Antibody variant with reduced biological activity

Abstract

To provide a structural variation (antibody variant) of nemolizumab, particularly a structural variation having a lower biological activity than nemolizumab, and provide a nemolizumab-containing composition comprising the variation, a method for reducing the variation, and an analytical method.SOLUTION: The present invention discloses an antibody variant of nemolizumab having a specific sequence. In addition, this disclosure provides a nemolizumab-containing composition comprising the antibody variant, a method for reducing the antibody variant, and an analytical method.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to antibody variants of nemolizumab, for example, antibody variants having reduced biological activity compared to nemolizumab. The present invention also relates to pharmaceutical compositions containing such antibody variants at reduced concentrations. The present invention further relates to methods for detecting and analyzing the antibody variants. [Background technology]

[0002] Antibodies are attracting attention as pharmaceuticals due to their high stability in plasma and minimal side effects. Numerous IgG-type antibody drugs are currently on the market, and numerous antibody drugs are currently under development. In general, antibody drugs contain various components, including the target substance (antibody), which is the active ingredient, as well as structural variants of the target substance (antibody variants). These structural variants are classified into target substance-related substances, which have efficacy and safety equivalent to the target substance, and target substance-derived impurities, which are not equivalent to the target substance. The production rate of some of these substances varies depending on the conditions of the drug substance manufacturing process. Because of this heterogeneity in antibody drugs, it is necessary to set release specifications and expiration date specifications for each component of the drug substance and formulation, thereby managing the quality of antibody drugs.

[0003] In recent years, monoclonal antibodies have been discovered that bind to interleukin-31 (IL-31) receptor A (IL-31RA) and inhibit the binding of IL-31 to IL-31RA (Patent Documents 1 to 3). Among these, the anti-IL-31RA antibody nemolizumab (CIM331) acts as an IL-31 antagonist, thereby inhibiting the function of IL-31, which is thought to be an pruritus-inducing cytokine. Clinical trials have been conducted in patients with atopic dermatitis, and the results have recently been reported (Non-Patent Document 1). However, it has not yet been reported whether antibody variants with reduced biological activity compared to nemolizumab are produced in the production of nemolizumab drug substances and formulations, and the proportion of such variants. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2007 / 142325 [Patent Document 2] WO2009 / 072604 [Patent Document 3] WO2010 / 064697 [Non-patent literature]

[0005] [Non-Patent Document 1] N Engl J Med 2020; 383:141-150 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention has been made in consideration of the above-mentioned circumstances, and aims to provide structurally modified forms of nemolizumab (antibody variants), nemolizumab-containing compositions containing the antibody variants and methods for producing the same, methods for reducing the antibody variants, and methods for analyzing the antibody variants. [Means for solving the problem]

[0007] As a result of intensive research to achieve the above object, the present inventors have succeeded in identifying multiple antibody variants (structurally modified forms of nemolizumab) contained in pharmaceutical compositions containing nemolizumab as an active ingredient. Furthermore, they have found that some of these antibody variants have lower biological activity (inhibitory activity of IL-31 signaling by binding to the IL-31 receptor) than nemolizumab. Furthermore, they have discovered a method for efficiently detecting and analyzing these antibody variants.

[0008] The present disclosure is based on these findings and relates, in one non-limiting embodiment, to: [1] An antibody variant having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3), (a) an amino acid residue D at the first position from the N-terminus of said sequence; (b) an amino acid residue D at the fourth position from the N-terminus of said sequence; or (c) an amino acid residue D at the fifth position from the N-terminus of the sequence; is a succinimide residue or an isoaspartic acid residue. [2] The antibody variant of [1], wherein the sequence is a CDR sequence. [2.2] The antibody variant of [1], wherein the sequence is a CDR3 sequence. [3] The antibody variant of [1], wherein the sequence is contained in a heavy chain. [4] The antibody having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3), (1) An antibody comprising a heavy chain variable region comprising CDR1 set forth in SEQ ID NO: 1, CDR2 set forth in SEQ ID NO: 2, and CDR3 set forth in SEQ ID NO: 3, and a light chain variable region comprising CDR1 set forth in SEQ ID NO: 4, CDR2 set forth in SEQ ID NO: 5, and CDR3 set forth in SEQ ID NO: 6; (2) An antibody comprising a heavy chain variable region set forth in SEQ ID NO: 7 and a light chain variable region set forth in SEQ ID NO: 8, or (3) An antibody comprising a heavy chain set forth in SEQ ID NO: 9 and a light chain set forth in SEQ ID NO: 10 The antibody variant of [1], wherein: [5] The antibody variant of [1], wherein the antibody having a variable region comprising the amino acid sequence DGYDDGPYTLET (sequence number 3) is nemolizumab. [6] An antibody variant according to any one of [1] to [5], which, when separated using anion exchange chromatography, has a shorter retention time than the antibody having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3). [7] An antibody variant of any of [1] to [6], which has lower biological activity than the antibody having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3). [1.2.0] A pharmaceutical composition comprising any one of the antibody variants of [1] to [7]. [1.2.1] A pharmaceutical composition comprising an antibody variant of any of [1] to [7], wherein the proportion of isoaspartic acid residue-containing antibody variants in the pharmaceutical composition relative to the total of the antibody variant and unmodified antibody is 36% or less, and / or the proportion of isoaspartic acid residue-containing peptides derived from the antibody variant in the pharmaceutical composition after enzymatic digestion relative to the total of the isoaspartic acid residue-containing peptides and corresponding unmodified peptides is 20% or less. [1.2.2] A pharmaceutical composition comprising any of the antibody variants of [1] to [7], wherein the proportion of isoaspartic acid residue-containing antibody variants in the pharmaceutical composition relative to the total of the antibody variant and unmodified antibody is 27.8% or less, and / or the proportion of isoaspartic acid residue-containing peptides derived from the antibody variant in the pharmaceutical composition after enzymatic digestion relative to the total of the isoaspartic acid residue-containing peptides and corresponding unmodified peptides is 15% or less. [1.2.3] A pharmaceutical composition comprising an antibody variant of any of [1] to [7], wherein the proportion of isoaspartic acid residue-containing antibody variants in the pharmaceutical composition relative to the total of the antibody variant and unmodified antibody is 17.9% or less, and / or the proportion of isoaspartic acid residue-containing peptides derived from the antibody variant in the pharmaceutical composition after enzymatic digestion relative to the total of the isoaspartic acid residue-containing peptides and corresponding unmodified peptides is 9.4% or less. [1.2.4] A pharmaceutical composition comprising any one of the antibody variants of [1] to [7], wherein when the pharmaceutical composition is separated using anion exchange chromatography, the ratio of the area of ​​the peak containing the antibody variant containing isoaspartic acid residues to the total peak area is 40.3 area% or less, 30.4 area% or less, or 19.3 area% or less. [1.2.5] Any of the pharmaceutical compositions [1.2.1] to [1.2.4] containing nemolizumab. [1.2.6] A pharmaceutical composition comprising any one of the antibody variants of [1] to [7], wherein the proportion of succinimide residue-containing antibody variants in the pharmaceutical composition relative to the sum of the antibody variant and unmodified antibody is 36% or less, and / or the proportion of succinimide residue-containing peptides derived from the antibody variant in the pharmaceutical composition relative to the sum of the succinimide residue-containing peptides and corresponding unmodified peptides after enzymatic digestion is 20% or less. [1.2.7] A pharmaceutical composition comprising an antibody variant according to any one of [1] to [7], wherein the proportion of succinimide residue-containing antibody variants in the pharmaceutical composition relative to the sum of the antibody variant and unmodified antibody is 27.8% or less, and / or the proportion of succinimide residue-containing peptides derived from the antibody variant in the pharmaceutical composition relative to the sum of the succinimide residue-containing peptides and corresponding unmodified peptides after enzymatic digestion is 15% or less. [1.2.8] A pharmaceutical composition comprising any of the antibody variants of [1] to [7], wherein the proportion of succinimide residue-containing antibody variants in the pharmaceutical composition relative to the sum of the antibody variants and unmodified antibodies is 18.7% or less, and / or the proportion of succinimide residue-containing peptides derived from the antibody variant in the pharmaceutical composition relative to the sum of the succinimide residue-containing peptides and corresponding unmodified peptides after enzymatic digestion is 9.8% or less. [1.2.9] A pharmaceutical composition comprising any one of the antibody variants of [1] to [7], wherein when the pharmaceutical composition is separated using anion exchange chromatography, the ratio of the area of ​​the peak containing the succinimide residue-containing antibody variant to the total peak area is 29.1 area% or less, 21.6 area% or less, or 13.8 area% or less. [1.2.10] Any of the pharmaceutical compositions [1.2.6] to [1.2.9] containing nemolizumab. [1.2.11] Any of the pharmaceutical compositions [1.2.0] to [1.2.9], which are pharmaceutical compositions for use in one or both of the treatment and prevention of at least one of atopic dermatitis, dialysis-related pruritus, and other pruritus. [1.2.12] A method for treating or preventing at least one of atopic dermatitis, dialysis-related pruritus, and other pruritus, comprising administering any one of the pharmaceutical compositions of [1.2.0] to [1.2.9]. [1.3.1] A method for detecting an antibody variant of any of [1] to [7], comprising a step of separating a sample containing an antibody having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) or an enzyme-treated version of the antibody by affinity chromatography, ion exchange chromatography, normal phase chromatography, reverse phase chromatography, hydrophilic interaction chromatography (HILIC), hydrophobic interaction chromatography (HIC), charge-based separation, size exclusion chromatography (SEC), gel permeation chromatography (GPC), or a combination thereof. [1.3.2] The detection method according to [1.3.1], wherein the ion exchange chromatography is anion exchange chromatography. [1.3.3] The detection method of [1.3.2], characterized in that the anion exchange chromatography is performed using a column with an inner diameter of 4.6-7.5 mm, a length of 35-150 mm, and a particle size of 2.5-10 μm, at a column temperature of 25-50°C, and elution with a mobile phase of pH 6.0-10.0. [1.3.4] The detection method according to [1.3.2] or [1.3.3], characterized in that the anion exchange chromatography is performed using a column with an inner diameter of 7.5 mm, a length of 75 mm, and a particle size of 2.5 μm at a column temperature of 40°C, and elution is performed with a mobile phase containing 250 mmol / L sodium chloride at pH 7.5. [1.3.5] Any of the detection methods [1.3.1] to [1.3.4], comprising a step of quantifying peptides by mass spectrometry and / or ultraviolet absorption measurement following the step of separating the sample containing the enzyme-treated antibody. [1.3.6] A detection method according to any one of [1.3.1] to [1.3.5], characterized in that any one of the antibody variants according to [1] to [7] is used as a standard substance (standard product). [1.4.1] A method for producing a composition containing nemolizumab, comprising a step of carrying out any of the detection methods described in [1.3.1] to [1.3.6]. [1.4.2] A method for producing a composition containing nemolizumab, comprising a step of purifying a solution containing nemolizumab by affinity chromatography, ion exchange chromatography, hydrophilic interaction chromatography (HILIC), hydrophobic interaction chromatography (HIC), charge-based separation, size exclusion chromatography (SEC), gel permeation chromatography (GPC), or a combination thereof. [1.4.3] The manufacturing method of [1.4.2], wherein the purifying step comprises purifying a nemolizumab-containing composition obtained from nemolizumab-producing cells by affinity chromatography. [1.4.4] A manufacturing method according to [1.4.3], characterized in that the hold time of the nemolizumab-containing solution after purification by affinity chromatography is within 72 hours, and the content of any of the antibody variants [1] to [7] in the composition is lower than when the hold time is longer. [1.4.5] The production method according to [1.4.4], wherein the retention time of the nemolizumab-containing solution after the affinity chromatography purification is within about 24 hours. [1.4.6] A manufacturing method according to any one of [1.4.1] to [1.4.5], characterized in that any one of the antibody variants according to [1] to [7] is used as a standard substance (standard product). [1.5.1.1] A method for suppressing the content of any of the antibody variants [1] to [7], comprising a step of purifying by affinity chromatography an antibody-containing composition obtained from cells that produce an antibody having a variable region containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3), wherein the content of any of the antibody variants [1] to [7] in the composition is lower than in a case that does not include the purification step. [1.5.1.2] The method of [1.5.1.1], characterized in that the retention time of the nemolizumab-containing solution after purification by affinity chromatography is within 48 hours, and the content of any of the antibody variants [1] to [7] in the composition is lower than when the retention time is longer. [1.5.1.3] The method of [1.5.1.2], characterized in that the retention time of the nemolizumab-containing solution after purification by affinity chromatography is within approximately 24 hours. [1.5.1.4] Any of the methods [1.5.1.1] to [1.5.1.3], characterized in that any of the antibody variants [1] to [7] is used as a standard substance (standard product). [1.6.1.1] A composition comprising any one of the antibody variants of [1] to [7], wherein the antibody variant has a shorter retention time than nemolizumab when separated using anion exchange chromatography and contains more isoaspartic acid residues than nemolizumab. [1.6.1.2] The composition of [1.6.1.1], wherein the content of the antibody variant is 40.3 area% or less, 30.4 area% or less, or 19.3 area% or less, and the composition contains nemolizumab. [1.6.2.1] A composition comprising any one of the antibody variants of [1] to [7], wherein the antibody variant has a shorter retention time than nemolizumab when separated using anion exchange chromatography and contains more succinimide residues than nemolizumab. [1.6.2.2] A composition of [1.6.2.1], wherein the content of the antibody variant is 29.1 area% or less, 21.6 area% or less, or 13.8 area% or less, and the composition contains nemolizumab. [2.1.1.1] An antibody variant of nemolizumab that is composed of two heavy chains and two light chains and has a smaller molecular weight than nemolizumab. [2.1.1.2] An antibody variant of nemolizumab, characterized by having a structure formed by two heavy chains bonded together by the cysteines at position 224 (EU numbering) in the heavy chain of nemolizumab. [2.1.1.3] An antibody variant of nemolizumab, characterized by having a structure formed by two light chains bonded together by the cysteines at position 214 (EU numbering) in the light chain of nemolizumab. [2.1.1.4] An antibody variant of nemolizumab, characterized in that it has a structure formed by one light chain and one heavy chain, in which the cysteine ​​at position 214 (EU numbering) in the light chain of nemolizumab is bonded to the cysteine ​​at position 224 (EU numbering) in the heavy chain of nemolizumab, and the cysteines at positions 227 and 230 (EU numbering) in the heavy chain are bonded within one heavy chain. [2.1.1.5] An antibody variant of nemolizumab, characterized by having a structure formed by a single light chain in which the cysteine ​​at position 214 (EU numbering) in the light chain of nemolizumab is linked to a free cysteine. [2.1.1.6] An antibody variant of nemolizumab, characterized in that it is composed of two heavy chains and one light chain of nemolizumab, in which the two heavy chains are bonded to each other via two cysteines at positions 227 and 230 (EU numbering), and further in that the cysteine ​​at position 224 (EU numbering) in the first heavy chain is bonded to the cysteine ​​at position 214 (EU numbering) in the light chain, and further in that the cysteine ​​at position 224 (EU numbering) in the second heavy chain is bonded to a free cysteine. [2.1.2] A pharmaceutical composition comprising any one of the antibody variants [2.1.1.1] to [2.1.1.6], wherein the proportion of the antibody variant in all antibody molecules in the pharmaceutical composition is 11.0 CPA% or less. [2.2.1] An antibody variant of nemolizumab, characterized in that the asparagine at position 55 in the Kabat numbering of the nemolizumab heavy chain is deamidated. [2.2.2] A pharmaceutical composition comprising an antibody variant of [2.2.1], wherein after enzymatic digestion of the pharmaceutical composition, the proportion of deamidated peptides derived from the antibody variant relative to the total of the deamidated peptides and corresponding unmodified peptides is 2.0% or less. [Effects of the Invention]

[0009] The present inventors have succeeded in identifying structurally modified antibody variants (antibody variants) with significantly lower biological activity than nemolizumab. Such antibody variants are useful as standard substances (reference standards) for detecting / analyzing the variants in the quality evaluation of pharmaceutical compositions containing nemolizumab. Furthermore, the present inventors have discovered a method for controlling (reducing) the production of aspartic acid isomerization variants, which are such antibody variants, and a method for measuring the content of these variants (variant detection / analysis methods). These methods have higher throughput than conventional methods and are important technologies for improving and controlling the quality of nemolizumab. Furthermore, the nemolizumab-containing pharmaceutical compositions of the present invention, which have a low content of these antibody variants, are useful as means for treating and / or preventing atopic dermatitis, dialysis-related pruritus, and other pruritus. [Brief explanation of the drawings]

[0010] [Figure 1A] 1 is a chromatogram showing the results of separation of a nemolizumab-containing solution by anion exchange high performance liquid chromatography under the conditions described in Example 1. A TSK DEAE NPR column (inner diameter 4.6 mm, length 35 mm, particle size 2.5 μm) was used, and separation was performed under salt concentration adjustment condition 1 shown in Table 1. [Figure 1B] 1 is a chromatogram showing the results of separation of a nemolizumab-containing solution by anion exchange high performance liquid chromatography under the conditions described in Example 1. A TSK DEAE NPR column (inner diameter 7.5 mm, length 75 mm, particle size 2.5 μm) was used, and separation was performed under salt concentration adjustment condition 2 shown in Table 2. [Figure 2] FIG. 1 is a schematic diagram showing the structures of HH dimer and LL dimer, which are variants of nemolizumab. [Figure 3] This is a scatter plot in which the content of isoAsp isomers (top) or Asu isomers (bottom) in peptide units in samples after storing HCCF or affinity pools at 23°C for various times is plotted on the X axis, and the area percentage of Pre-Region 1 (top) or Pre-Region 2 (bottom) in AE-HPLC is plotted on the Y axis. [Figure 4] FIG. 1 is a schematic diagram showing the structure of HL form, a variant of nemolizumab. [Figure 5]FIG. 1 is a schematic diagram showing the structures of nemolizumab variants, cysteinylated HHL body (left) and cysteinylated light chain (right). DETAILED DESCRIPTION OF THE INVENTION

[0011] One embodiment of the present invention relates to antibody variants with reduced biological activity (e.g., inhibitory activity of IL-31 signaling via binding to the IL-31 receptor). These antibody variants were identified as aspartic acid isomerization variants (variants in which at least one aspartic acid residue in nemolizumab has been changed to a succinimide residue (Asu form) and variants in which at least one aspartic acid residue in nemolizumab has been changed to an isoaspartic acid residue (isoAsp form)) in analysis of the nemolizumab drug substance by the present inventors. As used herein, "antibody variant" may also be referred to as a structurally altered form of the original antibody, or an isomerized form, isomerization variant, or isomer.

[0012] Nemolizumab is a humanized IgG2 antibody that exhibits inhibitory activity of IL-31 signaling by binding to the IL-31 receptor and is composed of a heavy chain and a light chain that recognize the IL-31 receptor. Specifically, nemolizumab comprises an H-chain variable region comprising CDR1 set forth in SEQ ID NO: 1, CDR2 set forth in SEQ ID NO: 2, and CDR3 set forth in SEQ ID NO: 3, and an L-chain variable region comprising CDR1 set forth in SEQ ID NO: 4, CDR2 set forth in SEQ ID NO: 5, and CDR3 set forth in SEQ ID NO: 6. More specifically, nemolizumab comprises an H-chain variable region set forth in SEQ ID NO: 7 and an L-chain variable region set forth in SEQ ID NO: 8. Even more specifically, nemolizumab comprises an H-chain set forth in SEQ ID NO: 9 and an L-chain set forth in SEQ ID NO: 10. Note that, in this specification, nemolizumab and its variants are described separately, and therefore, the term "nemolizumab" does not include nemolizumab variants (e.g., aspartic acid isomerization variants).

[0013] Known methods for defining CDRs include the method of Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Ed (1991), Bethesda, MD), the method of Chothia et al. (Science (1986) 233, 755-758), and a method based on antigen-antibody contact regions (J Mol Biol (1996) 262, 732-745). Specifically, CDRs according to each method are defined as follows: CDR Kabat Chothia Contact L1 L24-L34 L24-L34 L30-L36 L2 L50-L56 L50-L56 L46-L55 L3 L89-L97 L89-L97 L89-L96 H1 H31-H35B H26-H32 / 34 H30-H35B (Kabat numbering) H1 H31-H35 H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H52-H56 H47-H58 H3 H95-H102 H95-H102 H93-H101

[0014] In the present disclosure, "the biological activity of an antibody variant is reduced" means that the biological activity is lower than that of a comparable antibody (e.g., nemolizumab), and preferably is statistically significantly lower. In one embodiment, the biological activity of the antibody variant of the present invention is lower by 10% or more, for example, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 59% or more, 60% or more, 65% or more, 70% or more, 75% or more, 79% or more, 80% or more, 85% or more, or 90% or more, compared to the biological activity of a comparable antibody (e.g., nemolizumab). In one aspect, the biological activity (relative biological activity) of the antibody variants of the present invention relative to the biological activity of a comparable antibody (e.g., the CIM331 standard, i.e., nemolizumab) is 90% or less, for example, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 41% or less, 40% or less, 35% or less, 30% or less, 25% or less, 21% or less, 20% or less, 15% or less, 10% or less, or 5% or less.

[0015] In one embodiment, the biological activity of nemolizumab or its antibody variant is neutralizing activity against the IL-31 receptor. In one embodiment, the neutralizing activity against the IL-31 receptor is activity that inhibits the binding of IL-31 to the IL-31 receptor. In one embodiment, the neutralizing activity against the IL-31 receptor is activity that inhibits IL-31 signaling through binding to the IL-31 receptor (e.g., activity that suppresses IL-31-dependent cell proliferation). As described in WO2010 / 064697, nemolizumab (CIM331) has neutralizing activity against human IL-31RA and cynomolgus monkey IL-31RA. Such neutralizing activity can be evaluated, for example, by the method described in WO2010 / 064697.

[0016] One method for determining whether nemolizumab or its antibody variants inhibit IL-31 signaling is to examine whether nemolizumab or its antibody variants inhibit the binding of IL-31 to the IL-31 receptor. Examples of methods for such determination include ELISA, flow cytometry-based assays, and surface plasmon resonance-based assays. For example, in the case of ELISA, a system is prepared in which IL-31 receptor (or IL-31RA) protein is immobilized on a plate, and the amount of IL-31 protein bound thereto is detected with a secondary antibody such as an enzyme-labeled anti-IL-31 antibody. Nemolizumab or its antibody variants are added to the system, and the amount of detected IL-31 protein is measured to determine whether nemolizumab or its antibody variants inhibit the binding of IL-31 to the IL-31 receptor. Alternatively, whether nemolizumab or its antibody variants inhibit IL-31 signaling can be confirmed by examining whether nemolizumab or its antibody variants inhibit physiological activities induced by the action of IL-31 on cells. The physiological activities are not particularly limited as long as they can be measured quantitatively or qualitatively by some method, and examples include cell proliferation activity, protein phosphorylation activity, and gene / protein expression induction activity. For example, cells expressing IL-31 receptor on their surface and whose proliferation activity is induced in response to external IL-31 stimulation are prepared, and nemolizumab or its antibody variants are added to the cells. The inhibition of IL-31 signaling by nemolizumab or its antibody variants can be assessed by measuring whether the IL-31-induced cell proliferation activity is reduced. Such cells may be natural cells that naturally express IL-31 receptor, or genetically engineered cells artificially expressing IL-31 receptor. A suitable example of a genetically engineered cell is Ba / F3 cells expressing IL-31 receptor. As another alternative method, the method described in Dillon et al. (Nat Immunol (2004) 5, 752-760) can also be used.

[0017] In the present disclosure, the degree to which nemolizumab or its antibody variants inhibit IL-31 signaling is not limited, but may be, for example, 10% or more, preferably 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more.

[0018] The term "antibody" is used in the broadest sense and may be a monoclonal antibody, polyclonal antibody, dimer, multimer, multispecific antibody (e.g., bispecific antibody), antibody derivative, or modified antibody, as long as it exhibits the desired biological activity (e.g., antigen-binding activity) (Miller K et al. J Immunol. 2003, 170(9), 4854-61). The antibody may be a mouse antibody, human antibody, humanized antibody, chimeric antibody, or may be derived from another species or artificially synthesized. The antibodies disclosed herein may be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. The immunoglobulin may be derived from any species (e.g., human, mouse, or rabbit). The terms "antibody," "immunoglobulin," and "immunoglobulin" are used interchangeably and broadly. Furthermore, antibodies are not limited to antibody molecules composed of two heavy chains and two light chains, but may also be antibody fragments with smaller molecular weights. For example, the definition of an antibody herein also includes molecules having a structure formed by two heavy chains, a structure formed by two light chains, a structure formed by one light chain and one heavy chain, a structure formed by one light chain linked to a free cysteine, and a structure formed by two heavy chains and one light chain. In light of this broad definition, antibody variants of the present invention are also included in the definition of an antibody. Therefore, the term "(whole) antibody molecule in a pharmaceutical composition" herein can include both antibodies and their variants.

[0019] The antibody may be a recombinant antibody produced using genetic engineering technology. Recombinant antibodies can be obtained by cloning the DNA encoding the antibody from antibody-producing cells such as hybridomas or sensitized lymphocytes that produce antibodies, incorporating it into a vector, and introducing this into a host (host cell) for production.

[0020] In the present invention, antibodies can be produced by methods known to those skilled in the art. Specifically, DNA encoding the antibody of interest is incorporated into an expression vector. The DNA is incorporated into the expression vector so that it is expressed under the control of an expression control region, such as an enhancer or promoter. Next, host cells are transformed with this expression vector to express the antibody. An appropriate combination of host and expression vector can be used.

[0021] The antibodies obtained in this manner can be isolated and purified from inside or outside the host cells (e.g., from the culture medium). Antibody separation and purification can be performed using methods commonly used for antibody purification, and are not limited in any way. The antibodies and antibody variants of the present invention can be separated and purified by appropriately selecting and combining, for example, chromatography columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. For example, various matrices can be used for separation and purification using chromatography columns, such as anion exchange matrices, cation exchange matrices, anti-human IgG affinity matrices, and protein L matrices.

[0022] Without being limited to a particular theory, the antibody variants of the present invention may be structural variants of an antibody molecule (e.g., nemolizumab) having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) that arise during the manufacturing process (e.g., the purification step) of the antibody molecule. In one aspect, the antibody variants of the present invention have at least one (e.g., one, two, or three) amino acid residues changed compared to an antibody molecule (e.g., nemolizumab) having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3). In one embodiment, the antibody variants of the present invention have one to three (i.e., one, two, or three) amino acid residues changed compared to an antibody molecule (e.g., nemolizumab) having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3). In certain embodiments, the antibody variants of the present invention have at least one (e.g., one, two, or three) amino acid residues altered in the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) compared to an antibody molecule (e.g., nemolizumab) having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3). In certain embodiments, the antibody variants of the present invention have one to three, i.e., one, two, or three, amino acid residues altered in the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) compared to an antibody molecule (e.g., nemolizumab) having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3).

[0023] In another aspect, the antibody variants of the present invention have a different combination of SS bonds (bonds between cysteine ​​residues or bonds between a cysteine ​​residue and a free cysteine) compared to an antibody molecule (e.g., nemolizumab) having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3). In one embodiment of this aspect, the antibody variants of the present invention have the same amino acid sequence as an antibody molecule (e.g., nemolizumab) having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3), but a different combination of SS bonds (bonds between cysteine ​​residues or bonds between a cysteine ​​residue and a free cysteine) and a different molecular weight. In another embodiment, the antibody variants of the present invention have a different amino acid sequence and a different combination of SS bonds (bonds between cysteine ​​residues or bonds between a cysteine ​​residue and a free cysteine) compared to an antibody molecule (e.g., nemolizumab) having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3), and a different molecular weight.

[0024] In one aspect, the present invention relates to antibody variants having the following characteristics: one or more aspartic acid (Asp) residues in the heavy chain CDR of an antibody (e.g., nemolizumab) having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) are changed to succinimide (Asu) or isoaspartic acid (isoAsp) residues; - Compared to the aforementioned antibodies (e.g., nemolizumab), its biological activity (IL-31 signaling inhibitory activity via binding to the IL-31 receptor) is extremely low; The amount of antibody produced increases depending on the hold time of the antibody-containing solution after purification of the antibody (e.g., nemolizumab) by affinity chromatography until the next step. Antibody variants having such characteristics may be referred to herein as aspartic acid isomerization variants or aspartic acid isomers. In a specific embodiment, the antibody is nemolizumab, and the amino acid sequence is the heavy chain CDR3 sequence of nemolizumab.

[0025] In one embodiment, the aspartic acid isomerization variant of the present invention is a variant of an antibody having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3), (a) an amino acid residue D (Asp) at the first position from the N-terminus of the sequence; or (b) an amino acid residue D (Asp) at the fourth position from the N-terminus of the sequence; or (c) an amino acid residue D (Asp) at the fifth position from the N-terminus of the sequence; is a succinimide residue (sometimes referred to herein as a succinimide or Asu form), or an isoaspartic acid residue (sometimes referred to herein as an isoaspartic acid or isoAsp form). The position of the succinimide residue in the succinimide form is preferably the first or fifth position from the N-terminus of the sequence (SEQ ID NO: 3), and particularly preferably the fifth position from the N-terminus of the sequence. In a particular embodiment, the aspartic acid isomerization variant of the present invention is a variant of an antibody having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3), (a2) amino acid residues D (Asp) at the first and fifth positions from the N-terminus of the sequence or (b2) amino acid residues D (Asp) at the fourth and fifth positions from the N-terminus of the sequence or (c2) amino acid residues D (Asp) at the first and fourth positions from the N-terminus of the sequence is a succinimide (Asu) residue or an isoaspartic acid (isoAsp) residue. Examples of such antibody variants include those an antibody variant wherein position 1 is an isoAsp residue and position 5 is an Asu residue; an antibody variant wherein position 1 is an Asu residue and position 5 is isoAsp; antibody variants in which positions 1 and 5 are Asu residues; an antibody variant in which positions 1 and 5 are isoAsp residues; an antibody variant wherein position 4 is an isoAsp residue and position 5 is an Asu residue; an antibody variant wherein position 4 is an Asu residue and position 5 is an isoAsp residue; antibody variants in which positions 4 and 5 are Asu residues; an antibody variant in which positions 4 and 5 are isoAsp residues; an antibody variant wherein position 1 is an isoAsp residue and position 4 is an Asu residue; an antibody variant wherein position 1 is an Asu residue and position 4 is an isoAsp residue; an antibody variant in which positions 1 and 4 are Asu residues; or Antibody variants with isoAsp residues at positions 1 and 4 and preferably, an antibody variant in which positions 1 and 4 are isoAsp residues; an antibody variant in which positions 1 and 5 are isoAsp residues; an antibody variant in which positions 4 and 5 are isoAsp residues; an antibody variant wherein position 1 is an isoAsp residue and position 5 is an Asu residue; or an antibody variant wherein position 4 is an isoAsp residue and position 5 is an Asu residue; Examples include: In a particular embodiment, the antibody is nemolizumab and the amino acid sequence is the sequence of the heavy chain CDR3 of nemolizumab.

[0026] In another aspect, the present invention also relates to a method for detecting and analyzing aspartic acid isomerization variants. In one embodiment, the method of the present invention comprises a step of separating a sample containing an antibody having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) or an enzyme-treated antibody by affinity chromatography, ion exchange chromatography, normal-phase chromatography, reverse-phase chromatography, hydrophilic interaction chromatography (HILIC), hydrophobic interaction chromatography (HIC), charge-based separation, size-exclusion chromatography (SEC), gel permeation chromatography (GPC), or a combination thereof. In a specific embodiment, the method of the present invention comprises a step of enzymatically treating the antibody-containing sample prior to the separation step. Enzymes used to treat the antibody-containing sample include, but are not limited to, trypsin and Lys-C. In a specific embodiment, the method of the present invention comprises a step of identifying and / or quantifying peptides by mass spectrometry and / or ultraviolet absorption measurement following the step of separating the enzyme-treated antibody-containing sample.

[0027] In the methods of the present invention, detection and analysis can be performed using as an indicator the presence or absence of a structural change in aspartic acid in the CDR having the amino acid sequence of SEQ ID NO: 3. The structural change can be detected, for example, using as an indicator a shift in molecular weight or a shift in retention time due to the structural change in LCMS analysis. Alternatively, the structural change can be detected using as an indicator the difference in the degree of separation by ion exchange chromatography. For example, when separated by anion exchange chromatography, the aspartic acid isomerization variants of the present invention are separated in a more basic region (a region with a shorter retention time than the main peak) than the main peak of nemolizumab, the main component.

[0028] In one embodiment of the method of the present invention, the ion exchange chromatography is anion exchange chromatography. In one embodiment, the anion exchange chromatography is characterized by using a column with an inner diameter of 4.6-7.5 mm, a length of 35-150 mm, and a particle size of 2.5-10 μm, eluting with a mobile phase of pH 6.0-10.0 at a column temperature of 25-50° C. In a particular embodiment, the anion exchange chromatography is characterized by using a column with an inner diameter of 7.5 mm, a length of 75 mm, and a particle size of 2.5 μm, eluting with a mobile phase containing 250 mmol / L sodium chloride at pH 7.5, eluting with a column temperature of 40° C.

[0029] In one embodiment, the method of the present invention comprises the steps of performing one or more analyses selected from the group consisting of quantitative analysis, qualitative analysis, and structural analysis using as a standard (i) an aspartic acid isomerization variant of the present invention, (ii) a peptide containing isoAsp or Asu residues obtained by enzymatic digestion of the aspartic acid isomerization variant, or (iii) a peptide comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3), in which one, two, or three of the first, fourth, and fifth positions from the N-terminus of the sequence have been substituted with succinimide (Asu) or isoaspartic acid (isoAsp) residues. An example of the peptide (iii) is the peptide in which one of the first, fourth, and fifth positions from the N-terminus of the amino acid sequence of SEQ ID NO: 3 has been substituted with an Asu or isoAsp residue. Another example of the peptide (iii) is a peptide in which two of the first, fourth, and fifth positions from the N-terminus of the amino acid sequence of SEQ ID NO: 3 are changed to Asu residues or isoAsp residues, a peptide in which the first position is an isoAsp residue and the fifth position is an Asu residue; a peptide in which the first position is an Asu residue and the fifth position is isoAsp; peptides with Asu residues at positions 1 and 5; peptides in which positions 1 and 5 are isoAsp residues; a peptide in which the fourth position is an isoAsp residue and the fifth position is an Asu residue; a peptide in which the fourth position is an Asu residue and the fifth position is an isoAsp residue; peptides with Asu residues at the fourth and fifth positions; peptides with isoAsp residues at the fourth and fifth positions; a peptide in which the first position is an isoAsp residue and the fourth position is an Asu residue; a peptide in which the first position is an Asu residue and the fourth position is an isoAsp residue; a peptide in which the first and fourth positions are Asu residues; or Peptides with isoAsp residues at positions 1 and 4 and preferably, peptides in which the first and fourth positions are isoAsp residues; peptides in which positions 1 and 5 are isoAsp residues; peptides with isoAsp residues at the fourth and fifth positions; a peptide in which the first position is an isoAsp residue and the fifth position is an Asu residue; or a peptide in which the fourth position is an isoAsp residue and the fifth position is an Asu residue; Examples include:

[0030] In another aspect, the present invention enables the production and quality control of pharmaceutical compositions containing nemolizumab by implementing one or a combination of the above-mentioned detection methods and analysis methods. Thus, the present invention relates to a quality control method for pharmaceutical compositions containing nemolizumab, which includes implementing the above-mentioned detection methods and analysis methods or combining these methods. The present invention also relates to a production method for pharmaceutical compositions containing nemolizumab, which includes implementing one or more of such detection methods, analysis methods, and quality control methods.

[0031] In the production method of the present invention, a nemolizumab-containing pharmaceutical composition having a low content of aspartic acid isomerization variants can be produced by purifying a solution containing nemolizumab by affinity chromatography, ion exchange chromatography, hydrophilic interaction chromatography (HILIC), hydrophobic interaction chromatography (HIC), charge-based separation, size exclusion chromatography (SEC), gel permeation chromatography (GPC), or a combination thereof. Such nemolizumab-containing pharmaceutical compositions having a low content of aspartic acid isomerization variants are useful as a means for treating and / or preventing diseases for which nemolizumab exerts a therapeutic and / or preventive effect, such as atopic dermatitis, dialysis-related pruritus, and other pruritus. In one aspect, the production method of the present invention includes a step of culturing nemolizumab-producing cells (e.g., CHO cells) into which a gene encoding nemolizumab has been incorporated. The culturing step can be performed under conditions known to those skilled in the art that are suitable for culturing nemolizumab-producing cells (e.g., CHO cells), and for example, a commercially available basal medium (basal medium for animal cell culture) can be used. In one embodiment, the production method of the present invention comprises the step of centrifuging and filtering a culture medium of nemolizumab-producing cells to obtain a nemolizumab-containing solution (Harvest Cell Culture Fluid: HCCF). Nemolizumab can be purified from the obtained nemolizumab-containing solution by a combination of common column chromatography, such as affinity chromatography, ion exchange chromatography, and hydrophobic chromatography. In a specific embodiment, the production method of the present invention comprises the step of purifying the nemolizumab-containing solution obtained from the nemolizumab-producing cells by affinity chromatography (e.g., protein A affinity chromatography). The hold time of the antibody-containing solution after purification by affinity chromatography until the next step is not particularly limited, but shortening this hold time can reduce the content of aspartic acid isomerization variants. Therefore, in order to maintain an acceptable level of biological activity of the antibody and antibody variants in the nemolizumab-containing pharmaceutical composition obtained by the manufacturing method of the present invention, it is preferable to keep the antibody-containing solution after purification by affinity chromatography for a predetermined retention time until the next step (e.g., within about 138 hours, within about 72 hours, within about 48 hours, or within about 24 hours).

[0032] In another aspect, the present invention relates to a pharmaceutical composition comprising an antibody (e.g., nemolizumab) having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) and a variant thereof (e.g., an aspartic acid isomerization variant), wherein the proportion of the variant in all antibody molecules (including the antibody and its variants) in the pharmaceutical composition is reduced. The pharmaceutical composition of the present invention, in which the proportion of such variants is reduced, can be suitably used for the treatment and / or prevention of atopic dermatitis, dialysis-related pruritus, and other pruritus. In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for use in the treatment and / or prevention of at least one of atopic dermatitis, dialysis-related pruritus, and other pruritus. The present invention also relates to a method for treating or preventing at least one of atopic dermatitis, dialysis-related pruritus, and other pruritus, comprising the step of administering the pharmaceutical composition of the present invention. In one embodiment, the pharmaceutical composition of the present invention is obtained by a purification step including purification by affinity chromatography (e.g., protein A affinity chromatography). As described above, for example, by shortening the retention time of the antibody-containing solution after purifying the antibody (e.g., nemolizumab) solution by protein A affinity chromatography until the next step (e.g., retention time at 18 to 28°C or 18 to 23°C, preferably room temperature, 18°C, or 23°C), the proportion of aspartic acid isomerization variants in all antibody molecules in the obtained composition can be kept low.

[0033] The proportion of aspartic acid isomerization variants in antibody molecules in the pharmaceutical compositions of the present invention can be evaluated by various methods, including the above-mentioned methods for detecting / analyzing aspartic acid isomerization variants. Known methods for analyzing proteins such as antibodies include, for example, peptide mapping, in which proteins are decomposed into peptide fragments by enzymatic digestion or the like, separated by various types of chromatography, and subjected to mass spectrometry or ultraviolet absorption measurement to obtain information on the amino acid sequence and post-translational modifications of the protein. As described in the Examples of the present application, the structure and content of aspartic acid isomerization variants of the present invention can be analyzed by peptide mapping.

[0034] In one embodiment, the proportion of aspartic acid isomerization variants in antibody molecules in the pharmaceutical composition of the present invention can be expressed as the proportion of succinimide residue-containing peptides and isoaspartic acid residue-containing peptides derived from aspartic acid isomerization variants obtained by enzymatic digestion of the pharmaceutical composition of the present invention relative to the sum of the peptides derived from the variants and the corresponding unmodified peptides (unmodified peptides in which the site of modification to succinimide residues or isoaspartic acid residues is aspartic acid and otherwise has the same amino acid sequence as the variant-derived peptides) (herein, this may also be referred to as the proportion of aspartic acid isomerization variants per peptide). Examples of digestive enzymes that can be used include trypsin and Lys-C. The proportion of isoaspartic acid forms (isoAsp forms) per peptide in the pharmaceutical composition of the present invention (the proportion relative to the aspartic acid form per peptide) is, for example, 25% or less, 20% or less, 15% or less, or 9.4% or less. Furthermore, the proportion of succinimide forms (Asu forms) in peptide units in the pharmaceutical composition of the present invention (proportion compared to aspartic acid forms in peptide units) is, for example, 25% or less, 20% or less, 15% or less, or 9.8% or less.

[0035] Furthermore, as described in Example 6 of the present application, the ratio of succinimide residue-containing peptides and isoaspartic acid residue-containing peptides derived from aspartic acid isomerization variants obtained after enzymatic digestion to the sum of the variant-derived peptides and corresponding unmodified peptides (unmodified peptides in which the modification site to a succinimide residue or isoaspartic acid residue is aspartic acid and otherwise has the same amino acid sequence as the variant-derived peptide) (the ratio of aspartic acid isomerization variants on a peptide-by-peptide basis) correlates with the ratio of the peak area of ​​the elution peak group of aspartic acid isomerization variants to the total peak area (peak area ratio) in anion exchange chromatography (AE-HPLC) (Figure 3). Therefore, the ratio of aspartic acid isomerization variants in antibody molecules in the pharmaceutical composition of the present invention can be represented by the ratio (peak area ratio) of the combined peak area of ​​the elution peak groups of succinimide form (Asu form) and isoaspartic acid form (isoAsp form) to the total peak area when the pharmaceutical composition of the present invention is analyzed by anion exchange chromatography (AE-HPLC). This evaluation method has higher throughput than detection / analysis using peptide mapping methods and is advantageous in the quality control of nemolizumab.

[0036] In one embodiment, when the pharmaceutical composition of the present invention is analyzed by anion exchange chromatography (AE-HPLC), the elution fractions of the isoaspartic acid form (isoAsp form) and succinimide form (Asu form) are in a more basic region (a region with a shorter retention time than the main peak) than the main peak containing nemolizumab, the main component of the pharmaceutical composition of the present invention, and this region is sometimes referred to as the Pre-Region herein. Of the eight peaks contained in this region (Pre-Region), the isoaspartic acid form (isoAsp form) is mainly eluted in a region containing three peaks close to the main peak containing nemolizumab (sometimes referred to as Pre-Region 1 herein), and the succinimide form (Asu form) is mainly eluted in a region containing the remaining five peaks (sometimes referred to as Pre-Region 2 herein). Therefore, when evaluated by the analytical method of the present invention using anion exchange chromatography (AE-HPLC), the proportions (proportions per antibody) of isoaspartic acid forms (isoAsp forms) and succinimide forms (Asu forms) of all antibody molecules in the pharmaceutical composition of the present invention can be expressed as the proportions of the peak areas of Pre-Region 1 and Pre-Region 2, respectively.

[0037] In one embodiment, the proportion of isoaspartic acid forms (isoAsp forms) in all antibody molecules in the pharmaceutical composition of the present invention (area proportion of Pre-Region 1) when assessed by the analytical method of the present invention using anion exchange chromatography (AE-HPLC) is, for example, 50.2 area% or less, 40.3 area% or less, 30.4 area% or less, or 19.3 area% or less. In one embodiment, the proportion of succinimide forms (Asu forms) in all antibody molecules in the pharmaceutical composition of the present invention (area proportion of Pre-Region 2) when assessed by the analytical method of the present invention using anion exchange chromatography (AE-HPLC) is, for example, 36.6 area% or less, 29.1 area% or less, 21.6 area% or less, or 13.8 area% or less.

[0038] Furthermore, as described in Example 6 of the present application, the theoretical maximum proportion of aspartic acid isomerization variants in antibody units can be calculated based on the proportion of aspartic acid isomerization variants in peptide units, based on a binomial distribution. The proportion of isoaspartic acid forms (isoAsp forms) in antibody molecules in the pharmaceutical composition of the present invention relative to the total of the isoAsp forms and unmodified antibodies, calculated based on the proportion of aspartic acid isomerization variants per peptide (proportion per antibody unit), is, for example, 43.8% or less, 36.0% or less, 27.8% or less, or 17.9% or less. Furthermore, the proportion of isoAsp forms in which two heavy chains contain isoAsp residues in antibody molecules in the pharmaceutical composition of the present invention, per antibody unit, is, for example, 6.3% or less, 4.0% or less, 2.3% or less, or 0.9% or less. The proportion of isoAsp forms in which one of the two heavy chains contains an isoAsp residue in antibody molecules in the pharmaceutical composition of the present invention, per antibody unit, is, for example, 37.5% or less, 32.0% or less, 25.5% or less, or 17.0% or less. Similarly, the proportion of succinimide forms (Asu forms) in antibody molecules in the pharmaceutical composition of the present invention relative to the sum of the Asu forms and unmodified antibody (proportion per antibody unit) is, for example, 43.8% or less, 36.0% or less, 27.8% or less, or 18.7% or less. Furthermore, the proportion of Asu forms in which two heavy chains contain Asu residues relative to the sum of the Asu forms and unmodified antibody in antibody molecules in the pharmaceutical composition of the present invention is, for example, 6.3% or less, 4.0% or less, 2.3% or less, or 1.0% or less. The proportion of Asu forms in which one of the two heavy chains contains an Asu residue relative to the sum of the Asu forms and unmodified antibody in antibody molecules in the pharmaceutical composition of the present invention is, for example, 37.5% or less, 32.0% or less, 25.5% or less, or 17.7% or less.

[0039] In another aspect, the present invention relates to a method for producing a pharmaceutical composition having a reduced content of aspartic acid isomerization variants, and a method for suppressing the production of aspartic acid isomerization variants. The amount of aspartic acid isomerization variants produced can be reduced by shortening the hold time of the antibody-containing solution after purifying an antibody-containing solution containing an antibody (e.g., nemolizumab) having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) by affinity chromatography (e.g., protein A affinity chromatography) until the next step. Thus, the above method comprises a step of purifying an antibody-containing composition obtained from cells producing an antibody (e.g., nemolizumab) having a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) by affinity chromatography (e.g., protein A affinity chromatography), and is characterized by a short hold time of the antibody-containing solution after purification by affinity chromatography until the next step. In one embodiment, the retention time of the antibody-containing solution after purification by affinity chromatography until the next step (e.g., retention time at 18 to 28°C or 18 to 23°C, preferably room temperature, 18°C, or 23°C) is within about 138 hours, for example, within about 72 hours, within about 48 hours, or within about 24 hours, preferably within about 48 hours, and more preferably within about 24 hours.

[0040] In another aspect, the present invention relates to a method for purifying an antibody-containing composition, the method comprising the step of purifying the antibody-containing composition (e.g., Harvest Cell Culture Fluid (HCCF) obtained by centrifuging and filtering a culture medium of antibody-producing cells) by affinity chromatography (e.g., Protein A affinity chromatography). In one embodiment of the method, the antibody has a variable region comprising the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3). In a specific embodiment, the antibody is nemolizumab. In one embodiment of the method, the retention time of the antibody-containing solution after purification by affinity chromatography until the next step (e.g., retention time at 18 to 28°C or 18 to 23°C, preferably room temperature, 18°C, or 23°C) is about 138 hours or less, for example, about 72 hours or less, about 48 hours or less, or about 24 hours or less, preferably about 72 hours or less, more preferably about 48 hours or less, and even more preferably about 24 hours or less.

[0041] In another aspect, the present invention relates to an antibody variant having any of the following structures: (1) A structure formed by two heavy chains of nemolizumab linked together via the cysteine ​​residues at position 224 (EU numbering) in the heavy chain (HH dimer); (2) A structure formed by two light chains of nemolizumab linked by the cysteine ​​residues at EU numbering position 214 (LL dimer); (3) A structure formed by one light chain and one heavy chain, in which the cysteine ​​at position 214 (EU numbering) in the light chain of nemolizumab is bound to the cysteine ​​at position 224 (EU numbering) in the heavy chain of nemolizumab, and the cysteines at positions 227 and 230 (EU numbering) in the heavy chain are further bound within one heavy chain (HL form); (4) A structure formed by a single light chain in which the cysteine ​​at EU numbering position 214 in the light chain of nemolizumab is bound to a free cysteine ​​(cysteinylated light chain); or (5) Nemolizumab is composed of two heavy chains and one light chain, with the two heavy chains bonded to each other via cysteines at positions 227 and 230 (EU numbering). Furthermore, the cysteine ​​at position 224 (EU numbering) in the first heavy chain is bonded to the cysteine ​​at position 214 (EU numbering) in the light chain, and the cysteine ​​at position 224 (EU numbering) in the second heavy chain is bonded to a free cysteine ​​(cysteinylated HHL form). An antibody variant having such a structure is a low molecular weight species (LMWS) that has a smaller molecular weight than nemolizumab, which is composed of two heavy chains and two light chains, and is sometimes referred to herein as a low molecular weight variant of nemolizumab. In yet another aspect, the present invention relates to a pharmaceutical composition comprising nemolizumab and its low molecular weight variant (LMWS), in which the proportion of the low molecular weight variant in all antibody molecules in the pharmaceutical composition is kept low (for example, the proportion is 11.0 CPA (Corrected Peak Area)% or less).

[0042] In another aspect, the present invention relates to an antibody variant of nemolizumab in which the asparagine at position 55 (Kabat numbering) in the heavy chain is deamidated, and a pharmaceutical composition comprising the antibody variant. In one embodiment, after enzymatic digestion of the pharmaceutical composition, the proportion of deamidated peptides derived from the antibody variant to the total of the deamidated peptides and the corresponding unmodified peptides is 2.0% or less.

[0043] As used in this specification, an embodiment expressed by the expression "comprising..." encompasses an embodiment expressed by the expression "essentially consisting of..." as well as an embodiment expressed by the expression "consisting of...".

[0044] The numerical values ​​described in this specification may vary within a certain range due to, for example, the equipment, measurement conditions, and the techniques of a person skilled in the art, and may vary, for example, by about 10% as long as the objectives of the present invention are achieved.

[0045] The contents of all patents and references expressly cited herein are hereby incorporated by reference in their entirety. The present invention is further illustrated by, but not limited to, the following examples. [Example]

[0046] [Example 1] Separation of nemolizumab-containing solution by anion exchange high-performance liquid chromatography (AE-HPLC) method [1.1] Preparation of nemolizumab-containing solution Nemolizumab (also known as CIM331; H chain, SEQ ID NO: 9; L chain, SEQ ID NO: 10), an anti-human IL-31RA antibody also described in WO2010 / 064697, was produced by a method known to those skilled in the art, in accordance with the description of the patent document. Briefly, a nemolizumab-containing solution (drug substance) was produced by a method including a step of culturing host cells containing a vector into which a gene encoding nemolizumab had been introduced, and a step of purifying the solution containing nemolizumab obtained in the culturing step. Furthermore, a nemolizumab formulation was produced using this drug substance by the method described in WO2021 / 100794.

[0047] [1.2] Separation of nemolizumab-containing solutions by anion-exchange high-performance liquid chromatography A solution containing nemolizumab was taken and mobile phase A was added to adjust the nemolizumab concentration to 0.5 mg / mL (total concentration of nemolizumab and its variants), and this was used as the sample solution. Mobile phase A was used as the blank solution. A separation test was performed using 20 μL of the sample solution and blank solution (10 μL when TSK DEAE NPR (inner diameter 4.6 mm, length 35 mm, particle size 2.5 μm) and salt concentration gradient condition 1 were used) by anion exchange high-performance liquid chromatography under the following conditions.

[0048] [Test conditions] Detector: UV-visible spectrophotometer (measurement wavelength 280 nm) Column: TSK DEAE NPR (inner diameter 4.6 mm, length 35 mm, particle size 2.5 μm), TSK DEAE NPR (inner diameter 7.5 mm, length 75 mm, particle size 2.5 μm) or equivalent Guard column: TSK guard column DEAE NPR (inner diameter 4.6 mm, length 5 mm, particle size 5.0 μm) Column temperature: constant temperature around 40℃ Mobile phase A: 25 mmol / L Tris buffer, pH 7.5 ± 0.1 Mobile phase B: 25 mmol / L Tris buffer containing 250 mmol / L sodium chloride, pH 7.5 ± 0.1 Flow rate: 1.0 mL / min Mobile phase delivery: The concentration gradient was controlled by changing the mixing ratio of mobile phase A and mobile phase B as follows:

[0049] [Table 1]

[0050] [Table 2]

[0051] Separation was performed using a TSK DEAE NPR column (inner diameter 4.6 mm, length 35 mm, particle size 2.5 μm) under the above-mentioned concentration adjustment condition 1, resulting in the chromatogram shown in Figure 1A. Separation was performed using a TSK DEAE NPR column (inner diameter 7.5 mm, length 75 mm, particle size 2.5 μm) under the above-mentioned concentration adjustment condition 2, resulting in the chromatogram shown in Figure 1B.

[0052] As shown in Figure 1A-B, the region with an earlier retention time (shorter retention time) than the Main Region (including the Main peak) (the region containing fractions eluted at lower salt concentrations than the Main Region) was defined as the Pre-Region, and the region with a later retention time (longer retention time) than the Main Region (the region containing fractions eluted at higher salt concentrations than the Main Region) was defined as the Post-Region. Furthermore, the peaks in the Pre-Region were defined as peaks b1 to b8 in descending order of their retention time relative to the Main Region, and the peaks in the Post-Region were defined as peaks a1 to a6 in descending order of their retention time relative to the Main Region. The Pre-Region was divided into Pre-Region 1 (the region containing peaks b1 to b3) and Pre-Region 2 (the region containing peaks b4 to b8) in descending order of their retention time.

[0053] Comparing Figure 1A and Figure 1B, Figure 1B showed better separation of the b1 peak and the main peak. Therefore, in the following separation tests, the conditions shown in Figure 1B (column: TSK DEAE NPR (inner diameter 7.5 mm, length 75 mm, particle size 2.5 μm), salt concentration gradient conditions: Table 2) were used.

[0054] Information on the nemolizumab variants contained in each peak of Pre-Region 1, Pre-Region 2, and Post-Region shown in Figure 1B is summarized in Table 3 below. The biological activities listed in Table 3 are the results evaluated as described in Example 5.

[0055] [Table 3] HC: heavy chain; LMWS: low molecular weight species; HMWS: high molecular weight species

[0056] [Example 2] Evaluation of aspartic acid isomerization variants (isoAsp and succinimide) in Pre-Region 2 Fractions containing each peak separated and obtained using the column and mobile phase described in Example 1 were diluted with a denaturant solution containing guanidinium chloride (pH 5.5) and then reduced with tris(2-carboxyethyl)phosphine. The buffer was then exchanged with a digestion buffer containing 2-morpholinoethanesulfonic acid and urea (pH 5.5), followed by digestion with trypsin. Formic acid was added to the resulting digestion reaction solution, which was then injected into a reverse-phase high-performance liquid chromatograph for separation. The peptides were identified using a mass spectrometer, and the modification ratios per peptide were calculated. The modification ratios of interest per peptide were determined from the peak areas in the MS chromatogram, and this was used as the proportion of the variant of interest per peptide. Since the peaks of some isoAsp-containing peptides overlapped with the peaks of unmodified peptides in the MS chromatogram, the modification ratios per peptide were calculated using the following formula: Percent modification on a peptide basis (%) = (modified peptide of interest) *1 (peak area of ​​the unmodified peptide corresponding to the modified peptide of interest) ÷ (peak area of ​​the modified peptide of interest) + (peak area of ​​the unmodified peptide corresponding to the modified peptide of interest) *2 (peak area of ​​peptides containing some isoAsp residues) × 100 (*1: Refers to peptides containing isoAsp residues or succinimide residues derived from aspartic acid isomerization variants) (*2: Refers to an unmodified native peptide that has the same amino acid sequence as the modified peptide except for the modified site.)

[0057] The analysis revealed that the highest level of succinimide residue-containing peptides was detected in the fraction containing the b8 peak, which is part of Pre-Region 2. Specifically, in the b8 peak, Asp103 (Asp residue at position 99 in the Kabat numbering) was modified to a succinimide residue (Asu103) at a rate of 54.0%. On the other hand, the rate of modification of Asp99 (Asp residue at position 95 in the Kabat numbering) to a succinimide residue was low (2.7%), and modification of Asp102 (Asp residue at position 98 in the Kabat numbering) to a succinimide residue and modification of Asp99, Asp102, and Asp103 to isoAsp residues were not detected. TIFF2023159309000004.tif61161

[0058] Thus, compared to the main peak containing the main component nemolizumab (Asu99 and Asu102: Not detected, Asu103: 3.4%) separated and obtained using the column and mobile phase described in Example 1, it was confirmed that the aspartic acid isomerization variant (Asu form) was mainly eluted in Pre-Region 2.

[0059] [Example 3] Evaluation of aspartic acid isomerization variants (isoAsp and succinimide forms) in Pre-Region 1 As in Example 2, fractions containing each peak separated and obtained using the column and mobile phase described in Example 1 were diluted with a denaturant solution containing guanidinium chloride (pH 5.5) and then reduced with tris(2-carboxyethyl)phosphine. The buffer was then exchanged with a digestion buffer containing 2-morpholinoethanesulfonic acid and urea (pH 5.5), followed by digestion with trypsin. Formic acid was added to the resulting digestion reaction solution, which was then injected into a reverse-phase high-performance liquid chromatograph for separation. The peptides were identified using a mass spectrometer, and the modification ratios were calculated for each peptide. The modification ratios of interest for each peptide were determined from the peak areas in the MS chromatogram, and this was used as the proportion of the variant of interest for each peptide. Since the peaks of some isoAsp-containing peptides overlapped with the peaks of unmodified peptides in the MS chromatogram, the modification ratios for each peptide were calculated using the following formula: Percent modification on a peptide basis (%) = (modified peptide of interest) *1 (peak area of ​​the unmodified peptide corresponding to the modified peptide of interest) ÷ (peak area of ​​the modified peptide of interest) + (peak area of ​​the unmodified peptide corresponding to the modified peptide of interest) *2 (peak area of ​​peptides containing some isoAsp residues) × 100 (*1: Refers to peptides containing isoAsp residues or succinimide residues derived from aspartic acid isomerization variants) (*2: Refers to an unmodified native peptide that has the same amino acid sequence as the modified peptide except for the modified site.)

[0060] Theoretically, the following three types of structures are conceivable for isoAsp forms (top row: modification of Asp99 to an isoAsp residue, middle row: modification of Asp102 to an isoAsp residue, and bottom row: modification of Asp103 to an isoAsp residue), but it has not been determined at which Asp site is the actual isomerization occurring. If the molecular weight changes along with the structural change, it is possible to identify the amino acid site where the structure has changed by tandem mass spectrometry (MSMS analysis) using peptide mapping. However, because isoAsp has the same molecular weight as Asp, such identification by MSMS is difficult. TIFF2023159309000005.tif153169

[0061] The analysis revealed that, among all the AE-HPLC peaks, the highest levels of isoAsp residue-containing peptides (11.6% and 2.7%) were detected in the fraction containing the b1 peak, which is a part of Pre-Region 1. In addition, the proportions of Asp99 to succinimide residue (Asu99) in the b1 peak were 1.1%, Asp102 to succinimide residue (Asu102) was 0.2%, and Asp103 to succinimide residue (Asu103) was 4.7%. Thus, compared to the main peak containing the main component nemolizumab (modification to an isoAsp residue at any of Asp99, Asp102, and Asp103 was 0.5%), which was separated and obtained using the column and mobile phase described in Example 1, it was confirmed that the aspartic acid isomerization variants (isoAsp forms) were mainly eluted in Pre-Region 1.

[0062] Example 4: Evaluation of low molecular weight species (LMWS) in Pre-Region 1 [4.1] Evaluation by non-reduced LC-ESI-MS The fractions containing each peak separated and obtained using the column and mobile phase described in Example 1 were diluted with 0.1% formic acid, injected into a high-performance liquid chromatograph, desalted using a desalting column, and then the LMWS were identified using a mass spectrometer. As a result, it was identified that the peaks b2 and b3, which are part of Pre-Region 1, contain HH dimers and LL dimers.

[0063] [4.2] Non-reduced tryptic peptide mapping evaluation Fractions containing each peak separated using the column and mobile phase described in Example 1 were diluted with a denaturant solution containing guanidinium chloride at pH 7.0 and denatured. Subsequently, cysteine ​​side chains were capped with iodoacetic acid, buffer exchanged into a digestion buffer containing Tris, urea, and EDTA at pH 7.0, and digested with trypsin. Formic acid was added to the resulting digestion solution, which was then injected into a reverse-phase high-performance liquid chromatography column for separation. The peptides were identified using a mass spectrometer. The results indicated that the HH dimer has a structure in which C224 of the heavy chain, which forms a disulfide bond with C214 of the normal light chain, is linked between the heavy chains. Furthermore, the LL dimer is presumed to have a structure in which C214 of the light chain, which forms a disulfide bond with C224 of the normal heavy chain, is linked between the light chains (Figure 2).

[0064] [4.3] Non-reduced CE-SDS evaluation Fractions containing each peak separated and obtained using the column and mobile phase described in Example 1 were buffer exchanged with 0.1 mol / L sodium phosphate buffer, then mixed with an SDS solution containing N-ethylmaleimide and heated to denature and cap the cysteine ​​side chains. A dye reaction solution containing potassium cyanide and FQ-dye was then added to the solution, and the mixture was heated to label with the fluorescent dye. An SDS solution was added to the resulting solution, and analysis was performed by capillary electrophoresis-SDS. As a result, the contents of HH dimer and LL dimer in the b2 and b3 peaks were 18.4 CPA (Corrected Peak Area)% and 8.6 CPA%, respectively.

[0065] [Example 5] Evaluation of biological activity of aspartic acid isomerization variants (isoAsp and succinimide) The biological activity of each variant was evaluated by measuring the growth inhibitory activity of BaF / hIL-31R cells (BaF cells expressing human IL-31R), which proliferate in an IL-31-dependent manner. Specifically, FBS-RPMI medium containing IL-31 was added to fractions containing each peak separated and obtained using the column and mobile phase described in Example 1 to prepare dilutions of various concentrations. The dilutions were added to each well of a 96-well microplate, and a BaF / hIL-31R cell suspension was added to each well, followed by incubation in a CO2 incubator at 37°C for 24 hours. Filtered alamarBlue was then added, and the cells were further incubated in a CO2 incubator at 37°C for 3 hours, after which the absorbance (OD ) was measured using a microplate reader. 570 -OD 600 ) was measured.

[0066] As a result, when the main peak was used as the standard (normalized to 100%), the biological activity of the b8 peak was 21% of that of the main peak, indicating that the biological activity of the succinimide derivative (Asu103 variant equivalent to 54.0%) contained primarily in the b8 peak was significantly reduced. Furthermore, the biological activity of the b1 peak (11.6% isoAsp content) was shown to be 41% lower than that of the main peak. This suggests that the Asp isomerization in this isoAsp form is a chemical change in the CDR, and therefore strongly suggests that it affects biological activity. (Harris RJ. Heterogeneity of recombinant antibodies: linking structure to function. Dev Biol (Basel) 2005; 122:117-27; Dick, LWJ, et al., Identification and measurement of isoaspartic acid formation in the complementarity determining region of a fully human monoclonal antibody. J Chromatogr B Analyt Technol Biomed Life Sci, 2009;877:3841-9)

[0067] [Example 6] Confirmation that AE-HPLC can be used as a control analysis method for aspartic acid isomerization variants (isoAsp and succinimide forms) During the purification process of a nemolizumab-containing solution obtained by culturing host cells expressing nemolizumab, the retention time of the antibody-containing solution after the harvesting step (HCCF) by centrifugation and filtration after production culture and the subsequent purification step by protein A affinity chromatography (affinity pool) until the next step (sample retention time after each purification step) was examined to determine the effect on the content of aspartic acid isomerization variants (isoAsp and succinimide forms). The isoAsp and succinimide forms were determined for various samples (HCCF (after 0 / 72 hours at 23°C), affinity pool (after 0 / 24 / 48 / 72 / 138 hours at 23°C)). The content of the variants relative to the unmodified form was determined on a peptide-by-peptide basis by digesting each sample with trypsin and separating it by HPLC, followed by quantification using MS intensities in MS analysis, as in Examples 2 and 3. The content of the variants on a peptide-by-peptide basis was calculated using the following formula: Variant content (intensity%) on a peptide basis = (modified peptide of interest) *1 MS intensity of *2 )÷{(MS intensity of the modified peptide of interest)+(unmodified peptide corresponding to the modified peptide of interest) *3 MS intensity) × 100 (*1: Refers to peptides containing isoAsp residues or succinimide residues derived from aspartic acid isomerization variants) (*2: MS intensity is the intensity of the MS spectrum derived from the peptide.) (*3: Refers to an unmodified native peptide that has the same amino acid sequence as the modified peptide except for the modified site.) As a result, a significant time-dependent increase in aspartic acid isomerization variants (isoAsp and succinimide forms) was observed in the affinity pool (Table 4).

[0068] The above samples (HCCF (after 0 / 72 hours at 23°C) and affinity pool (after 0 / 24 / 48 / 72 / 138 hours at 23°C)) were separated by AE-HPLC as in Example 1, and the area percentages of each region in the HPLC chromatogram were determined. The correlations between the isoAsp content per peptide and the area percentage of Pre-Region 1 by AE-HPLC, and between the succinimide content per peptide and the area percentage of Pre-Region 2 by AE-HPLC were confirmed. It was found that the isoAsp content correlated with the area percentage of Pre-Region 1 (Figure 3). It was also found that the succinimide content correlated with the area percentage of Pre-Region 2 (Figure 3), demonstrating that AE-HPLC can be used as a control analytical method for the content of these variants.

[0069] Furthermore, the area increase rate per hour for Pre-Region 2 in the affinity pool was 0.0552 area% / h, and the area increase rate for Pre-Region 1 was 0.0448 area% / h (Table 4). These high area increase rates per hour indicated that, in order to ensure the quality of nemolizumab, it is important to set an appropriate sample retention time after the affinity chromatography purification process until the next process.

[0070] [Table 4]

[0071] [Example 7] Method for suppressing the formation of aspartic acid isomerization variants (isoAsp and succinimide forms) in the production of drug substances [7.1] Pre-Region 1 The shelf-life specification for nemolizumab formulations was set at 19.3% or less of the area percentage of Pre-Region 1 on AE-HPLC, based on clinical data, biological activity, PK, safety, and predicted impact on immunogenicity. The isoAsp form is a modification commonly found in antibody drugs and was considered to have little impact on safety and immunogenicity. Regarding biological activity, the b1 peak was found to have low biological activity, at 45% of that of the CIM331 reference material (nemolizumab) (Table 3). However, an increase in the area percentage of Pre-Region 1 on AE-HPLC up to 19.3% was not considered to have a significant impact on the biological activity of the overall formulation.

[0072] Next, we estimated the increase in Pre-Region 1 over 36 months of storage based on long-term stability studies of nemolizumab formulations. The upper limit of the 95% confidence interval for the slope, calculated for storage time (M) and the area percentage (area%) of Pre-Region 1, was 0.154 area% / M. Therefore, the area percentage increase by 36M was estimated to be 5.5 area% (0.154 area% / M × 36M = 5.544 area%). Therefore, the release specification for the drug substance was set at an area percentage of 13.8 area% or less (19.3% - 5.5%). Furthermore, as mentioned above, the area increase rate of Pre-Region 1 per hour in the affinity pool was 0.0448 area% / h (Table 4), the highest area increase rate in the purification process. Therefore, it was found that an appropriate hold time for the affinity pool is important for quality assurance. These stability results indicated that in order to ensure appropriate quality, the retention time in the affinity pool (18°C-28°C) should be within 24 hours (an increase equivalent to 1.08 area%).

[0073] As described above, an approximate straight line was obtained based on the data plotted with the isoAsp content per peptide as the X-axis and the area fraction of Pre-Region 1 in AE-HPLC as the Y-axis (Figure 3). Using the obtained approximate straight line, the total isoAsp content per peptide corresponding to the area fraction of Pre-Region 1 of 19.3% was calculated, and the result was 9.4% as a percentage of the total of the isoAsp content and the corresponding unmodified peptide not modified with isoAsp and Asu residues (an unmodified peptide in which the modification sites for isoAsp and Asu residues are aspartic acid and the rest has the same amino acid sequence as the isoAsp content). Based on this content per peptide, the theoretical maximum isoAsp content per antibody was calculated using the following equation based on a binomial distribution: *1 was calculated. *1: Corresponds to when the Asu body is 0%. Peptide content: X (%) <9.4% Both heavy chains are isomerized to isoAsp residues: X 2 < (0.094) 2 x 100 = 0.00883 → 0.88% Native form in which both heavy chains are unmodified: (1-X) 2 <(1-0.094) x (1-0.094) = 0.8208 → 82.1%> One heavy chain isomerized to an isoAsp residue: X(1-X) + (1-X)X = 2X(1-X) <2 x 0.094 x (1-0.094) = 0.1703 → 17.0%>

[0074] Therefore, the total isoAsp content in peptide units of 9.4% (19.3% area fraction of Pre-Region 1 by AE-HPLC) was found to correspond to the total isoAsp content in antibody units of 17.9%, which is the sum of the maximum content of isoAsp residues in two heavy chains (0.88%) and the maximum content of isoAsp residues in one of the two heavy chains (17.0%).

[0075] [7.2] Pre-Region 2 The shelf-life specification for nemolizumab formulations was set at 13.8% or less of the area percentage of Pre-Region 2 on AE-HPLC, based on clinical data and the expected impact on biological activity, PK, safety, and immunogenicity. The Asu form is a modification commonly found in antibody drugs and was considered to have little impact on safety and immunogenicity. Regarding biological activity, the b8 peak was found to have low biological activity, at 23% of that of the CIM331 reference material (nemolizumab) (Table 3). However, an increase in the area percentage of Pre-Region 2 on AE-HPLC up to 13.8% was not considered to have a significant impact on the biological activity of the overall formulation.

[0076] Next, we estimated the increase in Pre-Region 2 over 36 months of storage based on long-term stability studies of nemolizumab formulations. The upper limit of the 95% confidence interval for the slope, calculated for storage time (M) and the area percentage (area%) of Pre-Region 2, was 0.123 area% / M. Therefore, the area percentage increase by 36M was estimated to be 4.4 area% (0.123 area% / M × 36M = 4.428 area%). Therefore, the release specification for the drug substance was set at an area percentage of 9.4 area% or less (13.8% - 4.4%). Furthermore, as mentioned above, the area increase rate of Pre-Region 2 per hour in the affinity pool was 0.0552 area% / h (Table 4), the highest area increase rate in the purification process. Therefore, it was found that an appropriate hold time for the affinity pool is important for quality assurance. These stability results indicated that in order to ensure appropriate quality, the retention time in the affinity pool (18°C-28°C) should be within 24 hours (an increase equivalent to 1.32 area%).

[0077] As described above, an approximate straight line was obtained based on the data plotted with the Asu form content on a peptide basis as the X-axis and the area fraction of Pre-Region 2 in AE-HPLC as the Y-axis (Figure 3). Using the obtained approximate straight line, the total Asu form content on a peptide basis corresponding to the area fraction of Pre-Region 2 of 13.8% was calculated, and the result was 9.8% as a percentage of the total of Asu forms and the corresponding unmodified peptide not modified with isoAsp and Asu residues (the modification sites for isoAsp and Asu residues are aspartic acid, and the rest of the peptide has the same amino acid sequence as the Asu form). Based on this content on a peptide basis, the theoretical maximum Asu form content on an antibody basis was calculated using the following equation based on a binomial distribution: *1 was calculated. *1: Corresponds to when the isoAsp isomer is 0%. Peptide content: X (%) <9.8% Both heavy chains are isomerized to Asu residues: X 2 < (0.098) 2 x 100 = 0.00960 → 0.96% Native form in which both heavy chains are unmodified: (1-X) 2 <(1-0.098) x (1-0.098) = 0.8136 → 81.4%> One heavy chain isomerized to Asu residue: 2X(1-X) <2 x 0.098 x (1-0.098) = 0.1767 → 17.7%

[0078] Therefore, the total Asu content on a peptide basis of 9.8% (13.8% area fraction of Pre-Region 2 in AE-HPLC) was found to correspond to a total Asu content of 18.6% on an antibody basis, which is the sum of the maximum content of Asu forms in which two heavy chains contain Asu residues (0.96%) and Asu forms in which one of the two heavy chains contains Asu residues (17.7%).

[0079] [Example 8] Evaluation of intra-CDR deamidation products in post-regions Fractions containing each peak separated and obtained using the column and mobile phase described in Example 1 were diluted with a denaturant solution containing guanidinium chloride (pH 7.0) and then reduced with dithiothreitol. Subsequently, cysteine ​​side chains were capped with iodoacetic acid, buffer exchanged with a digestion buffer containing Tris, urea, and EDTA (pH 7.0), and digestion was carried out with trypsin. Formic acid was added to the resulting digestion reaction solution, which was then injected into a reversed-phase high-performance liquid chromatograph for separation. Peptide identification and variant quantification were performed using a mass spectrometer.

[0080] As a result, of all the AE-HPLC peaks, the fraction containing the a6 peak, which is a part of the post-region peak, showed the highest level of deamidation at Asn55 in the CDR of the nemolizumab heavy chain, at 14.9%. Because the deamidation level in this peak was low, it was not possible to directly evaluate the effect of deamidation at Asn55 on biological activity by measuring biological activity. However, since CDRs play an important role in antigen binding, this strongly suggests that deamidation at Asn55 in the CDR affects biological activity. Harris, RJ, et al., Identification of multiple sources of charge heterogeneity in a recombinant antibody. J Chromatogr B Biomed Sci Appl, 2001. 752(2): p. 233-45 Yan B, Steen S, Hambly D, et al. Succinimide formation at Asn 55 in the complementarity determining region of a recombinant monoclonal antibody IgG1 heavy chain. J Pharm Sci 2009; 98:3509-21. Qi P, Volkin DB, Zhao H, et al. Characterization of the photodegradation of a human IgG1 monoclonal antibody formulated as a high-concentration liquid dosage form. J Pharm Sci 2009; 98:3117-30

[0081] Furthermore, when the drug substance stored at 40°C for 12 weeks was analyzed using the same method, the content of the above-mentioned deamidation was 1.6% per peptide, even in the drug substance stored at 40°C for 12 weeks, and it was estimated that the content would not increase significantly even during storage of the formulation at 30°C (e.g., 1.6% per peptide).

[0082] [Example 9] Evaluation of LMWS in the Post-Region [9.1] Evaluation by non-reduced LC-ESI-MS The fractions containing each peak separated and obtained using the column and mobile phase described in Example 1 were diluted with 0.1% formic acid, then injected into a high-performance liquid chromatograph, desalted using a desalting column, and the LMWS were identified using a mass spectrometer.

[0083] As a result, it was determined that the fraction containing the a2 and a3 peaks, which are part of the post-region peaks, contained HL bodies, and the fraction containing the a1 peak contained cysteinylated light chains and cysteinylated HHL bodies.

[0084] [9.2] Evaluation by non-reduced tryptic peptide mapping Fractions containing each peak separated and obtained using the column and mobile phase described in Example 1 were diluted with a denaturant solution containing guanidinium chloride at pH 7.0 for denaturation. Subsequently, cysteine ​​side chains were capped with iodoacetic acid, buffer exchanged with a digestion buffer containing Tris, urea, and EDTA at pH 7.0, and digestion was carried out with trypsin. Formic acid was added to the resulting digestion reaction solution, which was then injected into a reversed-phase high-performance liquid chromatograph for separation, and the peptides were identified using a mass spectrometer. As a result, it was concluded that the HL form contained in the a2 and a3 peaks has a structure in which Cys227 and Cys230 in one heavy chain form a disulfide bond (Figure 4). On the other hand, although the cysteinylation sites of the cysteinylated light chain and cysteinylated HHL body contained in the a1 peak could not be identified, they were presumed to be the structures shown in Figure 5.

[0085] [9.3] Evaluation based on non-reduced CE-SDS Fractions containing each peak separated and obtained using the column and mobile phase described in Example 1 were buffer exchanged with 0.1 mol / L sodium phosphate buffer, and then mixed with an SDS solution containing N-ethylmaleimide to cap the cysteine ​​side chains. A dye reaction solution containing potassium cyanide and FQ-dye was then added to the solution, and fluorescent dye labeling was performed. SDS solution was added to the resulting solution, and analysis was performed by capillary electrophoresis-SDS.

[0086] As a result, the content of HL isomers in the fraction containing the a2 and a3 peaks was determined to be 16.7 CPA%, and the content of cysteinylated light chains in the fraction containing the a1 peak was determined to be 14.5 CPA%.

[0087] [Example 10] Management of LMWS [10.1] Evaluation method for low molecular weight species (CE-SDS) The nemolizumab drug substance and formulation were each buffer-substituted with 0.1 mol / L sodium phosphate buffer, then mixed with an SDS solution containing N-ethylmaleimide to cap the cysteine ​​side chains. A dye reaction solution containing potassium cyanide and FQ-dye was then added to the solution, and fluorescent dye labeling was performed. SDS solution was added to the resulting solution, and separation was performed using capillary electrophoresis-SDS (CE-SDS). Peaks with shorter migration times than the nemolizumab drug substance and the main component of the formulation (nemolizumab) were defined as peaks with molecular weights smaller than nemolizumab. These peaks were selected as the targets for evaluation in the LMWS control analysis method.

[0088] [10.2] Content control of low molecular weight species (CE-SDS) The shelf-life specifications for the nemolizumab drug product were set at 11.0 CPA% or less for the LMWS content (including LMWS identified in fractions containing the b2 and b3 peaks and fractions containing the a1, a2, and a3 peaks by AE-HPLC) analyzed by CE-SDS based on clinical experience, biological activity, PK, safety, and anticipated effects on immunogenicity. Since no increase in LMWS content was observed in stability testing of the drug product, the release specifications for the drug substance and drug product were also set at 11.0 CPA% or less. [Industrial Applicability]

[0089] The present inventors have discovered antibody variants with lower biological activity than nemolizumab, and methods for analyzing the same. The antibody variants and analytical methods of the present invention are useful for evaluating the quality of nemolizumab drug substances and formulations, as well as for developing nemolizumab formulations with reduced content of the antibody variants of the present invention and for developing methods for suppressing the production of the antibody variants of the present invention. Furthermore, pharmaceutical compositions of the present invention containing nemolizumab and with reduced content of the antibody variants of the present invention are useful as means for treating and / or preventing atopic dermatitis, dialysis pruritus, and other pruritus.

Claims

1. A method for producing nemolizumab, comprising the steps of: Culturing host cells that express nemolizumab; a harvesting step in which the solution obtained by the culturing step is centrifuged and filtered to obtain a host cell culture solution; a purifying step of purifying the host cell culture medium by affinity chromatography; a holding step of holding the purified solution after the purification step is completed; Here, in the holding step, the holding time is 24 hours or less.

2. The method of claim 1, wherein the retention time is a retention time in an affinity pool (18°C-28°C).

3. A pharmaceutical composition comprising nemolizumab and a variant of nemolizumab, the ratio of the area of ​​a succinimide-containing peak to the total peak area when the pharmaceutical composition is separated using anion exchange chromatography is 13.8% or less; The pharmaceutical composition, wherein the nemolizumab variant is the variant shown in (A) below: (A) A variant of nemolizumab having a variable region comprising the amino acid sequence DGYDDGPYTLET (sequence number 3), in which the amino acid residue D at the fifth position from the N-terminus of the sequence is changed to a succinimide residue.

4. The pharmaceutical composition of claim 3, wherein the major component of the succinimide-containing peak is the nemolizumab variant shown in (A).

5. The pharmaceutical composition of claim 3, wherein the succinimide-containing peaks are five peaks with shorter retention times among eight peaks that appear in a region with shorter retention times than the main peak containing nemolizumab.

6. The detection in the anion exchange chromatography is carried out using an ultraviolet-visible spectrophotometer (measurement wavelength: 280 nm); The anion exchange chromatography is carried out using a separation column and a guard column; The separation column has an inner diameter of 7.5 mm, a length of 75 mm, and a particle size of 2.5 μm. The guard column has an inner diameter of 4.6 mm, a length of 5 mm, and a particle size of 5.0 μm. The column temperature of the separation column and guard column was kept constant at about 40°C, and the elution was performed with a mobile phase of pH 7.5 ± 0.1 at a flow rate of 1.0 mL / min. The mobile phase is Mobile phase A: 25 mmol / L Tris buffer, pH 7.5 ± 0.1; and Mobile phase B: 25 mmol / L Tris buffer containing 250 mmol / L sodium chloride, pH 7.5 ± 0.1 Including, The pharmaceutical composition according to any one of claims 3 to 5, wherein the delivery of the mobile phase is concentration gradient controlled by changing the mixing ratio of the mobile phase A and the mobile phase B to achieve the following salt concentration gradient conditions: 。

7. The pharmaceutical composition according to claim 6, wherein the column temperature of about 40°C is a column temperature of 40°C.

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