Antibody variants with reduced biological activity

Abstract

This invention provides structural variants (antibody variants) of nemolizumab, particularly structural variants with lower biological activity than nemolizumab. It also provides nemolizumab-containing compositions, methods for reducing the amount of the variant, and analytical methods. [Solution] An antibody variant of nemolizumab having a specific sequence is disclosed. The disclosure also provides a nemolizumab-containing composition containing the antibody variant, a method for reducing the antibody variant, and an analytical method.

Description

[Technical Field]

[0001] This invention relates to antibody variants of nemolizumab, for example, antibody variants that exhibit lower biological activity compared to nemolizumab. The invention also relates to pharmaceutical compositions with low concentrations of such antibody variants. Furthermore, the invention relates to methods for detecting and analyzing such antibody variants. [Background technology]

[0002] Antibodies are attracting attention as pharmaceuticals due to their high stability in plasma and low incidence of side effects. Among them, IgG-type antibody drugs have been launched in large numbers, and many more antibody drugs are currently under development. Generally, antibody drugs contain various components, including the active ingredient (antibody) and structural variations of the target substance (antibody variants). These structural variations are classified into target substance-related substances, which have equivalent efficacy and safety to the target substance, and target substance-derived impurities, which do not. The production rate of some of these variations can fluctuate depending on the manufacturing conditions of the active pharmaceutical ingredient. Because of this heterogeneity in antibody drugs, it is necessary to control the quality of antibody drugs by setting shipping specifications and expiration date specifications for each component of the active pharmaceutical ingredient and the finished product.

[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 1-3). In particular, the anti-IL-31RA antibody nemolizumab (CIM331) inhibits the function of IL-31, which is considered a pruritus-inducing cytokine, by acting as an IL-31 antagonist. Clinical trials have been conducted on 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 generated during the manufacturing of the active pharmaceutical ingredient and formulation of nemolizumab, or what their proportion is. [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 [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention has been made in view of the above circumstances, and aims to provide a structural variant of nemolizumab (antibody variant), a nemolizumab-containing composition containing the antibody variant, a method for producing the same, a method for reducing the antibody variant, and a method for analyzing the antibody variant. [Means for solving the problem]

[0007] As a result of diligent research to achieve the above objectives, the inventors succeeded in identifying multiple antibody variants (structural variations of nemolizumab) contained in pharmaceutical compositions containing nemolizumab as an active ingredient. Furthermore, they found that some of these antibody variants exhibit lower biological activity (inhibitory activity of IL-31 signaling by binding to the IL-31 receptor) compared to nemolizumab. In addition, they discovered a method for efficiently detecting and analyzing these antibody variants.

[0008] This disclosure is based on such findings and relates to the following in one non-limiting embodiment: [1] A variant of an antibody having a variable region containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3), (a) amino acid residue D at the first position from the N-terminus of the sequence, (b) amino acid residue D at the fourth position from the N-terminus of the sequence, (c) Amino acid residue D at the 5th position from the N-terminus of the above sequence An antibody variant in which the residue is either succinimide or isoaspartic acid. [2] An antibody variant of [1] wherein the sequence is a CDR sequence. [2.2] An antibody variant of [1] wherein the sequence is a CDR3 sequence. [3] An antibody variant of [1] wherein the sequence is included in the heavy chain. [4] The antibody having a variable region containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) (1) An antibody comprising a heavy chain variable region including CDR1 described in SEQ ID NO: 1, CDR2 described in SEQ ID NO: 2, and CDR3 described in SEQ ID NO: 3, and a light chain variable region including CDR1 described in SEQ ID NO: 4, CDR2 described in SEQ ID NO: 5, and CDR3 described in SEQ ID NO: 6. (2) An antibody comprising the heavy chain variable region described in SEQ ID NO: 7 and the light chain variable region described in SEQ ID NO: 8, or (3) An antibody containing the heavy chain described in SEQ ID NO: 9 and the light chain described in SEQ ID NO: 10. An antibody variant of [1] which is one of the following: [5] The antibody variant of [1] wherein the antibody having a variable region containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) is nemolizumab. [6] Any antibody variant from [1] to [5] that, when separated using anion exchange chromatography, has a shorter retention time than the antibody having a variable region containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3). [7] Any antibody variant from [1] to [6] that has lower biological activity than the antibody having a variable region containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3). A pharmaceutical composition comprising any antibody variant of [1.2.0] [1] to [7]. A pharmaceutical composition comprising any antibody variant of [1.2.1] [1] to [7], wherein the proportion of the isoaspartic acid residue-containing antibody variant in the pharmaceutical composition to the total of the antibody variant and the unmodified antibody is 36% or less, and / or the proportion of the isoaspartic acid residue-containing peptide derived from the antibody variant in the pharmaceutical composition after enzymatic digestion to the total of the isoaspartic acid residue-containing peptide and the corresponding unmodified peptide is 20% or less. A pharmaceutical composition comprising any antibody variant of [1.2.2] [1] to [7], wherein the proportion of the isoaspartic acid residue-containing antibody variant in the pharmaceutical composition to the total of the antibody variant and the unmodified antibody is 27.8% or less, and / or the proportion of the isoaspartic acid residue-containing peptide derived from the antibody variant in the pharmaceutical composition after enzymatic digestion to the total of the isoaspartic acid residue-containing peptide and the corresponding unmodified peptide is 15% or less. A pharmaceutical composition comprising any antibody variant of [1.2.3] [1] to [7], wherein the proportion of the isoaspartic acid residue-containing antibody variant in the pharmaceutical composition to the total of the antibody variant and the unmodified antibody is 17.9% or less, and / or the proportion of the isoaspartic acid residue-containing peptide derived from the antibody variant in the pharmaceutical composition after enzymatic digestion to the total of the isoaspartic acid residue-containing peptide and the corresponding unmodified peptide is 9.4% or less. A pharmaceutical composition comprising any one of the antibody variants [1] to [7] of [1.2.4], wherein the ratio of the area of the peak containing the isoaspartic acid residue-containing antibody variant to the total peak area when the pharmaceutical composition is separated using anion exchange chromatography is 40.3 area% or less, 30.4 area% or less, or 19.3 area% or less. 〔1.2.5〕A pharmaceutical composition according to any one of [1.2.1] to [1.2.4] and containing nemolizumab. 〔1.2.6〕A pharmaceutical composition comprising any one of the antibody variants [1] to [7], wherein the ratio of the succinimide residue-containing antibody variant in the pharmaceutical composition to the total of the antibody variant and the unmodified antibody is 36% or less, and / or the ratio of the succinimide residue-containing peptide derived from the antibody variant in the pharmaceutical composition after enzymatic digestion to the total of the succinimide residue-containing peptide and the corresponding unmodified peptide is 20% or less. 〔1.2.7〕A pharmaceutical composition comprising any one of the antibody variants [1] to [7], wherein the ratio of the succinimide residue-containing antibody variant in the pharmaceutical composition to the total of the antibody variant and the unmodified antibody is 27.8% or less, and / or the ratio of the succinimide residue-containing peptide derived from the antibody variant in the pharmaceutical composition after enzymatic digestion to the total of the succinimide residue-containing peptide and the corresponding unmodified peptide is 15% or less. 〔1.2.8〕A pharmaceutical composition comprising any one of the antibody variants [1] to [7], wherein the ratio of the succinimide residue-containing antibody variant in the pharmaceutical composition to the total of the antibody variant and the unmodified antibody is 18.7% or less, and / or the ratio of the succinimide residue-containing peptide derived from the antibody variant in the pharmaceutical composition after enzymatic digestion to the total of the succinimide residue-containing peptide and the corresponding unmodified peptide is 9.8% or less. A pharmaceutical composition comprising any one of the antibody variants of [1.2.9] [1] to [7], wherein the ratio of the area of the peak containing the antibody variant containing a succinimide residue to the total peak area when the pharmaceutical composition is separated using anion exchange chromatography is 29.1 area% or less, 21.6 area% or less, or 13.8 area% or less. 〔1.2.10〕A pharmaceutical composition comprising nemolizumab and any one of the pharmaceutical compositions of [1.2.6] to [1.2.9]. 〔1.2.11〕Any one of the pharmaceutical compositions of [1.2.0] to [1.2.9], which is a pharmaceutical composition for use in treating or preventing at least one of atopic dermatitis, dialysis pruritus, and other pruritus. 〔1.2.12〕A method for treating or preventing at least one of atopic dermatitis, dialysis pruritus, and other pruritus, comprising the step of administering any one of the pharmaceutical compositions of [1.2.0] to [1.2.9]. 〔1.3.1〕A method for detecting any one of the antibody variants of [1] to [7], comprising separating a sample containing an antibody having a variable region containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) or an enzymatically treated antibody thereof 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 of [1.3.1], wherein the ion exchange chromatography is anion exchange chromatography. 〔1.3.3〕The detection method of [1.3.2], wherein the anion exchange chromatography uses 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 elutes with a mobile phase having a pH of 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 eluting with a mobile phase containing 250 mmol / L of sodium chloride at pH 7.5. [1.3.5] A detection method according to any of [1.3.1] to [1.3.4], comprising the step of separating a sample containing the enzyme-treated antibody, followed by the step of quantifying the peptide by mass spectrometry and / or ultraviolet absorption measurement. A detection method according to any of the following (1.3.1) to (1.3.5), characterized in that one of the antibody variants in (1) to (7) of (1.3.6) is used as a standard substance (standard). A method for producing a composition containing nemolizumab, comprising the step of performing one of the detection methods described in [1.4.1], [1.3.1], to [1.3.6]. [1.4.2] A method for producing a composition containing nemolizumab, comprising the 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 method for producing [1.4.2], wherein the purification step includes purifying the nemolizumab-containing composition obtained from nemolizumab-producing cells by affinity chromatography. [1.4.4] The method for producing the product according to [1.4.3], characterized in that the retention time of the nemolizumab-containing solution after purification by affinity chromatography is 72 hours or less, and the content of any of the antibody variants [1] to [7] in the composition is lower compared to the case where the retention time is longer. [1.4.5] The method for producing the method of [1.4.4], characterized in that the retention time of the nemolizumab-containing solution after purification by affinity chromatography is within approximately 24 hours. A method for producing any of the products described in [1.4.1] to [1.4.5], characterized by using one of the antibody variants in [1] to [7] as a standard substance (standard). [1.5.1.1] A method for suppressing the content of any antibody variant [1] to [7], comprising the step of purifying 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) by affinity chromatography, wherein the content of any antibody variant [1] to [7] in the composition is lower compared to a case without 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 48 hours or less, and the content of any antibody variant [1] to [7] in the composition is lower compared to the case where 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. A method of any of the following: [1.5.1.1] to [1.5.1.3], characterized by using one of the antibody variants [1] to [7] as a standard substance (standard). A composition comprising any of the antibody variants in [1.6.1.1] [1] to [7], wherein the antibody variant has a shorter retention time than nemolizumab when separated by anion exchange chromatography and contains more isoaspartic acid residues than nemolizumab. [1.6.1.2] The composition according to [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. A composition comprising any of the antibody variants in [1.6.2.1] [1] to [7], wherein the antibody variant has a shorter retention time than nemolizumab when separated by anion exchange chromatography and contains more succinimide residues than nemolizumab. [1.6.2.2] The composition according to [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] A nemolizumab antibody variant having a smaller molecular weight than nemolizumab, consisting of two heavy chains and two light chains. [2.1.1.2] A nemolizumab antibody variant characterized by having a structure formed by two heavy chains, where the cysteine ​​groups at EU numbering position 224 of the nemolizumab heavy chain are linked together. [2.1.1.3] A nemolizumab antibody variant characterized by having a structure formed by two light chains linked at EU numbering position 214 of the nemolizumab light chain. [2.1.1.4] A nemolizumab antibody variant characterized by having a structure formed by one light chain and one heavy chain, in which the cysteine ​​at EU numbering position 214 of the light chain of nemolizumab is bound to the cysteine ​​at EU numbering position 224 of the heavy chain of nemolizumab, and further binding of the cysteines at EU numbering positions 227 and 230 of the heavy chain within a single heavy chain. [2.1.1.5] A nemolizumab antibody variant characterized by having a structure formed by a single light chain in which the cysteine ​​at EU numbering position 214 of the nemolizumab light chain is bound to a free cysteine. [2.1.1.6] A nemolizumab antibody variant characterized by having a structure in which two heavy chains and one light chain of nemolizumab are composed of two cysteine ​​molecules bound to each other at EU numbering positions 227 and 230, respectively, the cysteine ​​at EU numbering position 224 on the first heavy chain is bound to the cysteine ​​at EU numbering position 214 on the light chain, and the cysteine ​​at EU numbering position 224 on the second heavy chain is bound to a free cysteine ​​molecule. A pharmaceutical composition comprising any antibody variant of [2.1.2], [2.1.1.1] to [2.1.1.6], wherein the proportion of the antibody variant in the total antibody molecules in the pharmaceutical composition is 11.0 CPA% or less. [2.2.1] A nemolizumab antibody variant characterized by deamidation of asparagine at position 55 of the nemolizumab heavy chain Kabat number. A pharmaceutical composition comprising the antibody variant of [2.2.2] [2.2.1], wherein the proportion of the deamidated peptide derived from the antibody variant after enzymatic digestion of the pharmaceutical composition is 2.0% or less of the total of the deamidated peptide and the corresponding unmodified peptide. [Effects of the Invention]

[0009] The inventors have successfully identified a structural variant (antibody variant) that exhibits significantly lower biological activity compared to nemolizumab. Such antibody variants are useful as standard materials (standards) for detection and analysis of these variants in the quality evaluation of nemolizumab-containing pharmaceutical compositions. Furthermore, the inventors have discovered a method to control (reduce to a low level) the generation of such antibody variants, namely aspartate isomerized variants, and a method for measuring the content of such variants (variant detection / analysis method). These methods have higher throughput compared to conventional methods and are important technologies for improving and controlling the quality of nemolizumab. Moreover, the nemolizumab-containing pharmaceutical composition of the present invention, which has a low content of such antibody variants, is useful as a means for the treatment and / or prevention of atopic dermatitis, dialysis pruritus, and other pruritus. [Brief explanation of the drawing]

[0010] [Figure 1A] This chromatogram shows the results of separating the nemolizumab-containing solution by anion exchange high-performance liquid chromatography under the conditions described in Example 1. A TSK DEAE NPR column (4.6 mm inner diameter, 35 mm length, 2.5 μm particle size) was used, and the separation was performed under salt concentration adjustment condition 1 shown in Table 1. [Figure 1B] This chromatogram shows the results of separating the nemolizumab-containing solution by anion exchange high-performance liquid chromatography under the conditions described in Example 1. A TSK DEAE NPR column (7.5 mm inner diameter, 75 mm length, 2.5 μm particle size) was used, and the separation was performed under salt concentration adjustment condition 2 shown in Table 2. [Figure 2] This is a schematic diagram showing the structures of HH dimer and LL dimer, variants of nemolizumab. [Figure 3] This scatter plot shows the content of isoAsp (top) or Asu (bottom) peptide units in samples after holding HCCF or Affinity Pool at 23°C for various durations, plotted on the X-axis, and the area percentage of Pre-Region 1 (top) or Pre-Region 2 (bottom) of AE-HPLC on the Y-axis. [Figure 4] This is a schematic diagram showing the structure of the HL variant of nemolizumab. [Figure 5]This is a schematic diagram showing the structures of the cysteinylated HHL (left) and cysteinylated light chain (right) variants of nemolizumab. [Modes for carrying out 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 by binding to the IL-31 receptor). These antibody variants were identified by the inventors in their analysis of the nemolizumab drug substance as aspartate isomerized variants (variants in which at least one aspartate residue in nemolizumab is replaced with a succinimide residue (Asu variant) and a variant in which at least one aspartate residue is replaced with an isoaspartate residue (isoAsp variant)). In this specification, “antibody variant” may also be referred to as a structural change, isomer, isomerized variant, or isomer of the original antibody.

[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 includes a heavy chain variable region containing CDR1 described in SEQ ID NO: 1, CDR2 described in SEQ ID NO: 2, and CDR3 described in SEQ ID NO: 3, and a light chain variable region containing CDR1 described in SEQ ID NO: 4, CDR2 described in SEQ ID NO: 5, and CDR3 described in SEQ ID NO: 6. More specifically, nemolizumab includes a heavy chain variable region described in SEQ ID NO: 7 and a light chain variable region described in SEQ ID NO: 8. Even more specifically, nemolizumab includes a heavy chain described in SEQ ID NO: 9 and a light chain described in SEQ ID NO: 10. In this specification, nemolizumab and its variants are described separately, and therefore the term "nemolizumab" does not include nemolizumab variants (e.g., aspartate isomerized variants).

[0013] Known methods for defining CDR include those by Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Ed (1991), Bethesda, MD), Chothia et al. (Science (1986) 233, 755-758), and methods based on the antigen-antibody contact region (J Mol Biol (1996) 262, 732-745). Specifically, CDR according to each method is 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 this disclosure, reduced biological activity of an antibody variant means that its biological activity is lower than that of a comparative antibody (e.g., nemolizumab), and preferably statistically significantly lower. In one embodiment, the biological activity of the antibody variant of the present invention is 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 comparative antibody (e.g., nemolizumab). In one embodiment, the biological activity (relative biological activity) of the antibody variant of the present invention relative to the biological activity of a comparative antibody (e.g., CIM331 standard substance, 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, neutralizing activity against the IL-31 receptor is activity that inhibits the binding of IL-31 to the IL-31 receptor. In one embodiment, neutralizing activity against the IL-31 receptor is inhibitory activity of IL-31 signaling by 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 way to determine whether nemolizumab or its antibody variant inhibits IL-31 signaling is to examine whether nemolizumab or its antibody variant inhibits the binding of IL-31 to the IL-31 receptor. Methods for such measurements include assays using ELISA, flow cytometry, and surface plasmon resonance. 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 to it is detected with a secondary antibody such as an enzyme-labeled anti-IL-31 antibody. When nemolizumab or its antibody variant is added to this system, the amount of detected IL-31 protein is measured to determine whether nemolizumab or its antibody variant inhibited the binding of IL-31 to the IL-31 receptor. Alternatively, whether nemolizumab or its antibody variant inhibits IL-31 signaling can be confirmed by examining whether the physiological activity caused by IL-31 action on cells is inhibited by nemolizumab or its antibody variant. The said physiological activity is not particularly limited as long as it can be measured quantitatively or qualitatively by some method, but examples include cell proliferation activity, protein phosphorylation activity, and gene / protein expression induction activity. For example, if cells expressing the IL-31 receptor on their surface and in which proliferation activity is induced in response to external IL-31 stimulation are prepared, and nemolizumab or its antibody variant is added to them, the degree to which the IL-31-induced cell proliferation activity decreases can be measured to evaluate whether nemolizumab or its antibody variant inhibited the IL-31 signal. Such cells may be native cells that naturally express the IL-31 receptor, or genetically modified cells that artificially express the IL-31 receptor may be used. A suitable example of genetically modified cells is Ba / F3 cells expressing the IL-31 receptor. Another method is to use the method described in Dillon et al.'s paper (Nat Immunol (2004) 5, 752-760).

[0017] In this disclosure, the extent to which nemolizumab or its antibody variant inhibits IL-31 signaling is not limited, but may inhibit, 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 its broadest sense and may refer to monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antibody derivatives, and antibody modifiers, as long as they exhibit the desired biological activity (e.g., antigen-binding activity) (Miller K et al. J Immunol. 2003, 170(9), 4854-61). Antibodies may be mouse antibodies, human antibodies, humanized antibodies, chimeric antibodies, or derived from other species, or may be artificially synthesized. Antibodies disclosed herein may be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Immunoglobulins may be derived from any species (e.g., human, mouse, or rabbit). The terms "antibody," "immunoglobulin," and "immunoglobulin" are interchangeable and used in a broad sense. Furthermore, antibodies are not limited to antibody molecules composed of two heavy chains and two light chains, but may also be antibody fragments having a smaller molecular weight. For example, 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 bound to free cysteine, and a structure formed by two heavy chains and one light chain are also included in the definition of an antibody as specified herein. In light of this broad definition, the antibody variants of the present invention are also included in the definition of an antibody. Therefore, "(total) antibody molecules in a pharmaceutical composition" as specified herein may include both antibodies and their variants.

[0019] As antibodies, recombinant antibodies produced using genetic engineering technology can be used. Recombinant antibodies can be obtained by cloning the DNA encoding them from a hybridoma or antibody-producing cell such as a sensitized lymphocyte, incorporating it into a vector, and introducing this vector into a host (host cell) to induce production.

[0020] In the present invention, antibodies can be produced by methods known to those skilled in the art. Specifically, DNA encoding the target antibody is incorporated into an expression vector. At that time, the DNA is incorporated into the expression vector so that it is expressed under the control of expression regulatory regions, such as enhancers and promoters. Next, host cells are transformed with this expression vector to express the antibody. In this case, an appropriate combination of host and expression vector can be used.

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

[0022] While not limited to any particular theory, the antibody variant of the present invention may be a structural variant of an antibody molecule (e.g., nemolizumab) that has a variable region containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) and is produced during the manufacturing process (e.g., purification process) of said antibody molecule. In one aspect, the antibody variant of the present invention has at least one (e.g., one, two, or three) amino acid residues altered compared to an antibody molecule (e.g., nemolizumab) that has a variable region containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3). In one embodiment, the antibody variant of the present invention has one to three, i.e., one, two, or three amino acid residues altered compared to an antibody molecule (e.g., nemolizumab) that has a variable region containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3). In certain embodiments, the antibody variant of the present invention has 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 having a variable region containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) (e.g., nemolizumab). In certain embodiments, the antibody variant of the present invention has 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 having a variable region containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) (e.g., nemolizumab).

[0023] In another aspect, the antibody variant of the present invention differs from an antibody molecule having a variable region containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) (e.g., nemolizumab) in terms of the combination of disulfide bonds (binding between cysteine ​​residues or between a cysteine ​​residue and free cysteine). In one embodiment of this aspect, the antibody variant of the present invention has the same amino acid sequence as an antibody molecule having a variable region containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) (e.g., nemolizumab), but differs in the combination of disulfide bonds (binding between cysteine ​​residues or between a cysteine ​​residue and free cysteine) and also differs in molecular weight. In another embodiment, the antibody variant of the present invention differs from an antibody molecule having a variable region containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) (e.g., nemolizumab) in terms of the amino acid sequence, the combination of disulfide bonds (binding between cysteine ​​residues or between a cysteine ​​residue and free cysteine), and also differs in molecular weight.

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

[0025] In one embodiment, the aspartic acid isomerized variant of the present invention is an antibody variant having a variable region containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3), (a) The amino acid residue D(Asp) at the first position from the N-terminus of the sequence or (b) Amino acid residue D(Asp) at the fourth position from the N-terminus of the above sequence or (c) Amino acid residue D(Asp) at the 5th position from the N-terminus of the above sequence The antibody variant is one in which the residue is a succinimide residue (which may be referred to herein as the succinimide variant or the Asu variant), or one in which the residue is an isoaspartic acid residue (which may be referred to herein as the isoaspartic acid variant or the isoAsp variant). In the succinimide variant, the succinimide residue is preferably at the 1st or 5th position from the N-terminus of the sequence (SEQ ID NO: 3), and particularly preferably at the 5th position from the N-terminus of the sequence. In a particular embodiment, the aspartic acid isomerized variant of the present invention is an antibody variant having a variable region containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3), (a2) Amino acid residue D(Asp) at the 1st and 5th positions from the N-terminus of the above sequence or (b2) Amino acid residues D(Asp) at the 4th and 5th positions from the N-terminus of the above sequence or (c2) Amino acid residue D(Asp) at the 1st and 4th positions from the N-terminus of the above sequence The antibody variant is one in which the residue is succinimide (Asu) or isoaspartic acid (isoAsp). An example of such an antibody variant is one in which the amino acid sequence (SEQ ID NO: 3) from the N-terminus An antibody variant in which the first position is an isoAsp residue and the fifth position is an Asu residue; An antibody variant in which the first position is an Asu residue and the fifth position is isoAsp; Antibody variants in which the 1st and 5th positions are Asu residues; Antibody variants in which the 1st and 5th positions are isoAsp residues; An antibody variant in which the fourth position is an isoAsp residue and the fifth position is an Asu residue; An antibody variant in which the fourth position is an Asu residue and the fifth position is an isoAsp residue; Antibody variants in which the 4th and 5th positions are Asu residues; Antibody variants in which the 4th and 5th positions are isoAsp residues; An antibody variant in which the first position is an isoAsp residue and the fourth position is an Asu residue; An antibody variant in which the first position is an Asu residue and the fourth position is an isoAsp residue; Antibody variants in which the 1st and 4th positions are Asu residues; or Antibody variants where the 1st and 4th positions are isoAsp residues These include, preferably, Antibody variants in which the 1st and 4th positions are isoAsp residues; Antibody variants in which the 1st and 5th positions are isoAsp residues; Antibody variants in which the 4th and 5th positions are isoAsp residues; An antibody variant in which the first position is an isoAsp residue and the fifth position is an Asu residue; or An antibody variant in which the fourth position is an isoAsp residue and the fifth position is an Asu residue; These are some examples. 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 methods for detecting and analyzing aspartic acid isomerized variants. In one embodiment, the method of the present invention includes separating a sample containing an antibody having a variable region including the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) or an enzymatically 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 particular embodiment, the method of the present invention includes enzymatically treating the antibody-containing sample prior to the separation step. Examples of enzymes for treating the antibody-containing sample include, but are not limited to, trypsin and Lys-C. In a particular embodiment, the method of the present invention includes identifying and / or quantifying peptides by mass spectrometry and / or ultraviolet absorption spectroscopy, following the step of separating the enzymatically treated antibody-containing sample.

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

[0028] In one embodiment of the method of the present invention, ion exchange chromatography is anion exchange chromatography. In one embodiment, 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, at a column temperature of 25-50°C, and eluting with a mobile phase of pH 6.0-10.0. In a specific embodiment, 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, at a column temperature of 40°C, and eluting with a mobile phase of pH 7.5 containing 250 mmol / L of sodium chloride.

[0029] Furthermore, in one embodiment, the method of the present invention includes the step of using a peptide containing (i) an aspartic acid isomerized variant of the present invention, (ii) an isoAsp residue or an Asu residue obtained by enzymatic digestion thereof, or (iii) a peptide containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3), wherein one, two, or three aspartic acid (Asp, D) residues at the 1st, 4th, and 5th positions from the N-terminus of the sequence are changed to succinimide (Asu) residues or isoaspartic acid (isoAsp) residues, as a standard, and performing one or more analyses selected from the group consisting of quantitative analysis, qualitative analysis, and structural analysis. An example of the peptide in (iii) is a peptide in which one of the 1st, 4th, and 5th positions from the N-terminus of the amino acid sequence of SEQ ID NO: 3 is changed to an Asu residue or an isoAsp residue. Another example of the peptide in (iii) is a peptide in which two of the 1st, 4th, and 5th 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 an isoAsp residue; Peptides in which the 1st and 5th positions are Asu residues; Peptides whose 1st and 5th positions 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 in which the 4th and 5th positions are Asu residues; Peptides in which the 4th and 5th positions are isoAsp residues; 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; Peptides in which the 1st and 4th positions are Asu residues; or Peptides in which the 1st and 4th positions are isoAsp residues These include, preferably, Peptides in which the 1st and 4th positions are isoAsp residues; Peptides whose 1st and 5th positions are isoAsp residues; Peptides in which the 4th and 5th positions are isoAsp residues; 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; These are some examples.

[0030] In another aspect, the present invention allows for the manufacture and quality control of pharmaceutical compositions containing nemolizumab by implementing one or a combination thereof of the detection and analysis methods described above. Accordingly, the present invention relates to a method for quality control of pharmaceutical compositions containing nemolizumab, comprising the steps of implementing the detection and analysis methods described above, or a combination thereof. The present invention also relates to a method for producing pharmaceutical compositions containing nemolizumab, comprising the steps of implementing one or more of such detection, analysis, and quality control methods.

[0031] In the production method of the present invention, a nemolizumab-containing pharmaceutical composition with a low content of aspartate isomerized 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 a nemolizumab-containing pharmaceutical composition with a low content of aspartate isomerized variants is useful as a means for the treatment and / or prevention of diseases in which nemolizumab exerts therapeutic and / or preventive effects, such as atopic dermatitis, dialysis pruritus, and other pruritus. In one embodiment, the manufacturing method of the present invention includes the step of culturing nemolizumab-producing cells (e.g., CHO cells) into which a gene encoding nemolizumab has been incorporated. This culturing step can be carried out 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 culture medium (basal culture medium for animal cells) can be used. In one embodiment, the production method of the present invention includes the step of obtaining a nemolizumab-containing solution (Harvest Cell Culture Fluid: HCCF) by centrifuging and filtering a culture medium of nemolizumab-producing cells. Purification of nemolizumab from the obtained nemolizumab-containing solution can be carried out by a combination of general column chromatography, such as affinity chromatography, ion exchange chromatography, or hydrophobic chromatography. In a particular embodiment, the production method of the present invention includes the step of purifying the nemolizumab-containing solution obtained from 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 to the next step is not particularly limited, but by shortening this time, the content of aspartic acid isomerized variants can be reduced. Therefore, in order to maintain the biological activity of the antibody and antibody variant in the nemolizumab-containing pharmaceutical composition obtained by the manufacturing method of the present invention to an acceptable level, it is preferable to set the retention time of the antibody-containing solution after purification by affinity chromatography to the next step within a predetermined range (for example, 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 containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) and a variant thereof (e.g., an aspartic acid isomerized variant), wherein the proportion of the variant in the total antibody molecules (including the antibody and its variant) in the pharmaceutical composition is kept low. Such a pharmaceutical composition of the present invention, with a low proportion of the variant, can be suitably used in the treatment and / or prevention of atopic dermatitis, dialysis pruritus, and other pruritus. In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for use in one or both of the treatment and prevention of at least one of atopic dermatitis, dialysis pruritus, and other pruritus. The present invention also relates to a method for treating or preventing at least one of atopic dermatitis, dialysis 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 setting a short retention time (e.g., 18-28°C or 18-23°C, preferably at room temperature, 18°C, or 23°C) of the antibody-containing solution after purification of the antibody solution containing the antibody (e.g., nemolizumab) by protein A affinity chromatography to the next step, the proportion of aspartic acid isomerized variants in the total antibody molecules in the obtained composition can be kept low.

[0033] The proportion of aspartic acid isomerized variants in antibody molecules within the pharmaceutical composition of the present invention can be evaluated by various methods, including the aspartic acid isomerized variant detection / analysis method described above. As a method for analyzing proteins such as antibodies, peptide mapping is a known method in which proteins are broken down into peptide fragments by enzymatic digestion, separated by various chromatography techniques, and then subjected to mass spectrometry or ultraviolet absorption spectroscopy to obtain information on the amino acid sequence and post-translational modifications of the protein. As described in the examples of this application, the structure and content of the aspartic acid isomerized variant of the present invention can be analyzed by peptide mapping.

[0034] In one embodiment, the proportion of aspartic acid isomerized variants in antibody molecules within 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 the aspartic acid isomerized variant obtained by enzymatic digestion of the pharmaceutical composition of the present invention, relative to the total of the variant-derived peptides and the corresponding unmodified peptides (unmodified peptides in which the modification site to the succinimide residue or isoaspartic acid residue is aspartic acid, and the rest of the amino acid sequence is the same as that of the variant-derived peptide) (this may also be referred to as the proportion of aspartic acid isomerized variants on a peptide basis). For example, trypsin, Lys-C, etc., can be used as digestive enzymes. The proportion of isoaspartic acid forms (isoAsp forms) on a peptide basis within the pharmaceutical composition of the present invention (proportion compared to aspartic acid forms on a peptide basis) is, for example, 25% or less, 20% or less, 15% or less, or 9.4% or less. Furthermore, the proportion of succinimide (Asu) in peptide units in the pharmaceutical composition of the present invention (the proportion compared to aspartate 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 this application, the ratio of succinimide residue-containing peptides and isoaspartic acid residue-containing peptides derived from aspartic acid isomerized variants obtained after enzymatic digestion to the total of the variant-derived peptides and the corresponding unmodified peptides (unmodified peptides in which the modification site to the succinimide residue or isoaspartic acid residue is aspartic acid, and the rest of the amino acid sequence is the same as that of the variant-derived peptide) (the ratio of aspartic acid isomerized variants on a peptide basis) correlates with the ratio of the peak area of ​​the aspartic acid isomerized variant elution peak group to the total peak area (peak area ratio) in anion exchange chromatography (AE-HPLC) (Figure 3). Therefore, the ratio of aspartic acid isomerized variants in antibody molecules in the pharmaceutical composition of the present invention can be expressed by the ratio of the peak area of ​​the combined peak group from which succinimide (Asu) and isoaspartic acid (isoAsp) elutes when the pharmaceutical composition of the present invention is analyzed by anion exchange chromatography (AE-HPLC) to the total peak area (peak area ratio). This evaluation method offers higher throughput compared to detection / analysis by peptide mapping, making it advantageous for 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 isoaspartate (isoAsp) and succinimide (Asu) are in a basic region (a region with a shorter retention time compared to the main peak) compared to the main peak containing nemolizumab, the main component of the pharmaceutical composition of the present invention, and may be referred to herein as the Pre-Region. Of the eight peaks included in this region (Pre-Region), isoaspartate (isoAsp) mainly elutes in the region containing three peaks close to the main peak containing nemolizumab (may be referred to herein as Pre-Region 1), and succinimide (Asu) mainly elutes in the region containing the remaining five peaks (may be referred to herein as Pre-Region 2). Therefore, when evaluated by the analytical method of the present invention using anion exchange chromatography (AE-HPLC), the proportion of isoaspartate (isoAsp) and succinimide (Asu) in the total antibody molecules in the pharmaceutical composition of the present invention (proportion per antibody unit) can be expressed as the proportion of peak areas in Pre-Region 1 and Pre-Region 2, respectively.

[0037] In one embodiment, the proportion of isoaspartate (isoAsp) in the total antibody molecules in the pharmaceutical composition of the present invention (area percentage of Pre-Region 1), as evaluated 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 (Asu) in the total antibody molecules in the pharmaceutical composition of the present invention (area percentage of Pre-Region 2), as evaluated 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 this application, based on the proportion of aspartic acid isomerized variants at the peptide level, the theoretical maximum proportion of aspartic acid isomerized variants at the antibody level can be calculated based on the binomial distribution. Based on the proportion of aspartic acid isomerized variants at the peptide level, the proportion of isoaspartic acid variants (isoAsp variants) in antibody molecules within the pharmaceutical composition of the present invention, relative to the total of the isoAsp variants and unmodified antibodies (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 variants in antibody molecules within the pharmaceutical composition of the present invention, where two heavy chains contain isoAsp residues, is, for example, 6.3% or less, 4.0% or less, 2.3% or less, or 0.9% or less. The proportion of isoAsp variants in antibody molecules within the pharmaceutical composition of the present invention, where one of the two heavy chains contains an isoAsp residue, is, for example, 37.5% or less, 32.0% or less, 25.5% or less, or 17.0% or less. Similarly, the ratio of succinimide (Asu) in the antibody molecule in the pharmaceutical composition of the present invention to the total of the Asu and unmodified antibodies (ratio 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 ratio of Asu in the antibody molecule in the pharmaceutical composition of the present invention, in which two heavy chains contain an Asu residue, to the total of the Asu and unmodified antibodies is, for example, 6.3% or less, 4.0% or less, 2.3% or less, or 1.0% or less. The ratio of Asu in the antibody molecule in the pharmaceutical composition of the present invention, in which one of the two heavy chains contains an Asu residue, to the total of the Asu and unmodified antibodies 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 in which the content of aspartic acid isomerized variants is kept low, and a method for suppressing the generation of aspartic acid isomerized variants. The amount of aspartic acid isomerized variants generated can be kept low by setting a short retention time for the antibody-containing solution after purification of an antibody-containing solution containing an antibody (e.g., nemolizumab) having a variable region including the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) by affinity chromatography (e.g., protein A affinity chromatography) to the next step. Therefore, the above method includes a step of purifying an antibody-containing composition obtained from cells that produce an antibody (e.g., nemolizumab) having a variable region including the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3) by affinity chromatography (e.g., protein A affinity chromatography), and is characterized by a short retention time for the antibody-containing solution after purification by affinity chromatography to 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 18-28°C or 18-23°C, preferably at room temperature, 18°C, or 23°C) is within approximately 138 hours, for example, within approximately 72 hours, within approximately 48 hours, or within approximately 24 hours, preferably within approximately 48 hours, and more preferably within approximately 24 hours.

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

[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 (HH dimer) where cysteine ​​molecules at EU numbering position 224 of the nemolizumab heavy chain are linked together; (2) A structure formed by two light chains linked at EU numbering position 214 of the nemolizumab light chain (LL dimer); (3) A structure formed by one light chain and one heavy chain (HL form), in which the cysteine ​​at EU numbering position 214 on the light chain of nemolizumab binds to the cysteine ​​at EU numbering position 224 on the heavy chain of nemolizumab, and further binding of the cysteines at EU numbering positions 227 and 230 on the heavy chain within a single heavy chain; (4) A structure formed by a single light chain in which the cysteine ​​at EU numbering position 214 of the nemolizumab light chain 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 linked at two cysteine ​​positions, EU numbering 227 and 230, respectively. Furthermore, the cysteine ​​at EU numbering 224 on the first heavy chain is linked to the cysteine ​​at EU numbering 214 on the light chain, and the cysteine ​​at EU numbering 224 on the second heavy chain is linked to a free cysteine ​​(Cysteinylated HHL isomer). Antibody variants having such a structure are low molecular weight variants (LMWS: Low molecular weight species) having a smaller molecular weight than nemolizumab, which is composed of two heavy chains and two light chains, and may be referred to herein as low molecular weight variants of nemolizumab. In another aspect, the present invention relates to a pharmaceutical composition comprising nemolizumab and its low molecular weight variant (LMWS), wherein the proportion of the low molecular weight variant in the total 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 in which the asparagine at position 55 of the Kabat numbering in the heavy chain of nemolizumab is deamidated, and to a pharmaceutical composition comprising the antibody variant. In one embodiment, the proportion of the deamidated peptide derived from the antibody variant after enzymatic digestion of the pharmaceutical composition is 2.0% or less of the total of the deamidated peptide and the corresponding unmodified peptide.

[0043] In this specification, the expression "comprising" includes the expressions "essentially consisting of" and "consisting of".

[0044] The numerical values ​​described herein may vary within a certain range due to factors such as equipment, measurement conditions, and the skills of those skilled in the art, and may vary by, for example, about 10%, as long as the objectives of the present invention can be achieved.

[0045] All patents and references expressly cited herein are incorporated herein by reference. The present invention is further illustrated by the following embodiments, but is not limited to those embodiments. [Examples]

[0046] [Example 1] Separation of nemolizumab-containing solution by anion exchange high-performance liquid chromatography (AE-HPLC) [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 prepared by a method known to those skilled in the art, in accordance with the description in the aforementioned patent document. Briefly, a nemolizumab-containing solution (API) was prepared by a method including the steps of culturing host cells containing a vector into which the gene encoding nemolizumab has been introduced, and purifying the nemolizumab-containing solution obtained in the culture step. Furthermore, a nemolizumab formulation was prepared using this API by the method described in WO2021 / 100794.

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

[0048] [Test conditions] Detector: UV-Vis absorbance 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°C 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] When a TSK DEAE NPR column (4.6 mm inner diameter, 35 mm length, 2.5 μm particle size) was used for separation under the above concentration adjustment condition 1, the chromatogram shown in Figure 1A was obtained. On the other hand, when a TSK DEAE NPR column (7.5 mm inner diameter, 75 mm length, 2.5 μm particle size) was used for separation under the above concentration adjustment condition 2, the chromatogram shown in Figure 1B was obtained.

[0052] As shown in Figures 1A-B, the region with a shorter retention time than the Main Region containing the Main peak (the region containing fractions eluted at a lower salt concentration than the Main Region) was defined as the Pre-Region, and the region with a longer retention time than the Main Region (the region containing fractions eluted at a higher salt concentration than the Main Region) was defined as the Post-Region. Furthermore, the peaks in the Pre-Region were defined as b1-b8 in order of proximity to the Main Region (i.e., in order of slowest retention time), and the peaks in the Post-Region were defined as a1-a6 in order of proximity to the Main Region (i.e., in order of fastest retention time). The Pre-Region was further divided into Pre-Region 1 (the region containing peaks b1-b3) and Pre-Region 2 (the region containing peaks b4-b8) in order of slowest retention time.

[0053] Comparing Figure 1A and Figure 1B, Figure 1B showed better separation of the b1 peak and the Main peak. Therefore, the following separation tests used the conditions 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).

[0054] Table 3 below summarizes the information on nemolizumab variants contained in the Pre-Region 1, Pre-Region 2, and Post-Region peaks shown in Figure 1B. The biological activity listed in Table 3 is the result of evaluation 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 isomerized variants (isoAsp and succinimide) in Pre-Region 2 The fractions containing each peak, separated and obtained using the column and mobile phase described in Example 1, were diluted with a pH 5.5 denaturing agent solution containing guanidium chloride, and then reduced with tris(2-carboxyethyl)phosphine. Subsequently, the buffer was changed to a pH 5.5 digestion buffer containing 2-morpholinoethanesulfonic acid and urea, and then digestion was carried out with trypsin. Formic acid was added to the obtained digestion reaction solution, and the solution was injected into reverse-phase high-performance liquid chromatography for separation. Peptides were identified and the modification ratio at the peptide level was calculated using a mass spectrometer. The modification ratio of the target peptide at the peptide level was determined from the peak area in the MS chromatogram, and this was taken as the ratio of the target variant at the peptide level. Since it was confirmed that the peaks of peptides containing some isoAsp residues overlapped with the peaks of unmodified peptides in the MS chromatogram, the modification ratio at the peptide level was calculated using the following formula. Modification rate at the peptide level (%) = (Modified peptide of interest) *1 (Peak area of ​​the modified peptide of interest) ÷ {(Peak area of ​​the modified peptide of interest) + (Unmodified peptide corresponding to the modified peptide of interest) *2 (Peak area of ​​[the original peptide] - (Peak area of ​​the peptide containing some isoAsp residues)) × 100 (*1: Refers to peptides containing isoAsp residues or succinimide residues derived from aspartic acid isomerized variants.) (*2: This refers to an unmodified native peptide that has the same amino acid sequence as the modified peptide except for the modification site.)

[0057] Analysis revealed that the highest levels of succinimide-containing peptides were detected in the fraction containing the b8 peak, which is part of Pre-Region 2, among all AE-HPLC peaks. Specifically, the modification of Asp103 (the Asp residue at Kabat numbering position 99) to the succinimide residue (Asu103) was detected at a rate of 54.0% in the b8 peak. On the other hand, the modification rate of Asp99 (the Asp residue at Kabat numbering position 95) to the succinimide residue was low (2.7%), and modifications of Asp102 (the Asp residue at Kabat numbering position 98) to the succinimide residue, as well as modifications of Asp99, Asp102, and Asp103 to the isoAsp residues, were not detected (Not detected). TIFF2026063423000004.tif61161

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

[0059] [Example 3] Evaluation of aspartic acid isomerized variants (isoAsp and succinimide) in Pre-Region 1 Similar to Example 2, the fractions containing each peak separated and obtained using the column and mobile phase described in Example 1 were diluted with a pH 5.5 denaturing agent solution containing guanidium chloride, and then reduced with tris(2-carboxyethyl)phosphine. Subsequently, the buffer was changed to a pH 5.5 digestion buffer containing 2-morpholinoethanesulfonic acid and urea, and then digestion was carried out with trypsin. Formic acid was added to the obtained digestion reaction solution, and the solution was injected into reverse-phase high-performance liquid chromatography for separation. Peptides were identified and the modification ratio at the peptide level was calculated using a mass spectrometer. The modification ratio of the target peptide at the peptide level was determined from the peak area in the MS chromatogram, and this was taken as the ratio of the target variant at the peptide level. Since it was confirmed that the peaks of peptides containing some isoAsp residues overlapped with the peaks of unmodified peptides in the MS chromatogram, the modification ratio at the peptide level was calculated using the following formula. Modification rate at the peptide level (%) = (Modified peptide of interest) *1 (Peak area of ​​the modified peptide of interest) ÷ {(Peak area of ​​the modified peptide of interest) + (Unmodified peptide corresponding to the modified peptide of interest) *2 (Peak area of ​​[the original peptide] - (Peak area of ​​the peptide containing some isoAsp residues)) × 100 (*1: Refers to peptides containing isoAsp residues or succinimide residues derived from aspartic acid isomerized variants.) (*2: This refers to an unmodified native peptide that has the same amino acid sequence as the modified peptide except for the modification site.)

[0060] Theoretically, three types of structures are possible for the isoAsp form (the top row shows modification of the isoAsp residue at Asp99, the middle row shows modification of the isoAsp residue at Asp102, and the bottom row shows modification of the isoAsp residue at Asp103), but it has not been determined which of these Asp sites actually undergoes isomerization. When molecular weight changes with structural changes, it is possible to identify the amino acid site where the structure has changed by tandem mass spectrometry (MSMS analysis) using peptide mapping, but since isoAsp has the same molecular weight as Asp, such identification by MSMS is difficult. TIFF2026063423000005.tif153169

[0061] Analysis revealed that the highest levels of isoAsp residue-containing peptides were detected in the fraction containing the b1 peak, which is part of Pre-Region 1, among all AE-HPLC peaks (two types of isoAsp residue-containing peptides at 11.6% and 2.7%). Furthermore, the percentage of Asp99 converted to a succinimide residue (Asu99) in the b1 peak was 1.1%, Asp102 converted to a succinimide residue (Asu102) was 0.2%, and Asp103 converted to a succinimide residue (Asu103) was 4.7%. Thus, compared to the Main peak containing the main component nemolizumab (with 0.5% modification of isoAsp residues at any of Asp99, Asp102, or Asp103), which was separated and obtained using the column and mobile phase described in Example 1, it was confirmed that the aspartic acid isomerized variant (isoAsp form) mainly eluted in Pre-Region 1.

[0062] [Example 4] Evaluation of Low Molecular Weight Species (LMWS) in Pre-Region 1 [4.1] Evaluation using 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 high-performance liquid chromatography, desalted using a desalting column, and then identified by mass spectrometry. As a result, it was identified that HH dimers and LL dimers were present in peaks b2 and b3, which are part of Pre-Region 1.

[0063] [4.2] Non-reduced tryptic peptide mapping evaluation Fractions containing each peak, separated and obtained using the column and mobile phase described in Example 1, were diluted with a pH 7.0 denaturing agent solution containing guanidium chloride and subjected to denaturation treatment. Subsequently, the cysteine ​​side chains were capped with iodoacetic acid, and the buffer was changed to a pH 7.0 digestion buffer containing Tris, urea, and EDTA, followed by a digestion reaction with trypsin. Formic acid was added to the resulting digestion reaction solution, and the solution was injected into reverse-phase high-performance liquid chromatography for separation. Peptides were identified using a mass spectrometer. As a result, it was identified that the HH dimer has a structure in which the C224 of the heavy chain, which normally forms a disulfide bond with the C214 of the light chain, is linked to the heavy chain itself. Furthermore, it was estimated that the LL dimer has a structure in which the C214 of the light chain, which normally forms a disulfide bond with the C224 of the heavy chain, is linked to the light chain itself (Figure 2).

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

[0065] [Example 5] Evaluation of the biological activity of aspartic acid isomerized variants (isoAsp and succinimide). The biological activity of each variant was evaluated by measuring its growth inhibitory activity in IL-31-dependently proliferating BaF / hIL-31R cells (BaF cells expressing human IL-31R). Specifically, fractions containing each peak separated and obtained using the column and mobile phase described in Example 1 were mixed with FBS-RPMI medium containing IL-31 to prepare dilutions of various concentrations. Each dilution was added to a 96-well microplate, and after adding BaF / hIL-31R cell suspension to each well, the cells were incubated in a 37°C CO2 incubator for 24 hours. Subsequently, filtered alamarBlue was added, and the cells were incubated in a 37°C CO2 incubator for another 3 hours, after which the absorbance (OD) was measured using a microplate reader. 570 -OD 600 ) was measured.

[0066] As a result, when normalized to the Main peak (Main peak: 100%), the biological activity of the b8 peak was 21% of that of the Main peak, indicating that the biological activity of the succinimide compound (equivalent to 54.0% of the Asu103 variant) mainly contained in the b8 peak was significantly attenuated. Furthermore, the biological activity of the b1 peak (containing 11.6% isoAsp) was shown to be attenuated to 41% compared to the Main peak. Since this isoAsp isomerization is a chemical change in the CDR, it is strongly suggested to affect 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 becomes a control analysis method for aspartic acid isomerization variants (isoAsp form and succinimide form) During the purification process of the solution containing nemolizumab obtained in the culturing process of the host cells expressing nemolizumab, the holding times until the next step of the antibody-containing solutions after the harvesting step (HCCF) by centrifugation and filtration after production culturing and after the subsequent purification step by protein A affinity chromatography (Affinity pool) (sample holding times after each purification step) were confirmed for the influence on the content of aspartic acid isomerization variants (isoAsp form and succinimide form). To do this, the contents of the isoAsp form and succinimide form of various samples (HCCF (after holding at 23°C for 0 / 72 hours), Affinity pool (after holding at 23°C for 0 / 24 / 48 / 72 / 138 hours)) were confirmed. The content of the variant compared to the unmodified form was determined in peptide units by performing digestion of each sample with trypsin and separation by HPLC, and then quantification using the MS intensity by MS analysis, similar to Examples 2 and 3. The content of the variant in peptide units was determined by the following calculation formula. Content of variant in peptide units (intensity %) = (MS intensity of the modified peptide *1 of interest *2 ) ÷ {(MS intensity of the modified peptide of interest) + (MS intensity of the unmodified peptide *3 corresponding to the modified peptide of interest)} × 100 (*1: Refers to a peptide containing an isoAsp residue or a succinimide residue derived from an aspartic acid isomerization variant) (*2: MS intensity is the intensity of the MS spectrum derived from the peptide.) (*3: Refers to the unmodified Native form peptide having the same amino acid sequence as the modified peptide except for the modification site in the modified peptide.) As a result, a significant time-dependent increase in aspartic acid isomerized variants (isoAsp and succinimide) was observed in the affinity pool (Table 4).

[0068] Furthermore, the above samples (HCCF (held at 23°C for 0 / 72 hours), Affinity pool (held at 23°C for 0 / 24 / 48 / 72 / 138 hours)) were separated by AE-HPLC in the same manner as in Example 1, and the area ratio of each region in the HPLC chromatogram was determined. The correlation between the isoAsp content at the peptide level and the area ratio of Pre-Region 1 in AE-HPLC, and between the succinimide content at the peptide level and the area ratio of Pre-Region 2 in AE-HPLC, were examined. It was found that the isoAsp content correlated with the area ratio of Pre-Region 1 (Figure 3). In addition, it was found that the succinimide content correlated with the area ratio of Pre-Region 2 (Figure 3), indicating that AE-HPLC can be used as a control analysis method for the content of these variants.

[0069] Furthermore, the area growth rate per hour for Pre-Region 2 in the affinity pool was 0.0552 area% / h, and the area growth rate for Pre-Region 1 was 0.0448 area% / h (Table 4). The high area growth rate per hour indicates that setting an appropriate sample retention time between the purification step by affinity chromatography and the next step is important for ensuring the quality of nemolizumab.

[0070] [Table 4]

[0071] [Example 7] Method for suppressing the generation of aspartic acid isomerized variants (isoAsp and succinimide) in the manufacture of the active pharmaceutical ingredient. [7.1] Pre-Region 1 The shelf-life specification for nemolizumab formulations was set at an area ratio of 19.3% or less in Pre-Region 1 on AE-HPLC, based on clinical performance, biological activity, PK, safety, and expected impact on immunogenicity. The IsoAsp variant is a commonly recognized modification for antibody drugs, and its impact on safety and immunogenicity was considered low. Regarding biological activity, the b1 peak showed low biological activity of 45% compared to the CIM331 standard substance (nemolizumab) (Table 3). However, an increase of up to 19.3% in the area ratio of Pre-Region 1 on AE-HPLC was not considered to have a significant impact on the overall biological activity of the formulation.

[0072] Next, the increase in Pre-Region 1 after 36 months of storage was estimated from long-term stability tests of the nemolizumab formulation. As a result, the upper limit of the 95% confidence interval for Slope obtained for the storage period (M) and the area percentage of Pre-Region 1 (area%) was 0.154 area% / M, and therefore the area percentage increase up to 36M was estimated to be 5.5 area% (0.154 area% / M × 36 M = 5.544 area%). For this reason, the shipment specification for the active pharmaceutical ingredient was set to an area percentage of 13.8 area% (19.3%-5.5%) or less. 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), and the area increase rate was highest in the purification process, indicating that setting an appropriate hold time in the affinity pool is important for quality assurance. These stability results indicate that, in order to ensure appropriate quality, a holding time of 24 hours (equivalent to an increase of 1.08 area%) or less in the affinity pool (18°C-28°C) is preferable.

[0073] As described above, an approximate straight line was obtained based on data plotted with the isoAsp content at the peptide level on the X-axis and the area percentage of Pre-Region 1 in AE-HPLC on the Y-axis (Figure 3). Using the obtained approximate straight line, the total isoAsp content at the peptide level corresponding to the area percentage of Pre-Region 1 (19.3%) was calculated, and it was found to be 9.4% as the ratio of the isoAsp content to the total of the isoAsp content and the corresponding unmodified peptide (an unmodified peptide in which the modification site at the isoAsp and Asu residues is aspartic acid, and the rest of the amino acid sequence is the same as that of the isoAsp content). Based on this content at the peptide level, the theoretical maximum isoAsp content at the antibody level was calculated using the following equation based on the binomial distribution. *1 I calculated it. *1: This corresponds to the case where the ASU content is 0%. Peptide content: X (%) <9.4%> isoAsp form:X 2 < (0.094) 2 x 100 = 0.00883 → 0.88%> Native form with both helical chains unmodified: (1-X) 2 <(1-0.094) x (1-0.094) = 0.8208 → 82.1%> isoAsp isomer, in which one of the heavy chains has been 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 of 9.4% at the peptide level (19.3% area ratio in Pre-Region 1 of AE-HPLC) corresponds to the total isoAsp content of 17.9% at the antibody level, which is the sum of the isoAsp content of 0.88% (where two heavy chains contain isoAsp residues) and the isoAsp content of 17.0% (where one of the two heavy chains contains isoAsp residues).

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

[0076] Next, the increase in Pre-Region 2 after 36 months of storage was estimated from long-term stability tests of the nemolizumab formulation. As a result, the upper limit of the 95% confidence interval for Slope obtained for the storage period (M) and the area percentage of Pre-Region 2 (area%) was 0.123 area% / M, and therefore the area percentage increase up to 36M was estimated to be 4.4 area% (0.123 area% / M × 36 M = 4.428 area%). For this reason, the shipment specification for the active pharmaceutical ingredient was set to an area percentage of 9.4 area% (13.8%-4.4%) or less. 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), and the area increase rate was highest in the purification process, indicating that setting an appropriate hold time in the affinity pool is important for quality assurance. These stability results indicate that, in order to ensure appropriate quality, the holding time in the affinity pool (18°C-28°C) should preferably be within 24 hours (equivalent to an increase of 1.32 area%).

[0077] As described above, a straight line was obtained based on data plotted with the Asu isomer content at the peptide level on the X-axis and the area percentage of Pre-Region 2 in AE-HPLC on the Y-axis (Figure 3). Using the obtained straight line, the total Asu isomer content at the peptide level corresponding to the area percentage of Pre-Region 2 (13.8%) was calculated, and it was found to be 9.8% as the ratio of the Asu isomer to the corresponding unmodified peptide (an unmodified peptide that has not undergone modification of isoAsp and Asu residues, with the modification site being aspartic acid, and the rest of the amino acid sequence being the same as the Asu isomer). Based on this content at the peptide level, the theoretical maximum Asu isomer content at the antibody level was calculated using the following equation based on the binomial distribution. *1 I calculated it. *1: This corresponds to the case where the isoAsp body is 0%. Peptide content: X (%) <9.8%> Asu-isomer:X 2 < (0.098) 2 x 100 = 0.00960 → 0.96%> Native form with both helical chains unmodified: (1-X) 2 <(1-0.098) x (1-0.098) = 0.8136 → 81.4%> Asu isomer in which one of the heavy chains has been isomerized to an Asu residue: 2X(1-X) <2 x 0.098 x (1-0.098) = 0.1767 → 17.7%>

[0078] Therefore, the total Asu content of 9.8% at the peptide level (13.8% area ratio in Pre-Region 2 of AE-HPLC) corresponds to the total Asu content of 18.6% at the antibody level, which is the sum of the Asu content containing Asu residues in both heavy chains (0.96%) and the Asu content containing Asu residues in one of the two heavy chains (17.7%).

[0079] [Example 8] Evaluation of deamidation bodies within CDRs in the post-region The fractions containing each peak, separated and obtained using the column and mobile phase described in Example 1, were diluted with a pH 7.0 denaturing agent solution containing guanidium chloride, and then reduced with dithiothreitol. Subsequently, the cysteine ​​side chains were capped with iodoacetic acid, and the buffer was changed to a pH 7.0 digestion buffer containing Tris, urea, and EDTA, followed by digestion with trypsin. Formic acid was added to the resulting digestion reaction solution, and the solution was injected into reverse-phase high-performance liquid chromatography for separation. Peptide identification and variant quantification were performed using a mass spectrometer.

[0080] As a result, among all the AE-HPLC peaks, the fraction containing the a6 peak, which is part of the Post-Region, showed the highest level of deamidation of the nemolizumab heavy chain at Asn55 within the CDR, at 14.9%. Because the level of deamidation at this peak was low, it was not possible to directly evaluate the impact of Asn55 deamidation on biological activity by measuring biological activity. However, since the CDR plays an important role in antigen binding, it is strongly suggested that deamidation at Asn55 within 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 active pharmaceutical ingredient (API) stored at 40°C for 12 weeks was analyzed using the same method, the content of the aforementioned deamidation was 1.6% in peptide units even in the API 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 (for example, 1.6% in peptide units).

[0082] [Example 9] Evaluation of LMWS in Post-Region [9.1] Evaluation using 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 high-performance liquid chromatography, desalted using a desalting column, and then identified by mass spectrometry.

[0083] As a result, it was identified that the fraction containing peaks a2 and a3, which are part of the Post-Region, contains the HL isomer, while the fraction containing peak a1 contains the Cysteinylated light chain and the Cysteinylated HHL isomer.

[0084] [9.2] Evaluation by Non-reduced tryptic peptide mapping The fractions containing each peak, separated and obtained using the column and mobile phase described in Example 1, were diluted with a pH 7.0 denaturing agent solution containing guanidium chloride and subjected to denaturation treatment. Subsequently, the cysteine ​​side chains were capped with iodoacetic acid, and the buffer was changed to a pH 7.0 digestion buffer containing Tris, urea, and EDTA, followed by a digestion reaction with trypsin. Formic acid was added to the resulting digestion reaction solution, and the solution was injected into reverse-phase high-performance liquid chromatography for separation. Peptides were identified using a mass spectrometer. As a result, it was concluded that the HL isomers contained in peaks a2 and a3 have a structure in which Cys227 and Cys230 within a single heavy chain form a disulfide bond (Figure 4). On the other hand, although the cysteinylation sites of the cysteinylated light chain and cysteinylated HHL isoform contained in the a1 peak could not be identified, they were presumed to be the structures shown in Figure 5.

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

[0086] As a result, the HL content in the fraction containing peaks a2 and a3 was identified as 16.7 CPA%, and the cysteinylated light chain content in the fraction containing peak a1 was identified as 14.5 CPA%.

[0087] [Example 10] Management of LMWS [10.1] Evaluation method for low molecular weight species (CE-SDS) The nemolizumab active pharmaceutical ingredient and formulation were buffered with 0.1 mol / L sodium phosphate buffer, then mixed with an SDS solution containing N-ethylmaleimide to cap the cysteine ​​side chains. Next, a dye reaction solution containing potassium cyanide and FQ-dye was added to the solution, and labeling with a fluorescent dye was performed. The resulting solution was then separated by capillary electrophoresis-SDS (CE-SDS). Peaks with shorter migration times than the main component (nemolizumab) of the nemolizumab active pharmaceutical ingredient and formulation were defined as peaks with a smaller molecular weight than nemolizumab. This peak group was set as the target for evaluation in the LMWS control analysis method.

[0088] [10.2] Content management of low molecular weight species (CE-SDS) The shelf-life specification for nemolizumab formulations was set at 11.0 CPA% or less for LMWS content (including LMWS identified in fractions containing b2 and b3 peaks and a1, a2, and a3 peaks by AE-HPLC) as analyzed by CE-SDS, based on clinical performance, bioactivity, PK, safety, and expected effects on immunogenicity. Since no increase in LMWS content was observed in the formulation stability test, the shipment specification for both the active pharmaceutical ingredient and the formulation was similarly set at 11.0 CPA% or less. [Industrial applicability]

[0089] The inventors have discovered an antibody variant with lower biological activity compared to nemolizumab, and a method for analyzing it. The antibody variant and analytical method of the present invention are useful for quality evaluation of the active pharmaceutical ingredient and formulation of nemolizumab, as well as for the development of nemolizumab formulations with a reduced content of the antibody variant of the present invention, and for the development of methods to suppress the generation of the antibody variant of the present invention. Furthermore, the pharmaceutical composition of the present invention, which contains nemolizumab and has a low content of the antibody variant of the present invention, is useful as a means for the treatment and / or prevention of atopic dermatitis, dialysis pruritus, and other pruritus.

Claims

1. A method for reducing the variant in a manufacturing process of a pharmaceutical composition comprising nemolizumab and a variant of nemolizumab, wherein the process comprises: Culture process of host cells expressing nemolizumab; A harvesting step in which the solution obtained in the culture step is centrifuged and filtered to obtain a host cell culture medium; A purification step in which the host cell culture medium is purified by affinity chromatography; After the purification process is completed, a holding process is performed to hold the purified solution; Includes, Here, in the holding process, the holding time is 24 hours or less. The aforementioned variant is the variant shown in (A) below, the method: (A) A variant of nemolizumab having a variable region containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3), wherein the amino acid residue D at the 5th position from the N-terminus of the sequence is changed to a succinimide residue.

2. The method according to claim 1, wherein the holding time is the holding time in an affinity pool (18°C-28°C).

3. A pharmaceutical composition comprising nemolizumab and a variant of nemolizumab, When the pharmaceutical composition is separated using anion exchange chromatography, the ratio of the area of ​​the succinimide-containing peak to the total peak area is 13.8% or less. The detection in the aforementioned anion exchange chromatography is performed using a UV-Vis spectrophotometer (measurement wavelength 280 nm). The anion exchange chromatography described above is performed 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, while the guard column has an inner diameter of 4.6 mm, a length of 5 mm, and a particle size of 5.0 μm. Here, the column is a DEAE column, and the separation column and guard column are eluted at a constant temperature of around 40°C 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 Includes, The delivery of the mobile phase is controlled by changing the mixing ratio of mobile phase A and mobile phase B to achieve the following salt concentration gradient conditions: The pharmaceutical composition wherein the nemolizumab variant is the variant shown in (A) below: (A) A variant of nemolizumab having a variable region containing the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 3), wherein the amino acid residue D at the 5th position from the N-terminus of the sequence is changed to a succinimide residue.

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

5. The pharmaceutical composition according to claim 3, wherein the succinimide-containing peaks are five of the eight peaks that appear in a region with a shorter retention time than the major peak containing nemolizumab, and the five peaks have shorter retention times.

6. The pharmaceutical composition according to claim 3, wherein the column temperature around 40°C is 40°C.

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