Method for analyzing impurity molecules in a composition containing a multispecific antigen-binding molecule

Abstract

The present disclosure provides an analysis method for measuring the content of light chain shifting molecules in a composition containing multi-specific antigen-binding molecules. This analysis method of the present disclosure includes: a step for treating a composition containing multi-specific antigen-binding molecules to prepare a plurality of types of F(ab) fragments; and a step for measuring the F(ab) fragments by a separation method based on electric charge or hydrophobic interactions to find the content (content ratio) of each of the fragments.

Description

Technical Field

[0001] The present disclosure relates to a method for analyzing impurity molecules in a composition containing a multispecific antigen-binding molecule. More specifically, the present disclosure relates to a method for quantifying the content of light chain exchange molecules generated by the mispairing of light chains in a composition containing a multispecific antigen-binding molecule, which involves processing the molecules contained in the composition to generate multiple types of F(ab) fragments, and measuring the F(ab) fragments to determine the content or content ratio of each fragment. process, and measuring the F(ab) fragments to determine the content or content ratio of each fragment. This includes the step of measuring.

Background Art

[0002] In a multispecific antigen-binding molecule with non-common light chains (especially a bispecific antibody), since it has two types of heavy chains (H1, H2) and two types of light chains (L1, L2), in addition to the target molecule (H1L1 / H2L2) in the culture process, nine impurity molecules are generated due to mispairing between heavy chains or between heavy chains and light chains (Figure 5). To suppress these impurity molecules, techniques such as the Knobs-into-holes technique (Patent Document 1) and techniques utilizing charge control between heavy chains or between heavy chain-light chains (Patent Documents 2 and 3) are known. Also, for the generated impurity molecules, techniques are known in which modifications are introduced into the amino acid residues of the heavy and light chains, and separation and removal are performed by chromatography using the pI difference between the target molecule and the impurity molecules (Patent Document 4). However, in the separation method using the pI difference, up to eight of the impurity molecules can be separated, but for the remaining one impurity molecule (H1L2 / H2L1, hereinafter referred to as "light chain exchange molecule") in which L1 and L2 that should pair with H1 and H2 are inverted, since the physical properties including the theoretical pI are common to the target molecule, it cannot be separated. Therefore, a method for accurately measuring the presence or absence, content, or content ratio of light chain exchange molecules in the composition obtained after the purification process is required for quality control.

[0003] Yin et al. disclose a method for analyzing impurity molecules containing light chain exchange molecules (Non-Patent Document 1). In this document, each molecule is separated into F(ab), mass spectrometry is performed, and an estimated value of the content of each molecule is calculated using theoretical calculations based on the mass spectrometry results and the probability of the heavy chain-light chain combination. However, since this method does not directly measure the content from the analysis results but only obtains an estimated value using theoretical calculations, accurate quantification of the light chain exchange molecule cannot be achieved.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] The invention in the present disclosure has been made in view of the above situation, and in a non-limiting aspect, an object is to provide an analytical method for distinguishing and quantifying a target multispecific antigen-binding molecule and a light chain exchange molecule contained in a composition.

Means for Solving the Problems

[0007] In a non-limiting aspect, as a result of intensive research, the inventors utilized the differences in the combinations of F(ab) fragments contained in multispecific antigen-binding molecules and light chain exchange molecules to find an analytical method for measuring the content (content ratio) of multispecific antigen-binding molecules and light chain exchange molecules contained in a composition. Specifically, by treating the molecules contained in the composition to generate multiple types of F(ab) fragments, separating each F(ab) fragment using the charge difference or the like, and measuring their respective contents (content ratios), it became possible to measure the presence or absence of the light chain exchange molecules in the composition, as well as the content or content ratio.

[0008] This disclosure is based on such findings and specifically includes the embodiments exemplified below. [1] A method for analyzing light chain exchange molecules in a composition containing a multispecific antigen-binding molecule, comprising: The multispecific antigen-binding molecule is a molecule (H1L1 / H2L2) comprising a first F(ab) (H1L1) containing a first heavy chain variable domain and a first light chain variable domain for a first antigen, and a second F(ab) (H2L2) containing a second heavy chain variable domain and a second light chain variable domain for a second antigen; The light chain exchange molecule is a molecule (H1L2 / H2L1) comprising a third F(ab) (H1L2) containing a first heavy chain variable domain and a second light chain variable domain, and a fourth F(ab) (H2L1) containing a second heavy chain variable domain and a first light chain variable domain; The analysis method comprises the following steps 1 to 2. 1) A step of treating a composition containing a multispecific antigen-binding molecule to generate two or more types of F(ab) fragments 2) A step of measuring two or more types of F(ab) fragments by a separation method based on charge or hydrophobic interaction to determine the content of light chain exchange molecules in the composition or the content ratio of light chain exchange molecules to the multispecific antigen-binding molecule [2] The method of [1], wherein step 1 comprises steps 1-1 and 1-2 below. Step 1-1: Cleaving the molecule contained in the composition to generate F(ab)’2 fragments Step 1-2: Cleaving the F(ab)’2 fragments to generate two or more F(ab) fragments [3] The method of [2], wherein Step 1-1 is a step of cleaving the Fc side of the hinge portion of the molecule contained in the composition with a protease. [4] The method of [3], wherein the protease is any one of a bacterial antibody-degrading enzyme, pepsin, ficin, or a combination thereof. [5] The method according to any one of [2] to [4], wherein Step 1-2 is a step of cleaving the disulfide bond of the F(ab)’2 fragment with a reducing agent. [6] The method of [5], wherein the reducing agent is any one of TCEP, 2-MEA, Cysteine, Dithiothreitol, 2-Mercaptoethanol, 3-mercapto-1,2-propanediol, TBP, or a combination thereof. [7] Step 1 is a step of cleaving the F(ab) side of the hinge portion of the molecule contained in the composition with a protease of [1]. [8] The method of [7], wherein the protease is any one of a bacterial antibody-degrading enzyme, papain, Lys-C, or a combination thereof thereof. [9] Step 2 is a step of measuring two or more F(ab) fragments by any one of cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography, or a combination thereof, according to any one of [1] to [8].

[10] The two or more F(ab) fragments generated in Step 1 each have a different isoelectric point, according to the method described in any one of [1] to [9].

[11] The multispecific antigen-binding molecule (H1L1 / H2L2) is a molecule in which the amino acid residues are modified so as to provide a difference in isoelectric point from the following impurities (1) to (8), according to any one of [1] to

[10] . (1) The homodimer (H1L1 / H1L1) of the first F(ab) (H1L1) containing the first heavy chain variable domain and the first light chain variable domain (2) The homodimer (H2L2 / H2L2) of the second F(ab) (H2L2) containing the second heavy chain variable domain and the second light chain variable domain (3) The homodimer (H1L2 / H1L2) of the third F(ab) (H1L2) containing the first heavy chain variable domain and the second light chain variable domain (4) The homodimer (H2L1 / H2L1) of the fourth F(ab) (H2L1) containing the second heavy chain variable domain and the first light chain variable domain (5) The heterodimer (H1L1 / H1L2) containing the first F(ab) (H1L1) containing the first heavy chain variable domain and the first light chain variable domain and the third F(ab) (H1L2) containing the first heavy chain variable domain and the second light chain variable domain (6) The heterodimer (H1L1 / H2L1) containing the first F(ab) (H1L1) containing the first heavy chain variable domain and the first light chain variable domain and the fourth F(ab) (H2L1) containing the second heavy chain variable domain and the first light chain variable domain (7) The heterodimer (H2L2 / H1L2) containing the second F(ab) (H2L2) containing the second heavy chain variable domain and the second light chain variable domain and the third F(ab) (H1L2) containing the first heavy chain variable domain and the second light chain variable domain (8) The heterodimer (H2L2 / H2L1) containing the second F(ab) (H2L2) containing the second heavy chain variable domain and the second light chain variable domain and the fourth F(ab) (H2L1) containing the second heavy chain variable domain and the first light chain variable domain

[12] The method of

[11] , wherein the impurities (1)-(8) are removed from the composition containing the multispecific antigen-binding molecule by a purification step utilizing the difference in isoelectric point.​​​​​​​​

[13] The method according to any one of [1] to

[12] , wherein the multispecific antigen-binding molecule is a bispecific antibody.

[14] The bispecific antibody has amino acid residues at positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 39, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 81, 82b, 83, 85, 86, 97, 105, 108, 110, and 112 according to Kabat numbering in the heavy-chain variable domain, and at least one amino acid residue selected from amino acid residues at positions 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, and 435 according to EU numbering in the heavy-chain constant domain is modified, the method of

[13] .

[15] The method according to any one of [1] to

[14] , wherein the composition containing the multispecific antigen-binding molecule or bispecific antibody is a pharmaceutical composition.

[16] The bispecific antibody is an antibody comprising an F(ab) that binds to factor IX of blood coagulation and / or activated factor IX of blood coagulation, and an F(ab) that binds to factor X of blood coagulation, the method of

[13] or

[14] .

[17] A quality control method for a pharmaceutical composition containing a multispecific antigen-binding molecule, comprising: The multispecific antigen-binding molecule is a molecule (H1L1 / H2L2) comprising a first F(ab) (H1L1) containing a first heavy-chain variable domain and a first light-chain variable domain against a first antigen, and a second F(ab) (H2L2) containing a second heavy-chain variable domain and a second light-chain variable domain against a second antigen, The light-chain exchange molecule is a molecule (H1L2 / H2L1) comprising a third F(ab) (H1L2) containing a first heavy-chain variable domain and a second light-chain variable domain, and a fourth F(ab) (H2L1) containing a second heavy-chain variable domain and a first light-chain variable domain, The quality control method comprises the following steps 1 to 3. 1) Treating a composition containing a multispecific antigen-binding molecule to generate two or more F(ab) fragments 2) Measuring two or more F(ab) fragments by a separation method based on charge or hydrophobic interaction, and determining the content of light chain exchange molecules in the composition or the content ratio of light chain exchange molecules to the multispecific antigen-binding molecule 3) Confirming that the content or content ratio of the light chain exchange molecules determined in step 2 is below a preset allowable value of the content / content ratio

[18] A method for evaluating the quality of a pharmaceutical composition containing a multispecific antigen-binding molecule, comprising The multispecific antigen-binding molecule is a molecule (H1L1 / H2L2) comprising a first F(ab) (H1L1) containing a first heavy chain variable domain and a first light chain variable domain for a first antigen, and a second heavy chain variable domain for a second antigen and a second F(ab) (H2L2) containing a second light chain variable domain The light chain exchange molecule is a molecule (H1L2 / H2L1) comprising a third F(ab) (H1L2) containing a first heavy chain variable domain and a second light chain variable domain, and a fourth F(ab) (H2L1) containing a second heavy chain variable domain and a first light chain variable domain The quality evaluation method comprises the following steps 1 to 3 1) Treating a composition containing a multispecific antigen-binding molecule to generate two or more F(ab) fragments 2) Measuring two or more F(ab) fragments by a separation method based on charge or hydrophobic interaction, and determining the content of light chain exchange molecules in the composition or the content ratio of light chain exchange molecules to the multispecific antigen-binding molecule 3) Confirming that the content or content ratio of the light chain exchange molecules determined in step 2 is below a preset allowable value of the content / content ratio

[19] Step 1 comprises the following steps 1-1 and 1-2 of the method of

[17] or

[18] 1-1) Cleaving the molecules contained in the composition to generate F(ab)'2 fragments Step of cleaving the F(ab)’2 fragment to generate two or more F(ab) fragments

[20] Step 1-1 is the method of

[19] , which is a step of cleaving the Fc side of the hinge portion of the molecule contained in the composition with a protease.

[21] The method of

[20] , wherein the protease is any one of a bacterial antibody-degrading enzyme, pepsin, ficin, or a combination thereof.

[22] Step 1-2 is the method of any one of

[19] to

[21] , which is a step of cleaving the disulfide bond of the F(ab)’2 fragment with a reducing agent.

[23] The method of

[22] , wherein the reducing agent is any one of TCEP, 2-MEA, Cysteine, Dithiothreitol, 2-Mercaptoethanol, 3-mercapto-1,2-propanediol, TBP, or a combination thereof.

[24] Step 1 is a step of cleaving the F(ab) side of the hinge portion of the molecule contained in the composition with a protease by the method of

[17] or

[18] .

[25] The method of

[24] , wherein the protease is any one of a bacterial antibody-degrading enzyme, papain, Lys-C, or a combination thereof thereof.

[26] Step 2 is a step of measuring two or more F(ab) fragments by any one of cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography, or a combination thereof, by the method of any one of

[17] to

[25] .

[27] The method described in any one of

[17] to

[26] , wherein the two or more F(ab) fragments generated in Step 1 each have a different isoelectric point.

[28] The multispecific antigen-binding molecule (H1L1 / H2L2) is a molecule in which amino acid residues are modified so as to have a difference in isoelectric point from the following impurities (1) to (8), by the method of any one of

[17] to

[27] . (1) Of the first F(ab) (H1L1) containing the first heavy chain variable domain and the first light chain variable domain Homodimer (H1L1 / H1L1) (2) The homodimer (H2L2 / H2L2) of the second F(ab) (H2L2) containing the second heavy chain variable domain and the second light chain variable domain (3) The homodimer (H1L2 / H1L2) of the third F(ab) (H1L2) containing the first heavy chain variable domain and the second light chain variable domain (4) The homodimer (H2L1 / H2L1) of the fourth F(ab) (H2L1) containing the second heavy chain variable domain and the first light chain variable domain (5) The first F(ab) (H1L1) containing the first heavy chain variable domain and the first light chain variable domain and the heterodimer (H1L1 / H1L2) containing the third F(ab) (H1L2) containing the first heavy chain variable domain and the second light chain variable domain (6) The first F(ab) (H1L1) containing the first heavy chain variable domain and the first light chain variable domain and the heterodimer (H1L1 / H2L1) containing the fourth F(ab) (H2L1) containing the second heavy chain variable domain and the first light chain variable domain (7) The second F(ab) (H2L2) containing the second heavy chain variable domain and the second light chain variable domain and the heterodimer (H2L2 / H1L2) containing the third F(ab) (H1L2) containing the first heavy chain variable domain and the second light chain variable domain (8) The second F(ab) (H2L2) containing the second heavy chain variable domain and the second light chain variable domain and the heterodimer (H2L2 / H2L1) containing the fourth F(ab) (H2L1) containing the second heavy chain variable domain and the first light chain variable domain The method of

[28] , wherein the composition containing the multispecific antigen-binding molecule has impurities (1) to (8) removed by a purification step utilizing the difference in isoelectric point. (30) The method according to any one of

[17] to

[29] , wherein the multispecific antigen-binding molecule is a bispecific antibody. (31) The method according to any one of

[17] to

[29] , wherein the multispecific antigen-binding molecule is a bispecific antibody. (32) The method according to any one of

[17] to

[29] , wherein the multispecific antigen-binding molecule is a bispecific antibody. (33) The method according to any one of

[17] to

[29] , wherein the multispecific antigen-binding molecule is a bispecific antibody. (34) The method according to any one of

[17] to

[29] , wherein the multispecific antigen-binding molecule is a bispecific antibody.

[31] The bispecific antibody is an antibody in which at least one amino acid residue selected from the amino acid residues at positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 39, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 81, 82b, 83, 85, 86, 97, 105, 108, 110, and 112 according to Kabat numbering in the heavy chain variable domain, and the amino acid residues at positions 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, and 435 according to EU numbering in the heavy chain constant domain is modified, the method of

[30] .

[32] A composition containing a multispecific antigen-binding molecule or a bispecific antibody is a pharmaceutical composition, the method of any one of

[17] to

[31] .

[33] The bispecific antibody is an antibody containing an F(ab) that binds to factor IX of blood coagulation and / or activated factor IX of blood coagulation and an F(ab) that binds to factor X of blood coagulation, the method of

[30] or

[31] .

[34] A pharmaceutical bulk substance whose quality is ensured by any one of the quality control methods or quality evaluation methods of

[17] to

[33] . [Advantages of the Invention]

[0009] In a non-limiting aspect, the analysis method of the present disclosure can accurately measure the content of the light chain exchange body molecule directly from the measurement results by cation exchange chromatography (CEX) or the like, which is different from the conventional analysis method of obtaining an estimated value using theoretical calculations. According to the analysis method of the present disclosure, it has become possible to accurately quantify whether a light chain exchange molecule is contained as an impurity in a composition containing a multispecific antigen-binding molecule as a target molecule, and if so, its content (content ratio). The molecular form of the multispecific antigen-binding molecule to be analyzed is not limited, and in addition to bispecific antibodies, it is also applicable to bispecific F(ab)'2 fragments, scFvs, etc. Further, it is also possible to use a multispecific antigen-binding molecule having two different F(abs), one of which has binding specificity for two or more antigens.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

[0011] I. Definition Antigen-binding molecule The term "antigen-binding molecule" as used herein, in its broadest sense, refers to a molecule that specifically binds to an antigenic determinant (epitope). In one embodiment, the antigen-binding molecule is an antibody, an antibody fragment, or an antibody derivative. In one embodiment, the antigen-binding molecule is a non-antibody protein, or a fragment thereof, or a derivative thereof.

[0012] As used herein, the term "specifically binds" refers to a state in which one of the specifically binding molecules does not show any significant binding to molecules other than the one or more molecules to which it binds. The term is also used when an antigen-binding domain is specific to a specific epitope among multiple epitopes contained in an antigen. When the epitope to which the antigen-binding domain binds is contained in multiple different antigens, an antigen-binding molecule having the antigen-binding domain can bind to various antigens containing the epitope.

[0013] In the present disclosure, "binding to the same epitope" refers to binding between two antigen-binding domains. This means that the epitopes overlap at least partially. The degree of overlap is not limited, but is at least 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, particularly preferably 90% or more, and most preferably 100%.

[0014] Antibody The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0015] "Binding activity" refers to the total strength of non-covalent interactions between one or more binding sites of a molecule (e.g., an antibody) and the binding partner of the molecule (e.g., an antigen). Here, the binding activity is not strictly limited to a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). For example, when the members of a binding pair reflect a 1:1 interaction in a monovalent manner, the binding activity refers to the intrinsic binding affinity ("affinity"). When the members of a binding pair are capable of both monovalent and multivalent binding, the binding activity is the sum of these binding forces. The binding activity of molecule X for its partner Y can generally be represented by the dissociation constant (KD) or "amount of analyte bound per unit amount of ligand". The binding activity can be measured by conventional methods known in the art, including those described herein.

[0016] In one aspect, the antigen-binding molecules and antibodies analyzed by the methods of the present disclosure are tested for their antigen-binding activity by known methods such as ELISA, Western blot, etc. obtained.

[0017] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies that make up that population are identical and / or bind to the same epitope, except for variant antibodies that may arise (e.g., variant antibodies that include naturally occurring mutations, or variant antibodies that occur during the production of a monoclonal antibody preparation. Such variants are usually present in a small amount). In contrast to polyclonal antibody preparations, which typically include different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the characteristic of an antibody being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of an antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by various methods including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, methods that include using transgenic animals that contain all or part of the human immunoglobulin locus, and may be made by a variety of techniques, and such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0018] A "native antibody" refers to immunoglobulin molecules with various structures that occur naturally. For example, a native IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N terminus to the C-terminus, each light chain has a variable light chain domain or light chain variable domain, also called the VL, followed by one constant domain (CL). It has a variable region (VL) that is exposed, followed by a constant light chain (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0019] The terms "full-length antibody", "complete antibody", and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to the native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0020] As used herein, the term "antigen-binding domain" refers to a region that specifically binds to and is complementary to part or all of an antigen. As used herein, an antigen-binding molecule comprises an antigen-binding domain. When the molecular weight of an antigen is large, the antigen-binding domain can bind only to a specific portion of the antigen. The specific portion is called an epitope. In one aspect, the antigen-binding domain includes an antibody fragment that binds to a specific antigen. The antigen-binding domain can be provided by the "variable domain" of one or more antibodies. In one non-limiting aspect, the antigen-binding domain includes a variable light chain region (VL) and a variable heavy chain region (VH) of an antibody. Examples of such antigen-binding domains include "scFv (single chain Fv)", "single chain antibody", "Fv", "scFv2 (single chain Fv 2)", "Fab", or "Fab'", etc. In another aspect, the antigen-binding domain includes a non-antibody protein or a fragment thereof that binds to a specific antigen. In certain aspects, the antigen-binding domain includes a hinge region.

[0021] Variable domain The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in binding the antigen-binding molecule or antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (VH and VL, respectively) typically each have four conserved It has a similar structure, including a framework region (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) One VH or VL domain would be sufficient to confer antigen-binding specificity . Furthermore, an antibody that binds to a particular antigen may be isolated by screening a complementary library of VL or VH domains using the VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0022] As used herein, the term "hypervariable region" or "HVR" refers to each region of the variable domain of an antibody that is hypervariable in sequence ("complementarity determining region" or "CDR") and / or forms a structurally defined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact"). Typically, an antibody contains six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 24-36 (H1), 52-56 (H2), and 94-102 (H3) as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991); (b) CDRs occurring at amino acid residues 24 - 34 (L1), 50 - 56 (L2), 89 - 97 (L3), 31 - 35b (H1), 50 - 65 (H2), and 95 - 102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contacts occurring at amino acid residues 27c - 36 (L1), 46 - 55 (L2), 89 - 96 (L3), 30 - 35b (H1), 47 - 58 (H2), and 93 - 101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732 - 745 (1996)); and (d) Combinations of (a), (b), and / or (c) that include HVR amino acid residues 46 - 56 (L2), 47 - 56 (L2), 48 - 56 (L2), 49 - 56 (L2), 26 - 35 (H1) , 26 - 35b (H1), 49 - 65 (H2), 93 - 102 (H3), and 94 - 102 (H3). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al. supra. "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain typically consist of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of HVRs and FRs typically appear in VH (or VL) in the following order: FR1 - H1(L1) - FR2 - H2(L2) - FR3 - H3(L3) - FR4. Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al. supra.

[0023] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain typically consist of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of HVRs and FRs typically appear in VH (or VL) in the following order: FR1 - H1(L1) - FR2 - H2(L2) - FR3 - H3(L3) - FR4. "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain typically consist of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of HVRs and FRs typically appear in VH (or VL) in the following order: FR1 - H1(L1) - FR2 - H2(L2) - FR3 - H3(L3) - FR4.

[0024] Constant region The constant region as one embodiment of the present invention is preferably an antibody constant region, more preferably an antibody constant region of IgG1, IgG2, IgG3, or IgG4 type, and even more preferably an antibody constant region of human IgG1, IgG2, IgG3, or IgG4 type. Further, the constant region as another embodiment of the present invention is preferably a heavy chain constant region, more preferably a heavy chain constant region of IgG1, IgG2, IgG3, or IgG4 type, and even more preferably a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 type. The amino acid sequences of human IgG1 constant region, human IgG2 constant region, human IgG3 constant region, and human IgG4 constant region are known. As the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, multiple allotype sequences due to gene polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No.91 - 3242, but any of them may be used in the present invention. Note that the constant region with modified amino acids of the present invention may contain other amino acid mutations or modifications as long as it contains the amino acid mutations of the present invention. In the present invention, any of them may be used. The constant region with modified amino acids of the present invention may contain other amino acid mutations or modifications as long as it contains the amino acid mutations of the present invention.

[0025] The term "hinge region" refers to the region that connects the CH1 domain and the CH2 domain in the wild - type antibody heavy chain, for example, from around position 216 to around position 230 according to the EU numbering system, or from around position 226 to around position 243 according to the Kabat numbering system, of the antibody heavy chain polypeptide part. In native IgG antibodies, the cysteine residue at position 220 of the EU numbering in the hinge region is known to form a disulfide bond with the cysteine residue at position 214 in the antibody light chain. Further, between two antibody heavy chains, it is known that the cysteine residues at position 226 and the cysteine residues at position 229 of the EU numbering in the hinge region form disulfide bonds with each other. The hinge region in the present specification includes not only the wild - type but also variants in which amino acid residues are substituted, added, or deleted in the wild - type. In native IgG antibodies, the cysteine residue at position 220 of the EU numbering in the hinge region is known to form a disulfide bond with the cysteine residue at position 214 in the antibody light chain. Further, between two antibody heavy chains, it is known that the cysteine residues at position 226 and the cysteine residues at position 229 of the EU numbering in the hinge region form disulfide bonds with each other. The hinge region in the present specification includes not only the wild - type but also variants in which amino acid residues are substituted, added, or deleted in the wild - type.

[0026] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes the Fc region of a native sequence and mutant antibody Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region or the glycine-lysine (Gly446-Lys447) may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991, according to the EU numbering system (also referred to as the EU index).

[0027] Antibody fragments "Antibody fragment" and "antibody fragment" refer to molecules other than the complete antibody that include a portion of the complete antibody that binds to an antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); single-chain Fab (scFab); single-domain antibodies; and multispecific antibodies formed from antibody fragments.

[0028] Fv (variable fragment) As used herein, the term "Fv (variable fragment)" refers to the variable region of the light chain of an antibody ​​It refers to the minimum unit of an antigen-binding domain derived from an antibody, which consists of a pair of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) of the antibody. In 1988, Skerra and Pluckthun inserted the antibody gene downstream of the bacterial signal sequence and found that by inducing the expression of the gene in Escherichia coli, it could be prepared from the periplasmic fraction of Escherichia coli in a homogeneous and active state (Science (1988) 240 (4855), 1038-1041). The Fv prepared from the periplasmic fraction had VH and VL associated in a manner that had binding to the antigen.

[0029] scFv, single-chain antibody, or sc(Fv)2 In this specification, the terms "scFv", "single-chain antibody", or "sc(Fv)2" mean an antibody fragment that contains variable regions derived from both the heavy chain and the light chain within a single polypeptide chain but lacks the constant region. Generally, a single-chain antibody further includes a polypeptide linker between the VH domain and the VL domain that enables it to form a desired structure that is thought to allow antigen binding. Single-chain antibodies are discussed in detail by Pluckthun in The Pharmacology of Monoclonal Antibodies, Volume 113, edited by Rosenberg and Moore, Springer-Verlag, New York, 269-315 (1994). Similarly, see International Patent Application Publication WO1988 / 001649 and U.S. Patent Nos. 4,946,778 and 5,260,203. In certain embodiments, a single-chain antibody can also be bispecific and / or humanized.

[0030] scFv is an antigen-binding domain in which VH and VL that constitute Fv are linked by a peptide linker (Proc. Natl. Acad. Sci. U.S.A. (1988) 85 (16), 5879-5883). The peptide The VH and VL can be held in a proximate state by a linker.

[0031] sc(Fv)2 is a single-chain antibody in which four variable regions of two VLs and two VHs are linked by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231 (1-2), 177-189). These two VHs and VLs may be derived from different monoclonal antibodies. For example, bispecific sc(Fv)2 that recognizes two epitopes present in the same antigen as disclosed in Journal of Immunology (1994) 152 (11), 5368-5374 is also preferably mentioned. sc(Fv)2 can be produced by methods known to those skilled in the art. For example, it can be produced by linking scFv with a linker such as a peptide linker.

[0032] As the constitution of the antigen-binding domains constituting sc(Fv)2 in the present specification, two VHs and two VLs are arranged in the order of VH, VL, VH, VL ([VH] linker -[VL] linker [VH] linker [VL]) starting from the N-terminal side of a single-chain polypeptide. However, the order of the two VHs and two VLs is not particularly limited to the above constitution, and they may be arranged in any order. For example, the following order constitutions can also be mentioned. [VL] linker [VH] linker [VH] linker [VL] [VH] linker [VL] linker [VL] linker [VH] [VH] linker [VH] linker [VL] linker [VL] [VL] linker [VL] linker [VH] linker [VH] [VL] linker [VH] linker [VL] linker [VH]

[0033] F(ab) As used herein, "F(ab)" (also referred to as Fab or Fab') consists of a light chain composed of VL (variable light region) and CL (constant light region), and a portion of the heavy chain composed of VH (variable heavy region) and CH1 (γ1 region in the constant heavy region), and the light chain and the said portion of the heavy chain can be structured such that they are bound by a disulfide bond at the C-terminal region. Also, F(ab) may include a part of the hinge region. F(ab) as an antibody fragment may be simply referred to as F(ab) or as an F(ab) fragment. Similarly, F(ab) as a part of an antigen-binding molecule (e.g., an antibody) is simply referred to as F(ab), but is also referred to as an F(ab) part or an F(ab) region to distinguish it from an F(ab) fragment. As used herein, F(ab) may or may not have antigen-binding activity. In an exemplary aspect, the two F(abs) contained in a multispecific antigen-binding molecule (H1L1 / H2L2) both have antigen-binding activity. In an exemplary aspect, the antigen-binding activity of one or both of the two F(abs) contained in a light chain exchange molecule (H1L2 / H2L1) of a multispecific antigen-binding molecule is weaker than the antigen-binding activity of the two F(abs) contained in the multispecific antigen-binding molecule. In certain aspects, one or both of the two F(abs) contained in a light chain exchange molecule of a multispecific antigen-binding molecule do not have antigen-binding activity.

[0034] F(ab’)2 As used herein, "F(ab’)2" (also referred to as F(ab)'2) refers to an antibody fragment having a structure in which two F(ab)s are linked (also referred to as an F(ab’)2 fragment) or a portion in an antigen-binding molecule (also referred to as an F(ab’)2 portion or F(ab’)2 region). The linkage of the two F(ab)s in F(ab’)2 is, for example, a disulfide bond in the immunoglobulin hinge region, but is not limited thereto. In one aspect, F(ab’)2 includes a portion of the hinge region. In one aspect, F(ab’)2 includes two light chains and two heavy chain portions in which disulfide bonds are formed between the two heavy chain portions so as to include a constant region of a portion of the CH1 domain and the CH2 domain. Included are two heavy chain portions including a constant region of a portion of the CH1 domain and the CH2 domain such that disulfide bonds are formed. As used herein, F(ab’)2 may or may not have antigen-binding activity. In an exemplary aspect, any F(ab’)2 included in a multispecific antigen-binding molecule has antigen-binding activity. In an exemplary aspect, the antigen-binding activity of F(ab’)2 included in a light chain exchange molecule of a multispecific antigen-binding molecule is weaker than the antigen-binding activity of F(ab’)2 included in the multispecific antigen-binding molecule. In certain aspects, F(ab’)2 included in a light chain exchange molecule of a bispecific antibody does not have antigen-binding activity.

[0035] Multispecific antigen-binding molecule In certain aspects, the antigen-binding molecules provided herein are multispecific antigen-binding molecules (e.g., bispecific antigen-binding molecules). A multispecific antigen-binding molecule is a monoclonal antigen-binding molecule having binding specificities for at least two different sites. In certain aspects, one of the binding specificities is for a particular antigen (e.g., Factor IX blood coagulation (FIX), epiregulin (EREG), IL-6 receptor (IL-6R), etc.) and the other is for any other antigen (e.g., Factor X blood coagulation (FX), GPC3, KLH, etc.). In certain aspects, the multispecific antigen-binding molecule may bind to two different epitopes on one antigen. Also, in certain aspects, the multispecific antigen-binding molecule has two or more ​ It contains one antigen-binding site having binding specificity for different antigens. As an example, a multi- specific antigen-binding molecule includes a first antigen-binding site for two or more different antigens and a second antigen-binding site for one antigen. The multi-specific antigen-binding molecule has two different antigen-binding sites and has binding specificity for three or more different antigens. A multi- specific antigen-binding molecule can be prepared as a full-length antibody or as an antibody fragment.

[0036] Methods for making multispecific antigen-binding molecules include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and knob-in-hole technology (see, e.g., U.S. Patent No. 5,731,168). Multispecific antigen-binding molecules can be made by manipulating electrostatic steering effects to create Fc heterodimeric molecules (WO2009 / 089004A1); cross-linking two or more antibodies or fragments (see U.S. Patent No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); creating antibodies having two specificities using leucine zippers (see Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); making bispecific antibody fragments using "diabody" technology (see Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991). They may also be made by preparing.

[0037] Generation of F(ab) fragment, F(ab’)2 fragment by treatment of the composition In certain embodiments, the compositions provided herein comprising the multispecific antigen-binding molecules are treated to generate F(ab) fragments. In such embodiments, the composition is "treated" or "To process" means to subject the composition to a treatment to cleave the multispecific antigen-binding molecule and / or the light chain exchange body molecule to generate F(ab), which can be carried out by methods known to those skilled in the art. "To cleave" a molecule refers to, for example, separating the bonds between amino acid residues or disulfide bonds in the molecule. As a method of treatment, for example, a protease can be added to the composition to cleave the multispecific antigen-binding molecule and / or the light chain exchange body molecule. The proteases include antibody-degrading enzymes derived from bacteria typified by GingisKHAN (registered trademark) (Kgp), FabRICATOR (registered trademark) (IdeS), FabRICATOR (registered trademark) Z (IdeZ), FabALACTICA (registered trademark) (IgdE), FabULOUS (registered trademark) (SpeB) (all from Genovis), as well as pepsin, papain, Lys-C, etc. can be used. By protease treatment, it is also possible to cleave the Fc side of the hinge region of the multispecific antigen-binding molecule and / or the light chain exchange body molecule, or to cleave the F(ab) side. When cleaving the F(ab) side, an F(ab) fragment is generated by the cleavage (see Figure 7). After generating the F(ab) fragment in this way, the F(ab) fragment can be obtained by adsorbing and removing the Fc fragment on a protein A column. On the other hand, when cleaving the Fc side, an F(ab')2 fragment is generated (see Figure 6). The F(ab')2 fragment can also be obtained by reacting the antibody with ficin under conditions for generating the F(ab')2 fragment. After partially digesting the antigen-binding molecule (e.g., a complete monoclonal antibody) with these proteases, the F(ab')2 fragment can be obtained by adsorbing and removing the Fc fragment on a protein A column. Next, the disulfide bond of the F(ab')2 fragment can be cleaved with a reducing agent to generate an F(ab) fragment (also called a Fab' fragment) (see Figure 6). The starting agent can use TCEP, 2-MEA, Cysteine, Dithiothreitol, 2-Mercaptoethanol, 3-mercapto-1,2-propanediol, TBP, etc.

[0038] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell (including the progeny of such a cell) into which foreign nucleic acid has been introduced. Host cells include "transformants" and "transformed cells", which include the original transformed cells and progeny derived from such cells regardless of the number of passages. The progeny may not be completely identical to the parent cell in terms of nucleic acid content and may contain mutations. Mutant progeny having the same function or biological activity as that used when the original transformed cell was screened or selected are also included herein.

[0039] As used herein, the term "vector" refers to a nucleic acid molecule that can increase another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures and vectors that are incorporated into the genome of the host cell into which they are introduced. A certain vector can result in the expression of a nucleic acid operably linked thereto. Such vectors are also referred to herein as "expression vectors".

[0040] Recombinant methods and constructs For example, as described in U.S. Patent No. 4,816,567, antigen-binding molecules can be produced using recombinant methods or constructs. In non-limiting examples of such production, an isolated nucleic acid encoding an antigen-binding molecule is used. Such nucleic acid may encode an amino acid sequence containing the VL of the antigen-binding molecule and / or an amino acid sequence containing the VH (e.g., the light chain and / or heavy chain of the antigen-binding molecule). One or more vectors containing such nucleic acid and the like. A vector (e.g., an expression vector) may be used. In an exemplary embodiment, a host cell containing such a nucleic acid or vector is used. In one such embodiment, the host cell contains a vector encoding a nucleic acid encoding an amino acid sequence comprising the VL of the antigen-binding molecule and an amino acid sequence comprising the VH of the antigen-binding molecule, or (2) a first vector containing a nucleic acid encoding an amino acid sequence comprising the VL of the antigen-binding molecule and a second vector containing a nucleic acid encoding an amino acid sequence comprising the VH of the antigen-binding molecule (e.g., is transformed). In one embodiment, the host cell is a eukaryotic cell (e.g., Chinese hamster ovary (CHO) cells) or lymphoid cells (e.g., Y0, NS0, Sp2 / 0 cells)). In one embodiment, provided is a method for producing an antigen-binding molecule, which includes culturing a host cell containing a nucleic acid encoding the antigen-binding molecule as described above under conditions suitable for the expression of the antigen-binding molecule, and optionally recovering the antigen-binding molecule from the host cell (or the host cell culture medium). For the recombinant production of an antigen-binding molecule, a nucleic acid encoding the antigen-binding molecule is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids will be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antigen-binding molecule).

[0041] Host cells suitable for cloning or expressing a vector encoding an antigen-binding molecule include the prokaryotic or eukaryotic cells described herein. For example, the antigen-binding molecule may be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (In addition, the expression of antibody fragments in E. coli is also described in these patents.)

[0042] in these patents.) See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp.245-254, which is hereby incorporated by reference in its entirety. After expression, the antigen-binding molecule may be isolated from the bacterial cell paste into the soluble fraction and further purified.

[0043] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts, including strains of fungi and yeast in which the glycosylation pathway has been "humanized" to result in the production of antigen-binding molecules with a partial or complete human glycosylation pattern, are suitable cloning or expression hosts for antigen-binding molecule-encoding vectors. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0044] Those derived from multicellular organisms (invertebrates and vertebrates) are also suitable host cells for the expression of glycosylated antigen-binding molecules. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjunction with insect cells, particularly for the transformation of Spodoptera frugiperda cells.

[0045] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429, which describe PLANTIBODIES™ technology for producing antigen-binding molecules in transgenic plants.

[0046] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension would be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 line transformed with SV40 (COS-7); human fetal kidney lines (293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977), etc.); baby hamster kidney cells (BHK); mouse cells avian cells (TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980), etc.); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); dog kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC5 cells; and FS4 cells, etc. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for the production of antigen-binding molecules, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In this specification, a first F(ab) comprising or consisting of a first heavy chain variable domain (VH contained in H1) and a first light chain variable domain (VL contained in L1) is designated H1L1, a second heavy chain variable do

[0047] Multispecific antigen-binding molecule (H1L1 / H2L2) and its light-chain exchange molecule (H1L2 / H2L1) ​ A second F(ab) comprising or consisting of a main (VH contained in H2) and a second light chain variable domain (VL contained in L2) is called H2L2, and a multispecific antigen-binding molecule comprising H1L1 and H2L2 is called H1L1 / H2L2. Further, a third F(ab) comprising or consisting of a first heavy chain variable domain (VH contained in H1) and a second light chain variable domain (VL contained in L2) is called H1L2, and the second heavy chain variable domain (VH contained in H2) and a fourth F(ab) comprising or consisting of a first light chain variable domain (VL contained in L1) is called H2L1, and a molecule comprising H1L2 and H2L1, that is, a light chain exchange molecule of H1L1 / H2L2, is called H1L2 / H2L1. Therefore, the light chain exchange molecule in the present specification is an impurity molecule generated by inversion (mispairing) of the combination of two different heavy chains and two different light chains constituting the target multispecific antigen-binding molecule. In one non-limiting aspect, the light chain exchange molecule H1L2 / H2L1 may be contained as an impurity in a composition comprising the multispecific antigen-binding molecule H1L1 / H2L2. In one aspect, the multispecific antigen-binding molecule H1L1 / H2L2 comprises a heavy chain portion (H1) comprising or consisting of a first heavy chain variable (VH) domain and a CH1 domain, and a light chain portion (L1) comprising or consisting of a first light chain variable (VL) domain and a CL domain, or consisting of these, a first F(ab) (called H1L1), and a second heavy chain variable (VH) domain and a CH1 domain, or a heavy chain portion (H2) consisting of these, and a second light chain variable (VL ) domain and a light chain portion (L2) comprising or consisting of a CL domain, or a second F(ab) (called H2L2) consisting of these. In one aspect, H1L2 / H2L1, which is a light chain exchange molecule of the multispecific antigen-binding molecule H1L1 / H2L2, comprises a heavy A third F(ab) (referred to as H1L2) comprising or consisting of a locking portion (H1) and a light chain portion (L2) comprising or consisting of a second variable light (VL) domain and a CL domain, and a heavy chain portion (H2) comprising or consisting of a second variable heavy (VH) domain and a CH1 domain, and a fourth F(ab) (referred to as H2L1) comprising or consisting of a light chain portion (L1) comprising or consisting of a first variable light (VL) domain and a CL domain. The first F(ab), H1L1, and the second F(ab), H2L2, may or may not each have antigen-binding activity. In a preferred exemplary embodiment, both H1L1 and H2L2 have antigen-binding activity. The third F(ab), H1L2, and the fourth F(ab), H2L1, may or may not each have antigen-binding activity. In a non-limiting embodiment, one or both of H1L2 and H2L1 have antigen-binding activity. In an exemplary embodiment, the antigen-binding activity of one or both of H1L2 and H2L1 is weaker than the antigen-binding activity of one or both of H1L1 and H2L2. In certain embodiments, one or both of H1L2 and H2L1 do not have antigen-binding activity.

[0048] Analysis method of a composition containing a multispecific antigen-binding molecule and a light-chain exchange molecule In one aspect, the present disclosure provides a method for analyzing impurity molecules (e.g., light chain exchange molecules) in a composition comprising a multispecific antigen-binding molecule. In one embodiment of this aspect, the present disclosure provides a method for detecting impurity molecules (e.g., light chain exchange molecules) in a composition comprising a multispecific antigen-binding molecule. In another embodiment, the present disclosure provides a method for measuring the content and / or content ratio of impurity molecules (e.g., light chain exchange molecules) in a composition comprising a multispecific antigen-binding molecule. In another aspect, the present disclosure provides a method for analyzing a multispecific antigen-binding molecule and an impurity molecule (e.g., a light chain exchange molecule) in a composition comprising the multispecific antigen-binding molecule and the impurity molecule. In one embodiment of this aspect, the present disclosure provides a method for detecting an impurity molecule in a composition comprising a multispecific antigen-binding molecule and an impurity molecule (e.g., a light chain exchange molecule). In one embodiment of this aspect, the present disclosure provides a method for measuring the content and / or content ratio of a multispecific antigen-binding molecule and an impurity molecule (e.g., a light chain exchange molecule) in a composition comprising the multispecific antigen-binding molecule and the impurity molecule. In another embodiment, the present disclosure provides a method for measuring the content and / or content ratio of a multispecific antigen-binding molecule in a composition comprising the multispecific antigen-binding molecule and an impurity molecule (e.g., a light chain exchange molecule). In another embodiment, the present disclosure provides a method for measuring the purity of a multispecific antigen-binding molecule in a composition comprising the multispecific antigen-binding molecule and an impurity molecule (e.g., a light chain exchange molecule).

[0049] In one embodiment, the method of the present disclosure comprises the following steps 1 to 2: 1) A step of treating a composition containing a multispecific antigen-binding molecule to generate a plurality of F(ab) fragments; and 2) A step of measuring the plurality of F(ab) fragments by a separation method based on charge or hydrophobic interaction to determine the content of the light chain exchange molecule in the composition or the content ratio of the light chain exchange molecule to the multispecific antigen-binding molecule.

[0050] In a specific embodiment, step 1 is a step of cleaving the F(ab) side of the hinge portion of the molecule contained in the composition with a protease. In a specific embodiment, step 1 comprises treating with a protease that cleaves the F(ab) region side of the immunoglobulin hinge region (the hinge portion of the antigen-binding molecule) under conditions for generating F(ab) fragments to generate F(ab) fragments. ​No. Non-limiting examples of such proteases include papain, Lys-C, GingisKHAN (registered trademark) (Genovis), FabALACTICA (registered trademark) (Genovis), and ficin. can be mentioned.

[0051] In another specific embodiment, step 1 includes the following steps 1-1 and 1-2: 1-1) a step of cleaving the molecule contained in the composition to generate an F(ab)'2 fragment; and 1-2) a step of cleaving the F(ab)'2 fragment to generate two or more F(ab) fragments. In a specific embodiment, step 1-1 is a step of cleaving the Fc side of the hinge portion of the molecule contained in the composition with a protease. In a specific embodiment, step 1-1 includes treatment with a protease that cleaves the Fc region side of the immunoglobulin hinge region (the hinge portion of the antigen-binding molecule) under conditions for generating an F(ab)'2 fragment. Non-limiting examples of such proteases include pepsin, FabRICATOR (registered trademark) (Genovis), FabRICATOR (registered trademark) Z (Genovis), FabULOUS (registered trademark) (Genovis), and ficin. In a specific embodiment, step 1-2 is a step of cleaving the disulfide bond of the F(ab)'2 fragment with a reducing agent. Non-limiting examples of such reducing agents include TCEP, 2-MEA , Cysteine, Dithiothreitol, 2-Mercaptoethanol, 3-mercapto-1,2-propanediol, and TBP. In one embodiment, the treatment with the reducing agent is performed under conditions for cleaving the disulfide bond in the hinge portion (immunoglobulin hinge region) of the F(ab)'2 fragment, and such conditions are known to those skilled in the art.

[0052] In certain embodiments, Step 2 can be performed by a separation method based on charge or hydrophobic interaction, which is known to those skilled in the art. Such separation methods include, for example, cation exchange (CEX ) chromatography, anion exchange (AEX) chromatography, hydrophobic interaction chrom atography (HIC), and reverse-phase chromatography, but are not limited thereto.

[0053] In certain embodiments, the two or more F(ab) fragments generated in Step 1 each have a different isoelectric point. In one embodiment, at least one amino acid residue constituting the F(ab) fragments of H1L1, H2L2, H1L2, and H2L1 is modified so that there are differences in the isoelectric points of the respective F(ab) fragments.

[0054] In certain embodiments, the multispecific antigen-binding molecule (H1L1 / H2L2) has the following impurities (1) to (8): (1) The homodimer (H1L1 / H1L1) of the first F(ab) (H1L1) containing the first heavy-chain variable domain and the first light-chain variable domain (2) The homodimer (H2L2 / H2L2) of the second F(ab) (H2L2) containing the second heavy-chain variable domain and the second light-chain variable domain (3) The homodimer (H1L2 / H1L2) of the third F(ab) (H1L2) containing the first heavy-chain variable domain and the second light-chain variable domain (4) The homodimer (H2L1 / H2L1) of the fourth F(ab) (H2L1) containing the second heavy-chain variable domain and the first light-chain variable domain (5) The first F(ab) (H1L1) containing the first heavy-chain variable domain and the first light-chain variable domain and the third F(ab) (H1L2) containing the first heavy-chain variable domain and the second light-chain variable domain Heterodimer (H1L1 / H1L2) (6) A first F(ab) (H1L1) comprising a first heavy chain variable domain and a first light chain variable domain and a fourth F(ab) (H2L1) comprising a second heavy chain variable domain and a first light chain variable domain. Heterodimer (H1L1 / H2L1) (7) A second F(ab) (H2L2) comprising a second heavy chain variable domain and a second light chain variable domain and a third F(ab) (H1L2) comprising a first heavy chain variable domain and a second light chain variable domain. Heterodimer (H2L2 / H1L2) (8) A second F(ab) (H2L2) comprising a second heavy chain variable domain and a second light chain variable domain and a fourth F(ab) (H2L1) comprising a second heavy chain variable domain and a first light chain variable domain. Heterodimer (H2L2 / H2L1) It is a molecule in which amino acid residues are modified so as to provide a difference in isoelectric point among them. In certain embodiments, the composition comprising the multispecific antigen-binding molecule has impurities (1)-(8) removed by a purification process utilizing the difference in isoelectric point.

[0055] In certain embodiments, the multispecific antigen-binding molecule is a bispecific antibody. In one embodiment, the bispecific antibody comprises first and second heavy chains having a pI (isoelectric point) difference. Such heavy chains comprise a heavy chain variable domain having a pI difference and / or a heavy chain constant domain having a pI difference. Examples of the heavy chain constant domain having a pI difference include the heavy chain constant regions of antibodies having a pI difference. The pI difference can be introduced into the first and second heavy chains by using the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4, which originally have a pI difference. Alternatively, a non-wild-type human constant region can be created by simultaneously modifying only the amino acids responsible for the isoelectric point difference between these subclasses, or adjacent amino acids that do not affect their isoelectric points, in the heavy chain constant regions of the first and second heavy chains, and a pI difference can be introduced into the two constant regions. Examples of the positions for modification to introduce a pI difference into the constant region include positions 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392 in the EU numbering of the heavy chain constant region. In addition, since a pI difference is generated by removing the sugar chain of the heavy chain constant region, position 297 of the sugar chain addition site is also listed as a position for modification to introduce a pI difference in the constant region. Similarly, for the heavy chain variable domain, the amino acid residues at specific positions in the heavy chain variable domains of the first and second heavy chains can be modified to introduce a pI difference between the two heavy chain variable domains. Examples of the heavy chain variable domain having such a pI difference include those in which the amino acid residue at a specific position in the first heavy chain variable domain has a charge, and the amino acid residue at the corresponding position in the second heavy chain variable domain has a charge opposite to that of the amino acid residue at the corresponding position in the first heavy chain variable domain, or has no charge. In certain embodiments, the bispecific antibody has Kabat numbering at positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 39, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 81, 82b An antibody in which at least one amino acid residue selected from the amino acid residues at positions 83, 85, 86, 97, 105, 108, 110, and 112, and the amino acid residues at positions 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, and 435 according to EU numbering in the heavy chain constant domain is modified. In certain embodiments, the bispecific antibody is an antibody comprising an F(ab) that binds to factor IX of blood coagulation and / or activated factor IX of blood coagulation, and an F(ab) that binds to factor X of blood coagulation.

[0056] In certain embodiments, a composition comprising a multispecific antigen-binding molecule or a bispecific antibody is a pharmaceutical composition.

[0057] In certain embodiments, the method of the present disclosure is a quality control method (or quality evaluation method) of a pharmaceutical composition, which includes the following steps 1 to 3. 1) A step of treating a composition containing a multispecific antigen-binding molecule to generate a plurality of F(ab) fragments ; 2) Measuring the plurality of F(ab) fragments by a separation method based on charge or hydrophobic interaction, and determining the content of light chain exchange body molecules in the composition or the content ratio of light chain exchange body molecules to the multispecific antigen-binding molecule; and 3) A step of confirming that the content or content ratio of the light chain exchange body molecules obtained in step 2 is not more than the allowable value of the content / content ratio of impurity molecules preset.

[0058] In certain embodiments, the present disclosure provides a pharmaceutical active ingredient with guaranteed quality by the above quality control method (or quality evaluation method).

[0059] All prior art documents cited in this specification are incorporated herein by reference.

Examples

[0060] ​Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited to these examples.

[0061] <Multispecific antigen-binding molecule and F(ab) of its light chain exchange molecule> In this example, a first heavy chain portion (H1) containing a first heavy chain variable (VH) domain and a CH1 domain, and a light chain portion (L1) containing a first light chain variable (VL) domain and a CL domain are included. The first F(ab) is called H1L1, and a second F(ab) containing a heavy chain portion (H2) containing a second heavy chain variable (VH) domain and a CH1 domain, and a light chain portion (L2) containing a second light chain variable (VL) domain and a CL domain is called H2L2. A homodimer containing only H1L1 in the Fab portion is designated as H1L1 / H1L1, and a homodimer containing only H2L2 is designated as H2L2 / H2L2. Also, a multispecific antigen-binding molecule containing H1L1 and H2L2 in the Fab portion is designated as H1L1 / H2L2. Further, a third F(ab) containing a heavy chain portion (H1) containing a first heavy chain variable (VH) domain and a CH1 domain, and a light chain portion (L2) containing a second light chain variable (VL) domain and a CL domain is designated as H1L2, and a fourth F(ab) containing a heavy chain portion (H2) containing a second heavy chain variable (VH) domain and a CH1 domain, and a light chain portion (L1) containing a first light chain variable (VL) domain and a CL domain is called H2L1. A homodimer containing only H1L2 in the Fab portion is designated as H1L2 / H1L2 and a homodimer containing only H2L1 is designated as H2L1 / H2L1. Also, a molecule containing H1L2 and H2L1 in the Fab portion, that is, a light chain exchange molecule of the multispecific antigen-binding molecule H1L1 / H2L2, is called H1L2 / H2L1. The amino acid sequences of each homodimer and multispecific antigen-binding molecule and light chain exchange molecule used in this example are shown in Table 1 below.

[0062]

Table 1

[0063] <Example 1> Separation of F(ab) fragments contained in anti-FIX(a) / anti-FX bispecific antibody and its impurity molecules (light chain exchange body molecules) Separation of F(ab) fragments Each homodimer consisting of the heavy and light chains of the anti-FIX(a) antibody and anti-FX antibody described in Table 1 The expression vector encoding them was introduced into HEK cells, cultured for 4 days, and then the culture supernatant was collected. Purification was performed using a Protein A column, and samples containing homodimers H1L1 / H1L1, H2L2 / H2L2, H1L2 / H1L2 , and H2L1 / H2L1 were prepared. Then, FabRICATOR (Genovis) was added to the sample and enzymatic treatment was performed. Furthermore, TCEP (Thermo Fisher Scientific) was added to a final concentration of 5 mM, and a sample containing F(ab) fragments of H1L1, H2L2, H1L2, and H2L1 was prepared. . For this sample, CEX analysis was performed according to the method shown in Example 5. The results are shown in Figure 1. Peaks derived from each F(ab) fragment were observed at different elution times. That is, H1L1, H2L2, H1L2, and H2L1, which are F(ab) domains contained in the anti-FIX(a) / anti-FX bispecific antibody and the light chain exchange body molecule, were eluted at different retention times.

[0064] <Example 2> Separation of F(ab) fragments contained in anti-EREG / anti-GPC3 bispecific antibody and its impurity molecules (light chain exchange body molecules) Each homoantibody consisting of the heavy and light chains of the anti-EREG antibody and anti-GPC3 antibody described in Table 1 The expression vector encoding them was introduced into HEK cells, cultured for 4 days, and then the culture supernatant was collected. Purification was performed using a Protein A column, and samples containing homodimers H1L1 / H1L1, H2L2 / H2L2, H1L2 / H1L2, and H2L1 / H2L1 were prepared. Then, GingisKHAN (Genovis) was added to the sample, and a sample containing F(ab) fragments of H1L1, H2L2, H1L2, and H2L1 was prepared. For this sample, CEX analysis was performed according to the method shown in Example 5. The results are shown in Figure 2. Peaks derived from each F(ab) fragment were observed at different elution times. That is, H1L1, H2L2, H1L2, and H2L1, which are F(ab) domains contained in the anti-EREG / anti-GPC3 bispecific antibody and the light chain exchange molecule, were eluted at different retention times.

[0065] <Example 3> Separation of F(ab) fragments contained in an anti-IL-6R / anti-KLH bispecific antibody and its impurity molecules (light chain exchange molecules) Each homobody consisting of the heavy and light chains of the anti-IL-6R antibody and the anti-KLH antibody described in Table 1 The expression vectors encoding them were introduced into HEK cells, cultured for 4 days, and then the culture supernatant was collected. Purification was performed using a Protein A column to prepare samples containing homodimers H1L1 / H1L1, H2L2 / H2L2, H1L2 / H1L2, and H2L1 / H2L1. Then, GingisKHAN (Genovis) was added to the sample to prepare a sample containing F(ab) fragments of H1L1, H2L2, H1L2, and H2L1. For this sample, CEX analysis was performed according to the method shown in Example 5. The results are shown in Figure 3. Peaks derived from each F(ab) fragment were observed at different elution times. That is, H1L1, H2L2, H1L2, and H2L1, which are F(ab) domains contained in the anti-IL-6R / anti-KLH bispecific antibody and the light chain exchange molecule, were eluted at different retention times.

[0066] <Example 4> Quantification of the content ratio of impurity molecules in a mixed sample of an anti-IL-6R / anti-KLH bispecific antibody and its impurity molecules (light chain exchange molecules) The expression vectors encoding the anti-IL-6R / anti-KLH bispecific antibody (H1L1 / H2L2) and its light chain exchange body molecule (H1L2 / H2L1) described in Table 1 were respectively introduced into HEK cells and cultured for 4 days. Subsequently, the culture supernatant was recovered. Purification was performed using a Protein A column, and anti-IL-6R / anti-KLH bispecific antibody H1L1 / H2L2 and light chain exchanger molecule H1L2 / H2L1 were prepared. These were mixed so that the mass ratio of the bispecific antibody to the light chain exchanger molecule was 90:10 to prepare a sample. GingisKHAN (Genovis) was added to this sample, and a sample containing F(ab) fragments of H1L1, H2L2, H1L2, and H2L1 decomposed from the bispecific antibody and the light chain exchanger molecule was prepared. For this sample, CEX analysis was performed according to the method shown in Example 5. The results are shown in Fig. 4 . Peaks derived from each F(ab) fragment of H1L1, H2L2, H1L2, and H2L1 were observed. The value obtained by dividing the peak area values of H1L2 and H2L1, which are derived from impurity molecules, by the total peak area value, that is, (peak area of H1L2 + peak area of H2L1) / (peak area of H1L1 + peak area of H2L2 + peak area of H1L2 + peak area of H2L1) was calculated according to the formula, and the content ratio of the impurity molecules was calculated to be 10%.

[0067] <Example 5> CEX Analysis of Samples Containing F(ab) Analysis was performed using a Prominence (Shimadzu Corporation) apparatus. 20 mM MES-NaOH, pH 5.5 was used as mobile phase A, and 20 mM MES-NaOH, 500 mM NaCl, pH 5.5 was used as mobile phase B. A ProPAC WCX-10 4×250 mm (Thermo Fisher Scientific) column was attached and equilibrated under the conditions of a flow rate of 0.5 mL / min, B 1%, and a column temperature of 25°C. 10 μg of each prepared sample containing F(ab) was injected into the column. Cation exchange chromatography (CEX) was performed by increasing B% at a rate of 1% / min while feeding at a flow rate of 0.5 mL / min . Peaks The detection was performed using the absorbance value at 280 nm.

Industrial Applicability

[0068] The method of the present disclosure can accurately measure the content or content ratio of impurity molecules (for example, light chain exchange body molecules) in a composition containing a multispecific antigen-binding molecule, and is useful compared to conventional analysis methods that only obtain estimated values using theoretical calculations.

Claims

1. A method for analyzing a light chain exchange molecule in a composition comprising a multispecific antigen-binding molecule, comprising: The multispecific antigen-binding molecule is a molecule (H1L1 / H2L2) comprising a first F(ab) (H1L1) comprising a first heavy chain variable domain and a first light chain variable domain for a first antigen, and a second F(ab) (H2L2) comprising a second heavy chain variable domain and a second light chain variable domain for a second antigen; The light chain exchange molecule is a molecule (H1L2 / H2L1) comprising a third F(ab) (H1L2) comprising a first heavy chain variable domain and a second light chain variable domain, and a fourth F(ab) (H2L1) comprising a second heavy chain variable domain and a first light chain variable domain; The analysis method is a method comprising the following steps 1 to 2. 1) A step of treating a composition comprising a multispecific antigen-binding molecule to generate two or more F(ab) fragments 2) Measuring two or more F(ab) fragments by a separation method based on charge or hydrophobic interaction, and directly determining the content of the light chain exchange molecule in the composition or the content ratio of the light chain exchange molecule to the multispecific antigen-binding molecule from the results of the measurement using the absorbance value

2. The method according to claim 1, wherein step 1 comprises the following steps 1-1 and 1-2. 1-1) A step of cleaving the molecule contained in the composition to generate an F(ab)'2 fragment 1-2) A step of cleaving the F(ab)'2 fragment to generate two or more F(ab) fragments

3. The method according to claim 2, wherein step 1-1 is a step of cleaving the Fc side of the hinge portion of the molecule contained in the composition with a protease.

4. The method according to claim 3, wherein the protease is any one of a bacterial-derived antibody-degrading enzyme, pepsin, ficin, or a combination thereof.

5. The method according to any one of claims 2 to 4, wherein step 1-2 is a step of cleaving the disulfide bond of the F(ab)'2 fragment with a reducing agent.

6. The method according to claim 5, wherein the reducing agent is any one of TCEP, 2-MEA, Cysteine, Dithiothreitol, 2-Mercaptoethanol, 3-mercapto-1,2-propanediol, TBP, or a combination thereof.

7. The method according to claim 1, wherein step 1 is a step of cleaving the F(ab) side of the hinge portion of the molecule contained in the composition with a protease.

8. The method according to claim 7, wherein the protease is any one of or a combination of a bacterial-derived antibody-degrading enzyme, papain, Lys-C.

9. The method according to any one of claims 1 to 8, wherein step 2 is a step of measuring two or more F(ab) fragments by any one of or a combination of cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, and reverse phase chromatography.

10. The method according to any one of claims 1 to 9, wherein the two or more F(ab) fragments generated in step 1 each have a different isoelectric point.

11. The method according to any one of claims 1 to 10, wherein the multispecific antigen-binding molecule (H1L1 / H2L2) is a molecule in which amino acid residues are modified so as to have a difference in isoelectric point from the following impurities (1) to (8). (1) A homodimer (H1L1 / H1L1) of a first F(ab) (H1L1) containing a first heavy chain variable domain and a first light chain variable domain (2) A homodimer (H2L2 / H2L2) of a second F(ab) (H2L2) containing a second heavy chain variable domain and a second light chain variable domain (3) A homodimer (H1L2 / H1L2) of a third F(ab) (H1L2) containing a first heavy chain variable domain and a second light chain variable domain (4) A homodimer (H2L1 / H2L1) of a fourth F(ab) (H2L1) containing a second heavy chain variable domain and a first light chain variable domain (5) A heterodimer (H1L1 / H1L2) containing a first F(ab) (H1L1) containing a first heavy chain variable domain and a first light chain variable domain and a third F(ab) (H1L2) containing a first heavy chain variable domain and a second light chain variable domain (6) A heterodimer (H1L1 / H2L1) containing a first F(ab) (H1L1) containing a first heavy chain variable domain and a first light chain variable domain and a fourth F(ab) (H2L1) containing a second heavy chain variable domain and a first light chain variable domain (7) A heterodimer (H2L2 / H1L2) containing a second F(ab) (H2L2) containing a second heavy chain variable domain and a second light chain variable domain and a third F(ab) (H1L2) containing a first heavy chain variable domain and a second light chain variable domain A heterodimer (H2L2 / H2L1) comprising a second F(ab) (H2L2) containing a second heavy chain variable domain and a second light chain variable domain, and a fourth F(ab) (H2L1) containing a second heavy chain variable domain and a first light chain variable domain

12. The method according to claim 11, wherein the composition containing the multispecific antigen-binding molecule has impurities (1) to (8) removed by a purification step utilizing the difference in isoelectric point.

13. The method according to any one of claims 1 to 12, wherein the multispecific antigen-binding molecule is a bispecific antibody.

14. The bispecific antibody is an antibody in which at least one amino acid residue selected from the amino acid residues at positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 39, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 81, 82b, 83, 85, 86, 97, 105, 108, 110 and 112 according to Kabat numbering in the heavy chain variable domain, and the amino acid residues at positions 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419 and 435 according to EU numbering in the heavy chain constant domain is modified. The method according to claim 13.

15. The method according to any one of claims 1 to 14, wherein the composition containing the multispecific antigen-binding molecule or the bispecific antibody is a pharmaceutical composition.

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