the concentration of cobalt was measured by ICP-MS as an indicator of the concentration of VB12.

[0157] As a result, there was no change in cobalt / antibody [%] before passing through the CEX (cation exchange) column and after elution from CEX (Table 4). This confirmed that VB12 contained in the antibody-containing composition could not be removed even by the additional chromatography step.

[0158] [Table 4]

[0159] Cobalt / antibody [%] Passing the column 0.29 After elution 0.31

[0160] Example 7. Additional study on the effect of VB12 on antibody coloration

[0161] Using the culture medium containing VB12 in the initial culture medium and the feed batch culture medium, an additional study on the adsorption of VB12 to the antibody was conducted on the antibody-containing composition (antibody concentration of about 30 mg / ml, colored (light red)) obtained by culturing under the same conditions as in Example 1, affinity column chromatography treatment, polishing step, and concentration.

[0162] Potassium cyanide was added to the composition containing the antibody at 0.1% (a condition without adding potassium cyanide was also implemented as a control), and the mixture was incubated at 37°C for 45 minutes. The samples were passed through a 5KD cutoff filter and the recovered samples after removing the antibodies were analyzed by reverse phase chromatography. As a result, no peak of VB12 (cyanocobalamin) was detected in the potassium cyanide non-treated sample (KCN-), while a peak showing VB12 (cyanocobalamin) was observed in the potassium cyanide treated sample (KCN+). Fig.19 , antibody A). Based on this result, it was confirmed that VB12 was released from the antibody by the potassium cyanide treatment, and it was confirmed that VB12 affected the coloring of the antibody.

[0163] Industrial Applicability

[0164] The present invention can be used for preparing drugs containing polypeptides.

Claims

1. A method for preparing a composition containing an antibody that inhibits coloration, comprising: a) feeding batch culturing eukaryotic cells containing a nucleic acid encoding an antibody in a cell culture medium without vitamin B12, wherein the fed-batch culturing is performed by an initial culture medium without vitamin B12 and a fed-batch culture medium without vitamin B12; b) a step of recovering a composition comprising antibodies from the culture, Wherein, the antibody comprises a heavy chain constant region containing the amino acid sequence of sequence number: 1 and a light chain constant region containing the amino acid sequence of sequence number:

2.

2. The method for preparing the composition according to claim 1, wherein: In the composition, the molar concentration ratio of vitamin B12 to antibody is lower than 0.26%.

3. The method for preparing the composition according to claim 1, wherein: The VCD (viable cell density) in the cell culture medium on day 7 from the start of fed-batch culture was 80×10 5 More than cells / mL.

4. The method for preparing the composition according to claim 1, wherein: The antibody is an IgG1 humanized antibody that binds to latent myostatin.

5. A method for preparing a pharmaceutical preparation containing an antibody, comprising preparing a composition containing an antibody according to the method according to any one of claims 1 to 4, and preparing the obtained composition into a pharmaceutical preparation.

6. The method according to claim 5, wherein: The eukaryotic cells are CHO cells.

Images