Antibody variants and isoforms with reduced biological activity

Abstract

Antibody variants and isoforms having less FVIII-like activity than emicizumab are provided.SOLUTION: To provide an antibody variant in which a specific amino acid residue in a variable region is cleaved and deleted (Q-CDR-ClippedVariant), and an antibody isoform in which a disulfide bond between heavy chains is hardly reduced under a mild reducing condition (Protecteddisulfideisoform).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to antibody variants and isoforms with reduced biological activity. For example, the present invention relates to antibody variants and isoforms of emicizumab with reduced blood coagulation factor VIII (FVIII)-like activity. The present invention also relates to pharmaceutical compositions containing reduced amounts of such antibody variants and isoforms. The present invention further relates to methods for detecting and analyzing the antibody variants and isoforms. [Background technology]

[0002] Antibodies have attracted attention as pharmaceuticals due to their high stability in plasma and minimal side effects. Among them, many IgG-type antibody drugs have been commercialized, and many antibody drugs are currently under development (Non-Patent Documents 1, 2, 3).

[0003] Hemophilia A is a bleeding disorder caused by a congenital hypofunction or deficiency of FVIII. Patients with hemophilia A are usually administered FVIII preparations for bleeding (on-demand administration). Recently, FVIII preparations have also been administered prophylactically to prevent bleeding events (Non-Patent Documents 1 and 2) (prophylactic administration). The blood half-life of FVIII preparations is approximately 12 to 16 hours. Therefore, for continuous prevention, FVIII preparations are administered to patients three times a week (Non-Patent Documents 3 and 4). In on-demand administration, additional FVIII preparations are administered at regular intervals as needed to prevent rebleeding. Furthermore, FVIII preparations are administered intravenously. Therefore, there has been a strong demand for drugs that are less invasive than FVIII preparations.

[0004] Occasionally, antibodies (inhibitors) against FVIII develop in hemophilia patients. Inhibitors counteract the effectiveness of FVIII preparations. Patients with inhibitors (inhibitor patients) are given bypassing agents to treat bleeding. Their mechanism of action is independent of FVIII's function, namely, its ability to catalyze the activation of blood coagulation factor X (FX) by activated blood coagulation factor IX (FIXa). Therefore, bypassing agents may not be able to adequately stop bleeding in some cases. Therefore, there has been a strong demand for drugs that can replace the function of FVIII and are not affected by the presence of inhibitors.

[0005] As a means of solving these problems, bispecific antibodies that substitute for the function of FVIII and their use have been reported (Patent Documents 1, 2, 3, and 4). Bispecific antibodies against FIXa and FX can exert FVIII-like activity and substitute for the function of FVIII by positioning both factors in close proximity (Non-Patent Document 5). It has been reported that the FVIII-like activity of such antibodies can be improved by optimizing their affinity for FIXa and FX (Non-Patent Document 6). One such antibody, emicizumab (ACE910), which has high FVIII-like activity, has been reported to exert hemostatic effects in a monkey hemophilia model (Non-Patent Documents 7 and 8), and clinical trials are being conducted in patients with hemophilia A. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO 2005 / 035754 [Patent Document 2] WO 2005 / 035756 [Patent Document 3] WO 2006 / 109592 [Patent Document 4] WO 2012 / 067176 [Non-patent literature]

[0007] [Non-Patent Document 1] Blood 58, 1-13 (1981) [Non-patent document 2] Nature 312, 330-337(1984) [Non-patent document 3] Nature 312, 337-342(1984) [Non-patent document 4] Biochim.Biophys.Acta 871, 268-278(1986) [Non-Patent Document 5] Nat Med. 2012 Oct;18(10):1570-4. [Non-patent document 6] PLoS One. 2013;8(2):e57479. [Non-Patent Document 7] J Thromb Haemost. 2014 Feb;12(2):206-213. [Non-patent document 8] Blood. 2014 Nov 13;124(20):3165-71. [Non-Patent Document 9] J. Appl. Cryst. 13, 577-584 (1980) [Non-Patent Document 10] IUCrJ. 2, 9-18 (2015) Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention has been made in view of the above circumstances, and aims to provide antibody variants or isoforms with low FVIII-like activity. [Means for solving the problem]

[0009] As a result of intensive research to achieve the above object, the present inventors have succeeded in identifying antibody variants and isoforms contained in pharmaceutical compositions containing emicizumab as an active ingredient, and have found that the FVIII-like activity of these antibody variants and isoforms is significantly lower than that of emicizumab.

[0010] The present invention is based on such findings and specifically provides the following [1] to

[21] . [1] An antibody variant having a variable region comprising the amino acid sequence SISPSGQSTYYRREVKG (SEQ ID NO: 2), (a) the amino acid residue R at the 12th position from the N-terminus of the sequence (the 61st position from the N-terminus of the emicizumab Q chain: position 60 according to the Kabat numbering); or (b) amino acid residues YYR at positions 10 to 12 from the N-terminus of the sequence (positions 59 to 61 from the N-terminus of the emicizumab Q chain: positions 58 to 60 in the Kabat numbering system) and the variable region is truncated at the deletion site. [2] The antibody variant of [1], wherein the sequence is a CDR sequence. [3] The antibody variant of [1], wherein the sequence is a CDR2 sequence. [4] The antibody variant of [1], wherein the sequence is contained in a heavy chain. [5] The antibody variant of [1], which is a bispecific antibody variant. [6] An antibody variant of [1], which is a variant of emicizumab. [7] A method for detecting an antibody variant described in any of [1] to [6], comprising a step of separating a sample containing an antibody having a variable region comprising the amino acid sequence SISPSGQSTYYRREVKG (SEQ ID NO: 2) 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. [8] The detection method according to [7], wherein any one of the antibody variants according to [1] to [6] is used as a standard. [8-2] The detection method of [8], which includes a step of performing one or more analyses selected from the group consisting of quantitative analysis, qualitative analysis, and structural analysis. [9] A pharmaceutical composition comprising the antibody variant of any one of [1] to [6], wherein the proportion of the antibody variant in the total antibody molecules in the pharmaceutical composition is 5% or less.

[10] The pharmaceutical composition according to [9], wherein the antibody is emicizumab.

[11] The pharmaceutical composition of [9], obtained by a purification process including purification by cation exchange chromatography (CEX).

[12] A method for inhibiting the production of any of the antibody variants of [1] to [6], comprising the step of culturing antibody-producing cells at a pH of 7.1 or higher and / or at a culture temperature of 36°C or lower. [12-2] The method of

[12] , wherein the culture conditions of the antibody-producing cells are changed during the culture to a pH of 7.1 or higher and / or a culture temperature of 36°C or lower.

[13] A bispecific antibody isoform comprising a first heavy chain (Q chain: SEQ ID NO: 10) and a second heavy chain (J chain: SEQ ID NO: 11), (1a) between a cysteine ​​at position 144 (EU numbering) in the first heavy chain (position 150 from the N-terminus of SEQ ID NO: 10) and a cysteine ​​at position 200 (EU numbering) in the second heavy chain (position 202 from the N-terminus of SEQ ID NO: 11); and (1b) between a cysteine ​​at position 200 (EU numbering) in the first heavy chain (position 206 from the N-terminus of SEQ ID NO: 10) and a cysteine ​​at position 144 (EU numbering) in the second heavy chain (position 146 from the N-terminus of SEQ ID NO: 11); forming a disulfide bond in (2a) between a cysteine ​​at position 226 (EU numbering) in the first heavy chain (position 229 from the N-terminus of SEQ ID NO: 10) and a cysteine ​​at position 229 (EU numbering) in the second heavy chain (position 228 from the N-terminus of SEQ ID NO: 11); and (2b) between the cysteine ​​at position 229 (EU numbering) in the first heavy chain (position 232 from the N-terminus of SEQ ID NO: 10) and the cysteine ​​at position 226 (EU numbering) in the second heavy chain (position 225 from the N-terminus of SEQ ID NO: 11). A bispecific antibody isoform that forms a disulfide bond in the

[14] The bispecific antibody isoform of

[13] , wherein a disulfide bond is formed between (1a) and (1b).

[15] A bispecific antibody isoform comprising a first heavy chain (Q chain: SEQ ID NO: 10) and a second heavy chain (J chain: SEQ ID NO: 11), characterized in that when separated using cation exchange chromatography, it is eluted in a more alkaline region than the bispecific antibody.

[16] Any of the bispecific antibody isoforms of

[13] to

[15] , which is an isoform of emicizumab.

[17] A method for detecting an antibody isoform according to any one of

[13] to

[16] , comprising a step of separating a sample containing a bispecific 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.

[18] The detection method according to

[17] , wherein any one of the bispecific antibody isoforms according to

[13] to

[16] is used as a standard. [18-2] The detection method of

[18] , which includes a step of performing one or more analyses selected from the group consisting of quantitative analysis, qualitative analysis, and structural analysis.

[19] A pharmaceutical composition comprising the bispecific antibody isoform of any one of

[13] to

[16] , wherein the proportion of the antibody isoform in all antibody molecules in the pharmaceutical composition is 2% or less.

[20] A method for reducing the content of any of the bispecific antibody isoforms according to

[13] to

[16] , comprising a step of purifying the antibody by cation exchange chromatography.

[21] An antibody isoform or variant of [1],

[13] , or

[15] , which has significantly reduced antibody biological activity.

[22] An isoform of an antibody or derivative thereof having two variable regions that recognize different epitopes, wherein the average Rg value is 3% or more, preferably 4% or more, more preferably 5% or more, more preferably 6% or more smaller than that of the antibody or derivative thereof, and / or the average Dmax value is 5% or more, preferably 6% or more, more preferably 7% or more, more preferably 7.5% or more smaller than that of the antibody or derivative thereof.

[23] An isoform of an antibody or derivative thereof having two variable regions that recognize different epitopes, wherein the average Rg value is 0.15 nm or more, preferably 0.2 nm or more, more preferably 0.25 nm or more, more preferably 0.3 nm or more smaller than that of the antibody or derivative thereof, and / or the average Dmax value is 0.5 nm or more, preferably 1.0 nm or more, more preferably 1.2 nm or more, more preferably 1.4 nm or more smaller than that of the antibody or derivative thereof.

[24] An isoform of

[22] or

[23] having a disulfide bond different from that of the antibody or its derivative.

[25] Any of the isoforms of

[22] to

[24] , wherein the antibody or derivative thereof is emicizumab (a bispecific antibody comprising a first heavy chain (Q chain: SEQ ID NO: 10), a second heavy chain (J chain: SEQ ID NO: 11), and a common light chain (SEQ ID NO: 12) that forms a pair with each of the first heavy chain and the second heavy chain).

[26] An isoform of emicizumab, wherein the average Rg value is 4.9 nm or less, preferably 4.8 nm or less, and / or the average Dmax value is 17.0 nm or less, preferably 16.5 nm or less.

[27] An isoform of

[25] or

[26] having disulfide bonds between the cysteine ​​at EU numbering position 144 (position 150 from the N-terminus of SEQ ID NO: 10) in the first heavy chain and the cysteine ​​at EU numbering position 200 (position 202 from the N-terminus of SEQ ID NO: 11) in the second heavy chain, and between the cysteine ​​at EU numbering position 200 (position 206 from the N-terminus of SEQ ID NO: 10) in the first heavy chain and the cysteine ​​at EU numbering position 144 (position 146 from the N-terminus of SEQ ID NO: 11) in the second heavy chain.

[28] A pharmaceutical composition comprising emicizumab and any one of the isoforms

[22] to

[27] , wherein the proportion of said isoform in all antibody molecules in said pharmaceutical composition is 2% or less.

[29] An isoform of a bispecific antibody (Q499-z121 / J327-z119 / L404-k; emicizumab) comprising a first heavy chain (Q chain: SEQ ID NO: 10), a second heavy chain (J chain: SEQ ID NO: 11), and a common light chain (SEQ ID NO: 12) paired with each of the first and second heavy chains, wherein the isoform has a molecular structure different from emicizumab in the amino acid residues at positions 146 to 174 (EU numbering) in the Q chain (positions 152 to 180 from the N-terminus of SEQ ID NO: 10) and the amino acid residues at positions 146 to 174 (EU numbering) in the J chain (positions 148 to 176 from the N-terminus of SEQ ID NO: 11).

[30] The isoform according to

[29] , wherein the difference in molecular structure is measured as a difference in deuterium exchange rate (%D) in HDX-MS measurement.

[31] A pharmaceutical composition comprising emicizumab and an isoform of

[29] or

[30] , wherein the proportion of the isoform in all antibody molecules in the pharmaceutical composition is 2% or less.

[0011] The present invention also provides the following [A1] to [A9]. [A1] The detection method of [7] or

[17] , which comprises a step of subjecting a sample containing an antibody and / or antibody variant or isoform to a reduction reaction, a hydrolysis reaction (digestion reaction), a protein denaturation reaction, or a combination thereof. [A2] The detection method of [A1], wherein the reduction reaction is carried out under mild reducing conditions (e.g., reduction with DTT in Tris buffer (pH 7.0) at 37°C). [A3] The detection method according to [A1], wherein the hydrolysis reaction is carried out using a site-specific cleavage enzyme (e.g., a sequence-specific protease such as IdeS protease, Lys-C, or papain). [A4] A detection method according to any one of [7],

[17] , or [A1] to [A3], comprising a step of separating a sample containing an antibody, an antibody variant or isoform, a reaction product thereof, or a combination 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. [A5] A detection method according to any one of [8],

[18] , or [A1] to [A4], which comprises a step of analyzing by SE-HPLC analysis, dynamic light scattering (DLS), SAXS measurement, electron microscope measurement, 3D modeling, SPR evaluation, HDX MS analysis, or a combination thereof. [A6] A quality control method for a pharmaceutical composition containing emicizumab, comprising steps of [7], [8],

[17] ,

[18] , or [A1] to [A5], or a combination of these methods. [A7] A method for producing a pharmaceutical composition containing emicizumab, comprising the steps of the method of [A6]. [A8] A method for purifying a composition containing emicizumab, characterized by comprising a step of cation exchange chromatography (CEX) in bind & elute mode. [A9] A method for producing a pharmaceutical composition containing emicizumab, comprising the steps of the purification method of [A8]. [Effects of the Invention]

[0012] The present inventors have succeeded in identifying antibody variants and isoforms contained in pharmaceutical compositions containing emicizumab as an active ingredient. The present inventors have also found that the FVIII-like activity of these antibody variants and isoforms is significantly lower than that of emicizumab. Therefore, pharmaceutical compositions containing emicizumab but with low contents of these antibody variants and isoforms are useful as a means for treating hemophilia. [Brief explanation of the drawings]

[0013] [Figure 1-1] Figure 1A shows the results of separation of emicizumab drug substance by CE-HPLC. The peak enclosed in a bold frame represents the Q-CDR-clipped variant. [Figure 1-2] Figure 1B is a schematic diagram showing the molecular structure of the Q-CDR-clipped variant. The numbers and the letters below them represent the amino acid residue positions counted from the N-terminus of the Q chain of emicizumab and the amino acid residues (single-letter code) at those positions, respectively. [Figure 2-1] Figure 2A shows the results of separation of emicizumab drug substance by CE-HPLC. The peak enclosed in a bold frame represents the protected disulfide isoform. [Figure 2-2]Figure 2B is a schematic diagram showing the molecular structure of a protected disulfide isoform. The numbers in the diagram and the letter C to the left of them represent the amino acid residue position counted from the N-terminus of the Q chain of emicizumab and the cysteine ​​residue at that position, respectively. Figure 2C is a diagram showing the content of protected disulfide isoforms in emicizumab drug substance. The content under various conditions (culture conditions of antibody-producing cells) is shown as the mean (Mean) and standard deviation (Std Dev) of the area % of the protected disulfide isoform peak in the CE-HPLC separation results shown in Figure 2A. [Figure 3] Figure 3 shows the results of reversed-phase high-performance liquid chromatography (RPLC) separation of emicizumab and protected disulfide isoforms after IdeS digestion and reduction. Figures 3A–D show the separation results of samples in which emicizumab (A and B) or protected disulfide isoforms (C and D) were reduced after IdeS digestion under conditions with or without denaturing agents (A and C: fully reducing conditions) or without denaturing agents (B and D: partially reducing conditions). Under fully reducing conditions (Figures 3A and C), peaks representing Q-chain Fd (Q-Fd), J-chain Fd (J-Fd), Q-chain Fc (Q-Fc), J-chain Fc (J-Fc), and L-chain (LC) were detected for both emicizumab and the protected disulfide isoforms. No differences in the reduction patterns were detected between emicizumab and the protected disulfide isoforms. On the other hand, under partial reduction conditions (Fig. 3B and D), in addition to the peaks representing Q chain Fd, J chain Fd, Q chain Fc, J chain Fc, and L chain, similar to those observed under fully reduced conditions, a unique peak representing a heterodimer of Q chain Fd and J chain Fd (J-Fd-Q-Fd) disulfide-bonded to each other was detected only for the protected disulfide isoform. [Figure 4]Figure 4 shows the results of reversed-phase high-performance liquid chromatography separation of emicizumab and protected disulfide isoforms after IdeS digestion (and denaturation). Figures 4A and 4B show the separation results of the protected disulfide isoform (A) or emicizumab (B) after IdeS digestion. Figures 4C and 4D show the separation results of the protected disulfide isoform (C) or emicizumab (D) after denaturation treatment after IdeS digestion. Regardless of whether denaturation treatment was performed or not, the F(ab')2 portion of the protected disulfide isoform (LC-J Fab-Q Fab-LC) separated with a longer retention time than the main component of emicizumab. [Figure 5] 5 shows the Q-CDR-clipped variant content in the culture supernatant when emicizumab-producing CHO cells were cultured under various culture conditions. The culture supernatant was purified using Protein A, and the sample was used to measure the Q-CDR-clipped variant content. The vertical axis represents the Q-CDR-clipped variant content (peak area %), and the horizontal axis represents various culture conditions. [Figure 6] 6 shows the Q-CDR-clipped variant content in the culture supernatant when emicizumab-producing CHO cells were cultured under various culture conditions. The culture supernatant was purified using Protein A, and the sample was used to measure the Q-CDR-clipped variant content. The vertical axis represents the Q-CDR-clipped variant content (peak area %), and the horizontal axis represents various culture conditions. [Figure 7]Figure 7 shows the results of CE-HPLC analysis of each fraction in the purification process, including the bind & elute mode step of cation exchange chromatography (CEX) of a solution of emicizumab antibody containing a Q-CDR clipped variant. "Load fraction" shows the results of CE-HPLC analysis of the antibody solution loaded onto a cation exchange column. "Wash fraction" shows the results of CE-HPLC analysis of the column adsorption fraction after passing a phosphate buffer containing 25 mmol / L sodium chloride adjusted to pH 7.2 through the column (after washing). "Elution fraction" shows the results of CE-HPLC analysis of the column adsorption fraction after passing a phosphate buffer containing 100 mmol / L sodium chloride adjusted to pH 6.5 through the column after washing. Compared to the load fraction and wash fraction, the peak of the Q-CDR clipped variant, which is located on the acidic side of the emicizumab antibody peak, disappeared in the elution fraction. [Figure 8] 8A to 8D show the results of SAXS analysis of the molecular structures of emicizumab (main) and protected disulfide isoform (BiAb3). Pair-distance distribution function [p(r)], Rg (nm), and Dmax (nm) represent the molecular pair-distance distribution function, radius of gyration, and maximum length, respectively. [Figure 9-1]Figure 9A shows the residual plot of deuterium exchange rate (%D) for emicizumab (the main component of cation-exchange high-performance liquid chromatography) and protected disulfide isoforms measured by HDX-MS (deuterium exchange times: 30 s, 60 s, 120 s, 240 s, 480 s, 960 s, 1920 s, and 3840 s). The bar graph shows the sum of the differences in the results for each deuterium exchange time for the Q, J, and L chains. Figure 9B shows areas where HDX-MS measurements suggest differences in molecular structure between emicizumab and protected disulfide isoforms. The difference in molecular structure between the two peptides was most pronounced in peptides containing amino acid residues at positions 146 to 174 (EU numbering) in the Q chain (positions 152 to 180 from the N-terminus of SEQ ID NO: 10) and positions 146 to 174 (EU numbering) in the J chain (positions 148 to 176 from the N-terminus of SEQ ID NO: 11) (indicated by an asterisk). [Figure 9-2] This is a continuation of Figure 9-1. DETAILED DESCRIPTION OF THE INVENTION

[0014] One embodiment of the present invention relates to antibody variants and isoforms with reduced biological activity (e.g., FVIII-like activity). These antibody variants and isoforms were identified as two types of structural variants (Q-CDR-clipped variant and protected disulfide isoform) in the analysis of emicizumab drug substance by the present inventors. As used herein, "antibody variant" and "antibody isoform" may also be referred to as mutants or isomers of an antibody molecule.

[0015] Emicizumab is a bispecific humanized IgG4 antibody consisting of anti-FIX(a) and anti-FX, which exhibits FVIII cofactor functional substitution activity. It is composed of two heavy chains (Q499 and J327) that recognize FIX(a) and FX, respectively, and a common light chain (L404).

[0016] Specifically, emicizumab is a bispecific antibody in which a first polypeptide and a third polypeptide form a pair, and a second polypeptide and a fourth polypeptide form a pair. The first polypeptide is an H chain comprising the amino acid sequence of H chain CDR1, 2, and 3 set forth in SEQ ID NOs: 1, 2, and 3 (Q499 H chain CDR); the second polypeptide is an H chain comprising the amino acid sequence of H chain CDR1, 2, and 3 set forth in SEQ ID NOs: 4, 5, and 6 (J327 H chain CDR); and the third polypeptide and the fourth polypeptide are a common L chain comprising the amino acid sequence of L chain CDR1, 2, and 3 set forth in SEQ ID NOs: 7, 8, and 9 (L404 L chain CDR). This bispecific antibody (Q499-z121 / J327-z119 / L404-k)

[0017] More specifically, emicizumab is a bispecific antibody in which a first polypeptide and a third polypeptide form a pair, and a second polypeptide and a fourth polypeptide form a pair, wherein the first polypeptide is an H chain comprising the amino acid sequence of the H chain variable region set forth in SEQ ID NO: 13, the second polypeptide is an H chain comprising the amino acid sequence of the H chain variable region set forth in SEQ ID NO: 14, and the third polypeptide and the fourth polypeptide are a common L chain comprising the amino acid sequence of the L chain variable region set forth in SEQ ID NO: 15.

[0018] More specifically, emicizumab is a bispecific antibody in which a first polypeptide and a third polypeptide form a pair and a second polypeptide and a fourth polypeptide form a pair, and the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 10, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 11, and the third polypeptide and the fourth polypeptide are a common L chain set forth in SEQ ID NO: 12 (Q499-z121 / J327-z119 / L404-k). Such antibodies can be obtained according to the methods described in, for example, WO2005 / 035756, WO2006 / 109592, WO2012 / 067176, and the like.

[0019] The antibody used in the present invention is not particularly limited as long as it binds to the desired antigen, and may be a polyclonal or monoclonal antibody, with monoclonal antibodies being preferred in that homogeneous antibodies can be stably produced.

[0020] It should be noted that the amino acids contained in the amino acid sequences described in the present invention may be modified after translation (for example, modification of N-terminal glutamine to pyroglutamic acid by pyroglutamylation is a modification well known to those skilled in the art), and even when such amino acids are post-translationally modified, they are naturally included in the antibodies used in the present invention.

[0021] In the present invention, the biological activity of an antibody, antibody variant, or antibody isoform is preferably FVIII-like activity. In the present invention, "FVIII-like activity" refers to activity that substitutes for the function of FVIII (FVIII cofactor substitution activity). In the present invention, "substituting for the function of FVIII" refers to recognizing FIX, FIXa, and FX and promoting the activation of FX by FIXa (promoting FXa production by FIXa). FXa production-promoting activity can be evaluated, for example, using an assay system consisting of FIXa, FX, the synthetic substrate S-2222 (a synthetic FXa substrate), and phospholipids. This assay system correlates with the severity and clinical symptoms of hemophilia A (Rosen S, Andersson M, Blomback M et al. Clinical applications of a chromogenic substrate method for determination of FVIII activity. Thromb Haemost 1985; 54: 811-23). The FVIII-like activity of antibodies such as emicizumab, antibody variants, and antibody isoforms can be evaluated according to the methods described in, for example, WO2005 / 035756, WO2006 / 109592, WO2012 / 067176, and the like.

[0022] In the present invention, "the biological activity of an antibody, antibody variant, or antibody isoform" means that the biological activity is reduced compared to the biological activity of a comparable antibody, and preferably that the reduction is statistically significant. In the present invention, "the biological activity of an antibody, antibody variant, or antibody isoform" means that the biological activity is reduced by 10% or more, for example, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more compared to the biological activity of a comparable antibody.

[0023] In the present invention, the terms "Q chain" and "J chain" refer to H chains (heavy chains) that contain variable regions capable of binding to FIX(a) and FX, respectively.

[0024] In the present invention, a "common L chain" refers to an L chain that can pair with two or more different H chains and exhibit binding ability to each of the antigens. Here, "different H chains" preferably refers to, but is not limited to, H chains of antibodies against different antigens, and refers to H chains that have different amino acid sequences. A common L chain can be obtained, for example, according to the method described in WO2006 / 109592.

[0025] The term "antibody" is used in the broadest sense and may be a monoclonal antibody, polyclonal antibody, dimer, multimer, multispecific antibody (e.g., bispecific antibody), antibody derivative, or modified antibody, as long as it exhibits the desired biological activity (Miller K et al. J Immunol. 2003, 170(9), 4854-61). Antibodies may be murine, human, humanized, chimeric, or derived from other species or may be artificially synthesized. The antibodies disclosed herein may be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. Immunoglobulins may be derived from any species (e.g., human, mouse, or rabbit). The terms "antibody," "immunoglobulin," and "immunoglobulin" are used interchangeably and broadly.

[0026] A "bispecific" antibody is an antibody that has two variable regions in the same antibody molecule, each recognizing a different epitope. A bispecific antibody may be an antibody that recognizes two or more different antigens, or an antibody that recognizes two or more different epitopes on the same antigen. Bispecific antibodies may include not only whole antibodies but also antibody derivatives.

[0027] The antibody may be a recombinant antibody produced using genetic engineering technology. Recombinant antibodies can be obtained by cloning the DNA encoding the antibody from antibody-producing cells such as hybridomas or sensitized lymphocytes that produce antibodies, incorporating it into a vector, and introducing this into a host (host cell) for production.

[0028] Bispecific antibodies are not limited to IgG types, but for example, IgG type bispecific antibodies can be secreted by hybrid hybridomas (quadromas) generated by fusing two types of IgG antibody-producing hybridomas (Milstein C et al. Nature 1983, 305: 537-540). Alternatively, the genes for the L and H chains constituting the two types of IgG of interest, a total of four genes, can be introduced into cells and co-expressed to secrete the antibodies.

[0029] The antibodies of the present invention can be produced by methods known to those skilled in the art. Specifically, DNA encoding the antibody of interest is incorporated into an expression vector. The vector is incorporated so that expression is under the control of an expression control region, such as an enhancer or promoter. Next, host cells are transformed with this expression vector to express the antibody. An appropriate combination of host and expression vector can be used.

[0030] The antibodies of the present invention obtained in this manner can be isolated from inside or outside the host cells (e.g., culture medium) and purified as substantially pure, homogeneous antibodies. Antibody separation and purification can be performed using methods commonly used for antibody purification, and are not limited in any way. For example, the method described in WO2013 / 086448 is known for separating IgG2 disulfide isoforms. The antibodies and antibody variants or antibody isoforms of the present invention can be separated and purified by appropriately selecting and combining, for example, chromatography columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. For example, various matrices can be used for separation and purification using chromatography columns, such as strong cation exchange matrices, weak cation exchange matrices, anti-human IgG affinity matrices, and protein L matrices.

[0031] In one aspect, the present invention relates to an antibody variant (sometimes referred to herein as a Q-CDR-Clipped Variant) having the following characteristics: ·Very low biological activity (FVIII-like activity) compared to emicizumab; The N-terminal and C-terminal fragments of the missing amino acid residue are linked by a disulfide bond (Figure 1B). The amount of antibody produced varies depending on the incubation time, temperature, and pH of the antibody-producing cells.

[0032] In one embodiment, the Q-CDR-Clipped Variant is a variant of an antibody having a variable region comprising the amino acid sequence SISPSGQSTYYRREVKG (SEQ ID NO: 2), (a) the amino acid residue R at the 12th position from the N-terminus of the amino acid sequence of SEQ ID NO: 2 (the 61st position from the N-terminus of the Q chain of emicizumab, i.e., position 60 according to the Kabat numbering); or (b) amino acid residues YYR at positions 10 to 12 from the N-terminus of the amino acid sequence of SEQ ID NO: 2 (positions 59 to 61 from the N-terminus of the Q chain of emicizumab: positions 58 to 60 in the Kabat numbering system) and the variable region is truncated at the deletion site. The Q-CDR-clipped variant is preferably a variant of a bispecific antibody, and particularly preferably a variant of emicizumab.

[0033] In another aspect, the present invention also relates to methods for detecting and analyzing Q-CDR-clipped variants. In one embodiment, the method for detecting Q-CDR-clipped variants comprises separating a sample containing an antibody having a variable region comprising the amino acid sequence SISPSGQSTYYRREVKG (SEQ ID NO: 2) 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 another embodiment, the method for analyzing Q-CDR-clipped variants comprises performing one or more analyses selected from the group consisting of quantitative analysis, qualitative analysis, and structural analysis using the Q-CDR-clipped variant as a standard. In such detection and analysis methods, the presence or absence of a deletion site in Fab (Q chain Fab) having the amino acid sequence of SEQ ID NO: 2 can be used as an indicator for detection and analysis. The deletion site can be detected, for example, by using a molecular weight shift resulting from the deletion in LCMS analysis. Furthermore, in Q-CDR-clipped variants, the N-terminal fragment and the C-terminal fragment of the deleted amino acid residue are linked by a disulfide bond. Therefore, differences in reduction patterns resulting from the presence or absence of the deletion site can be detected by analyzing samples subjected to a reduction reaction of the disulfide bond using various analytical techniques such as CE-HPLC, LCMS, and LC-UV. Structural analysis such as NMR measurement can also be used. Alternatively, the difference in the degree of separation by ion exchange chromatography can also be used as an indicator; for example, when separated by cation exchange chromatography, the Q-CDR-clipped variant is separated in the acidic region compared to the main peak of emicizumab. In another aspect, the present invention enables the production and quality control of a pharmaceutical composition containing emicizumab by implementing one or a combination of the above-mentioned detection methods and analytical methods. Thus, the present invention relates to a quality control method for a pharmaceutical composition containing emicizumab, which includes implementing the above-mentioned detection methods and analytical methods or combining these methods. The present invention also relates to the production of a pharmaceutical composition containing emicizumab, which includes implementing such a quality control method.

[0034] In another aspect, the present invention relates to a pharmaceutical composition comprising emicizumab and a Q-CDR-clipped variant, wherein the proportion of Q-CDR-clipped variants among the antibody molecules in the pharmaceutical composition is reduced. The pharmaceutical composition can be obtained by a purification process that includes purification by cation exchange chromatography (CEX). For example, an antibody solution containing emicizumab and a Q-CDR-clipped variant can be adsorbed onto a cation exchange column, and only acidic variants, including Q-CDR-clipped variants, can be specifically eluted and removed. The proportion of Q-CDR-clipped variants among all antibody molecules in the pharmaceutical composition can be evaluated by various methods, including the above-mentioned Q-CDR-clipped variant detection / analysis method. For example, it can be expressed by the ratio of the peak area of ​​the Q-CDR-clipped variant (peak area ratio) when the pharmaceutical composition is analyzed by cation exchange chromatography (CEX) or CE-HPLC. The proportion of Q-CDR-clipped variants (e.g., CEX peak area ratio) in all antibody molecules in the pharmaceutical composition is preferably 5% or less, for example, 5.0% or less, 4.0% or less, 3.0% or less, 2.0% or less, or 1.0% or less. The present invention also relates to a method for producing a pharmaceutical composition with a reduced content of Q-CDR-clipped variants, and a method for suppressing the production of Q-CDR-clipped variants. The amount of Q-CDR-clipped variants produced can be reduced by shortening the culture time of antibody-producing cells (e.g., within 15 days, preferably within 13 days), lowering the culture temperature (e.g., to 38°C or below, preferably to 37°C or below, and more preferably to 36°C or below), and / or increasing the culture pH (e.g., to 6.7 or higher, preferably to 6.9 or higher, and more preferably to 7.1 or higher) (Figure 4). Therefore, the above method is characterized by comprising a step of culturing antibody (e.g., emicizumab)-producing cells at a lower culture temperature (e.g., approximately 36°C or below) and a higher pH (e.g., approximately 7.1 or higher) than conventionally used for a certain period of time (e.g., approximately 15 days or less). In one embodiment, the above method comprises a step of culturing antibody-producing cells at a pH of 7.1 or higher and / or a culture temperature of 36°C or below. In a specific embodiment, the above method is characterized in that the culture conditions of the antibody-producing cells are shifted to culture conditions at pH 7.1 or higher and / or a culture temperature of 36°C or lower during the culture (e.g., from the second day of culture onwards). In another aspect, the present invention relates to a method for purifying a composition containing emicizumab, the method comprising a step of cation exchange chromatography (CEX) in bind & elute mode. The present invention also relates to a method for producing a pharmaceutical composition containing emicizumab, the method comprising a step of carrying out the purification method.

[0035] In another aspect, the present invention relates to an antibody isoform (sometimes referred to herein as a Protected Disulfide isoform) having the following characteristics: ·Very low biological activity (FVIII-like activity) compared to emicizumab; ·More hydrophobic than emicizumab; Compared to emicizumab, the inter-heavy chain disulfide bond (Figure 2B) is less susceptible to reduction under mild (partially reduced) conditions; - It is produced regardless of conditions (production parameters) such as the dissolved oxygen concentration and initial pH in the culture medium of antibody-producing cells, and the culture time before adding MTX (methotrexate). In one embodiment, the protected disulfide isoform is characterized in that it can bind to the antigens FIX(a) and FX but does not exhibit biological activity (FVIII-like activity). In a specific embodiment, the protected disulfide isoform is a structural isomer that has the same (normal) disulfide bonds as emicizumab, but has stronger hydrophobicity than usual due to a structural change in the Fab portion, making the disulfide bonds between the heavy chains less susceptible to reduction than usual.

[0036] In another embodiment, the protected disulfide isoform is a bispecific antibody isoform comprising a first heavy chain (Q chain: SEQ ID NO: 10) and a second heavy chain (J chain: SEQ ID NO: 11), (1a) between a cysteine ​​at position 144 (EU numbering) in the first heavy chain (position 150 from the N-terminus of SEQ ID NO: 10) and a cysteine ​​at position 200 (EU numbering) in the second heavy chain (position 202 from the N-terminus of SEQ ID NO: 11); and (1b) between a cysteine ​​at position 200 (EU numbering) in the first heavy chain (position 206 from the N-terminus of SEQ ID NO: 10) and a cysteine ​​at position 144 (EU numbering) in the second heavy chain (position 146 from the N-terminus of SEQ ID NO: 11); forming a disulfide bond in (2a) between a cysteine ​​at position 226 (EU numbering) in the first heavy chain (position 229 from the N-terminus of SEQ ID NO: 10) and a cysteine ​​at position 229 (EU numbering) in the second heavy chain (position 228 from the N-terminus of SEQ ID NO: 11); and (2b) between a cysteine ​​at position 229 (EU numbering) in the first heavy chain (position 232 from the N-terminus of SEQ ID NO: 10) and a cysteine ​​at position 226 (EU numbering) in the second heavy chain (position 225 from the N-terminus of SEQ ID NO: 11); It is a bispecific antibody isoform in which disulfide bonds are formed at the nucleus.

[0037] In certain embodiments, the protected disulfide isoform is (1a) between a cysteine ​​at position 144 (EU numbering) in the first heavy chain (position 150 from the N-terminus of SEQ ID NO: 10) and a cysteine ​​at position 200 (EU numbering) in the second heavy chain (position 202 from the N-terminus of SEQ ID NO: 11); (1b) between a cysteine ​​at position 200 (EU numbering) in the first heavy chain (position 206 from the N-terminus of SEQ ID NO: 10) and a cysteine ​​at position 144 (EU numbering) in the second heavy chain (position 146 from the N-terminus of SEQ ID NO: 11); (1c) between the cysteine ​​at EU numbering position 226 (position 229 from the N-terminus of SEQ ID NO: 10) in the first heavy chain and the cysteine ​​at EU numbering position 226 (position 225 from the N-terminus of SEQ ID NO: 11) in the second heavy chain; and (1d) between the cysteine ​​at position 229 (EU numbering) in the first heavy chain (position 232 from the N-terminus of SEQ ID NO: 10) and the cysteine ​​at position 229 (EU numbering) in the second heavy chain (position 228 from the N-terminus of SEQ ID NO: 11). forming a disulfide bond in (2a) between the cysteine ​​at position 226 (EU numbering) in the first heavy chain (the 229th position from the N-terminus of SEQ ID NO: 10) and the cysteine ​​at position 229 (EU numbering) in the second heavy chain (the 228th position from the N-terminus of SEQ ID NO: 11); (2b) between a cysteine ​​at position 229 (EU numbering) in the first heavy chain (position 232 from the N-terminus of SEQ ID NO: 10) and a cysteine ​​at position 226 (EU numbering) in the second heavy chain (position 225 from the N-terminus of SEQ ID NO: 11); (2c) between a cysteine ​​at position 144 (EU numbering) in the first heavy chain (position 150 from the N-terminus of SEQ ID NO: 10) and a cysteine ​​at position 200 (EU numbering) in the first heavy chain (position 206 from the N-terminus of SEQ ID NO: 10); and (2d) between a cysteine ​​at position 144 (EU numbering) in the second heavy chain (position 146 from the N-terminus of SEQ ID NO: 11) and a cysteine ​​at position 200 (EU numbering) in the second heavy chain (position 202 from the N-terminus of SEQ ID NO: 11); and particularly preferably an isoform of emicizumab.

[0038] In another aspect, the present invention also relates to methods for detecting and analyzing protected disulfide isoforms. In one embodiment, the method for detecting protected disulfide isoforms comprises separating a sample containing a bispecific antibody by affinity chromatography, ion exchange chromatography, normal-phase chromatography, reversed-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 another embodiment, the method for analyzing protected disulfide isoforms comprises performing one or more analyses selected from the group consisting of quantitative analysis, qualitative analysis, and structural analysis using protected disulfide isoforms as standards. Such detection and analysis methods can utilize differences in the structure of the inter-heavy chain disulfide bond region and / or Fab region between the protected disulfide isoform and emicizumab. These structural differences can be detected by various analytical techniques, for example, as described below.

[0039] For example, by analyzing a sample after digestion with IdeS protease under non-reducing conditions (which cleaves a single site below the hinge region of IgG to generate F(ab')2 and Fc fragments) using a reverse-phase column (e.g., a C4 column), peaks reflecting the difference in the three-dimensional structure or the strength of hydrophobicity between emicizumab and the protected disulfide isoform can be detected. Furthermore, by analyzing the sample after IdeS digestion and subsequent reduction under mild reducing conditions (e.g., reduction with DTT in Tris buffer (pH 7.0) at 37°C) using a reversed-phase column, peaks reflecting the difference in the ease of reduction of disulfide bonds between emicizumab and the protected disulfide isoform can be detected. When emicizumab and the protected disulfide isoform are subjected to a reduction reaction under conditions in which all disulfide bonds are reduced, no difference is detected in the results of reversed-phase column analysis, but when the reduction reaction is performed under the above-mentioned mild reducing conditions, a difference in the reduction pattern is detected in the results of reversed-phase column analysis. Without intending to be limited by a particular theory, it is thought that under the above-mentioned mild reducing conditions, emicizumab reduces both the disulfide bonds between the heavy chains and the disulfide bonds between heavy chains, whereas the protected disulfide isoform reduces only the disulfide bonds between the heavy chains and the disulfide bonds between heavy chains, but not the disulfide bonds between heavy chains.

[0040] Furthermore, when a sample is subjected to limited Lys-C digestion under non-denaturing conditions (e.g., in Tris buffer) (e.g., by stopping the Lys-C digestion reaction midway), the sample can be analyzed using a reverse-phase column (e.g., a C4 column) to detect peaks reflecting differences in the Lys-C digestion pattern due to differences in the conformation between emicizumab and the protected disulfide isoform. Although not intending to be limited by any particular theory, it is believed that Lys-C digestion under non-denaturing conditions results in preferential cleavage of sites that are easily accessible to Lys-C while maintaining the conformation, resulting in the detection of a Lys-C digestion pattern that reflects the differences in conformation.

[0041] Alternatively, by performing limited Lys-C digestion on a non-reduced sample (i.e., SS bonds are retained) after denaturation (e.g., treatment with 5 M guanidine at 37°C for 30 minutes) and then analyzing it using a reversed-phase column, peaks reflecting differences in Lys-C digestion patterns due to differences in the conformation between emicizumab and the protected disulfide isoform can be detected. Although not intending to be limited by any particular theory, it is thought that Lys-C digestion of a non-reduced sample after denaturation results in preferential cleavage of sites that are easily accessible to Lys-C, while retaining SS bonds (disulfide bonds) but not retaining the steric structure, resulting in the detection of a Lys-C digestion pattern that reflects differences in SS bonds. In addition to the reduction reaction, IdeS digestion, and limited Lys-C digestion under non-denaturing or denaturing conditions described above, various degradation reactions such as papain digestion can be used. Furthermore, in addition to reversed-phase chromatography using a C4 column or the like, various analytical techniques can be used, such as SE-HPLC analysis, dynamic light scattering (DLS), SAXS measurement, electron microscopy measurement, 3D modeling, SPR evaluation, and HDX MS analysis. In another aspect, the present invention enables the production and quality control of a pharmaceutical composition containing emicizumab by implementing one or a combination of the above-mentioned detection methods and analytical methods. Thus, the present invention relates to a quality control method for a pharmaceutical composition containing emicizumab, which includes implementing the above-mentioned detection methods and analytical methods or combining these methods. The present invention also relates to the production of a pharmaceutical composition containing emicizumab, which includes implementing such a quality control method.

[0042] In another aspect, the present invention relates to a pharmaceutical composition comprising emicizumab and a protected disulfide isoform, wherein the proportion of the protected disulfide isoform in the antibody molecules in the pharmaceutical composition is reduced. The proportion of the protected disulfide isoform in the total antibody molecules in the pharmaceutical composition can be evaluated by various methods, including the above-mentioned protected disulfide isoform detection / analysis method, and can be expressed, for example, by the proportion of the peak area of ​​the protected disulfide isoform (peak area ratio) when the pharmaceutical composition is analyzed by cation exchange chromatography (CEX) or CE-HPLC. The proportion of the protected disulfide isoform in the total antibody molecules in the pharmaceutical composition (e.g., CEX peak area ratio) is preferably 2% or less, for example, 2.0% or less, 1.5% or less, 1.0% or less, or 0.5% or less. In another aspect, the present invention relates to a method for purifying a composition containing emicizumab, the method comprising a step of cation exchange chromatography (CEX) in bind & elute mode. The present invention also relates to a method for producing a pharmaceutical composition containing emicizumab, the method comprising a step of carrying out the purification method.

[0043] In another aspect, the present invention discloses an isoform of emicizumab (protected disulfide isoform), which has the same heavy and light chain amino acid sequences as emicizumab but has a molecular structure with a smaller Rg (nm) and / or Dmax (nm) value than emicizumab. Such an isoform has a molecular structure in which the distance between the N-terminus of the J chain and the Q chain is shorter than that of emicizumab. Specifically, the average Rg value measured by a SAXS device is 3% or more, preferably 4% or more, more preferably 5% or more, and more preferably 6% or more smaller than that of emicizumab, and / or the average Dmax is 5% or more, preferably 6% or more, more preferably 7% or more, and more preferably 7.5% or more smaller than that of emicizumab. The mean Rg is at least 0.15 nm, preferably at least 0.2 nm, more preferably at least 0.25 nm, more preferably at least 0.3 nm, and / or the mean Dmax is at least 0.5 nm, preferably at least 1.0 nm, more preferably at least 1.2 nm, more preferably at least 1.4 nm, less than that of emicizumab. These isoforms also have a mean Rg of 4.9 nm or less, preferably 4.8 nm or less, and / or a mean Dmax of 17.0 nm or less, preferably 16.5 nm or less. Such Rg and Dmax values ​​are measured under the following conditions. (1) Antibody concentration: Antibody concentration 7.54 mg / mL (2) Solvent conditions: 150 mmol / L arginine, 20 mmol / L histidine-aspartic acid, pH6.0 (3) Temperature: 25℃ Here, the average Rg can be calculated by calculating Rg for each measurement from the Guinier plot and averaging them. The average Dmax can be calculated by calculating Dmax for each measurement from the x-intercept of p(r) and averaging them. The analysis methods for the Guinier plot and p(r) are as described in Example 9. In another aspect, the present invention discloses an isoform (protected disulfide isoform) of emicizumab, which has the same heavy and light chain amino acid sequences as emicizumab but has molecular structural differences compared to emicizumab in the amino acid residues at positions 146 to 174 (EU numbering) in the Q chain (positions 152 to 180 from the N-terminus of SEQ ID NO: 10) and the amino acid residues at positions 146 to 174 (EU numbering) in the J chain (positions 148 to 176 from the N-terminus of SEQ ID NO: 11). Such structural differences can be measured as differences in deuterium exchange rate (%D) in HDX-MS measurements, and specifically, can be confirmed as differences in deuterium exchange time for peptides containing amino acid residues in this region, as shown in Figure 9A. In another aspect, the present invention discloses a pharmaceutical composition comprising emicizumab and the isoform, wherein the proportion of the isoform in all antibody molecules in the pharmaceutical composition is 2% or less. In the above-mentioned antibody molecules, antibody variants, antibody isoforms, pharmaceutical compositions comprising antibody variants or isoforms, methods for analyzing antibody variants or isoforms, and methods for inhibiting the production of antibody variants or isoforms, the antibody is preferably a bispecific antibody, more preferably emicizumab (ACE910).

[0044] As used in this specification, an embodiment expressed by the expression "comprising..." encompasses an embodiment expressed by the expression "essentially consisting of..." as well as an embodiment expressed by the expression "consisting of...".

[0045] The numerical values ​​described in this specification may vary within a certain range due to, for example, the equipment, measurement conditions, and the techniques of a person skilled in the art, and may vary, for example, by about 10% as long as the objectives of the present invention are achieved.

[0046] The contents of all patents and references expressly cited herein are hereby incorporated by reference in their entirety. The present invention is further illustrated by, but not limited to, the following examples. [Example]

[0047] Example 1: Production of recombinant humanized bispecific monoclonal antibody (emicizumab antibody) For structural analysis of emicizumab isomers (antibody variants and isoforms), large amounts of emicizumab antibody were produced using the following method. CHO cells transfected with the gene encoding emicizumab were cultured as emicizumab-producing cells in a commercially available basal medium (basal medium for animal cell culture). The culture conditions were generally suitable for the cultivation of CHO cells. The expressed antibodies were purified by a combination of conventional column chromatography, such as affinity chromatography, ion exchange chromatography, and hydrophobic chromatography.

[0048] [Example 2] Separation of Q-CDR Clipped Variants and Protected Disulfide Isoforms (Cation Exchange High-Performance Liquid Chromatography) The sample solution, diluted with mobile phase A (composition described below), was injected into a cation exchange column (ProPac WCX-10, particle size 10 μm, internal diameter 4.0 mm, length 250 mm). Separation was performed by liquid chromatography using an acidic mobile phase (mobile phase A containing 9.6 mmol / L Tris, 6.0 mmol / L piperazine, and 11.0 mmol / L imidazole buffer, pH 6.0) and an alkaline mobile phase (mobile phase B containing 9.6 mmol / L Tris, 6.0 mmol / L piperazine, 11.0 mmol / L imidazole, and 150 mmol / L sodium chloride, pH 9.9) at a column temperature of 30 ± 5°C, a wavelength of 280 nm, and a flow rate of 1.0 mL / min. As a result, it was confirmed that the Q-CDR clipped variant was separated in the acidic region and the protected disulfide isoform in the alkaline region compared to the main peak corresponding to emicizumab (Figure 1A, Figure 2A).

[0049] [Example 3] Separation of Protected Disulfide Isoforms (IdeS Digestion, Partial Reduction, Reverse-Phase High-Performance Liquid Chromatography) The sample was diluted with phosphate buffer, digested with IdeS protease and PNGase-F, and then partially reduced with DTT in a denaturant-free Tris buffer. The resulting sample was then diluted with TFA and injected into a reversed-phase high-performance liquid chromatography (RPLC). The results showed that the major component of emicizumab, with the disulfide bond between the heavy and light chains reduced, was separated on the chromatograph, while the protected disulfide isoform, with the heavy chain remaining unreduced, was detected as a unique peak (Figure 3B and D).

[0050] [Example 4] Separation of Protected Disulfide Isoforms (IdeS Digestion, Denaturation, Reverse-Phase High-Performance Liquid Chromatography) The sample was diluted with buffer, digested with IdeS protease and PNGase-F, and then denatured in denaturing buffer. The sample was then diluted with TFA solution and injected into a reversed-phase high-performance liquid chromatography (RPLC) system for separation. The F(ab')2 portion of the protected disulfide isoform was found to have a longer retention time than the main component of emicizumab (Figure 4C and D).

[0051] [Example 5] Separation of Protected Disulfide Isoforms (IdeS Digestion, Reverse-Phase High-Performance Liquid Chromatography) The sample was diluted with buffer and digested with IdeS protease and PNGase-F. The diluted sample was then separated by reversed-phase high-performance liquid chromatography (RP-HPLC). The F(ab')2 portion of the protected disulfide isoform was found to have a longer retention time than the main component of emicizumab (Figures 4A and B).

[0052] [Example 6] Evaluation of the biological activity of Q-CDR clipped variants and protected disulfide isoforms Chromogenic assay The amount of activated blood coagulation factor X (FXa) produced by reacting emicizumab with phospholipids in the presence of FIXa and FX was quantified using a specific chromogenic substrate. Specifically, various concentrations of emicizumab dilutions were prepared by adding a solution containing trishydroxymethylaminomethane, sodium chloride, and BSA (TBSB) to the sample. The dilutions were then added to each well of a 96-well microplate. A coagulation factor solution containing FIXa, FX, calcium chloride, magnesium chloride, phospholipids, and TBSB was added to each well, shaken, and allowed to stand for 30 minutes. Subsequently, ethylenediaminetetraacetic acid solution was added to each well, shaken, and then a chromogenic substrate solution (N-benzoyl-L-isoleucyl-L-glutamyl-glycyl-L-arginine-p-nitroaniline hydrochloride and its methyl ester) was added to each well, shaken, and allowed to stand for 35 minutes. After shaking for 1-2 minutes, the absorbance (Abs) of each well was measured at 405 nm using a plate reader. A 4-parameter parallel-line logistic regression program was used to determine the specific activity of the sample relative to the standard solution from the absorbance values ​​obtained for each concentration of sample solution and standard solution. The Q-CDR clipped variant exhibited 18±1% bioactivity, and the protected disulfide isoform exhibited 8±0% bioactivity relative to the emicizumab standard solution.

[0053] Clotting assay In this study, human factor VIII-deficient plasma was used to measure the time to fibrin formation and clotting time, as measured by turbidity change, in a system reflecting the intrinsic coagulation activation mechanism. Specifically, emicizumab dilutions of various concentrations were prepared by adding a solution containing trishydroxymethylaminomethane, sodium chloride, and BSA to the sample. Factor VIII-deficient plasma was added to the dilution solution and incubated, followed by the addition of APTT reagent and incubation. Finally, calcium chloride solution was added and the clotting time was measured using a fully automated blood coagulation analyzer. The specific activity of the sample relative to the standard was calculated using a parallel line assay. The Q-CDR clipped variant and the protected disulfide isoform exhibited biological activity of 18±1% and 16±1%, respectively, relative to the standard emicizumab solution.

[0054] [Example 7] Evaluation of the effect of culture parameters on the ratio of Q-CDR clipped variants [Initial medium] Plant-derived hydrolysates, amino acids, etc. were added to commercially available basal medium, which was then dissolved and sterilized by filtration. [Fed-batch medium] Glucose, amino acids, etc. were added to commercially available basal medium, which was then dissolved and sterilized by filtration. 〔cell〕 Emicizumab-producing CHO cells (DXB-11 strain) into which a gene encoding emicizumab was introduced were used.

[0055] [Culture method] Add production medium (-10 to 10% of the standard concentration) to a 1 L-scale cell culture device, and incubate the above CHO cell line at 2 to 6 × 10 5 The cells were seeded at 100 cells / mL, and culture was initiated at a temperature of 36-38°C, a dissolved oxygen concentration of 40%, and an initial pH of 7.20. From days 1-3 after the start of culture, a fed-batch medium (-10-10% of the standard concentration) was added at a constant flow rate, and from day 3 after the start of culture, the pH was shifted to 6.70-7.10, and culture was continued for 13-15 days. Culture was performed under a total of 56 conditions according to an experimental design based on a central composite design with 12 center points (six factors: production medium concentration, feed medium concentration, initial cell density, temperature, timing of start of feed, and post-shift pH). For all conditions, culture fluid was sampled on days 13, 14, and 15 of culture (168 samples in total), centrifuged (3000 rpm, 5 minutes), and the supernatant was purified with Protein A and used to measure the proportion of Q-CDR clipped variants.

[0056] [Analysis method] Viable cell count and viability were measured using trypan blue staining. Q-CDR clipped variants were detected as peaks by cation exchange high-performance liquid chromatography using a cation column (ProPac WCX-10).

[0057] 〔result〕 The results are shown in Figures 5 and 6. The Q-CDR clipped variant ratio was affected by temperature and post-shift pH. Within the range tested (temperature 36-38°C, post-shift pH 6.70-7.10), incubation at 36°C reduced the Q-CDR clipped variant ratio, while incubation at a post-shift pH of 7.10 reduced the Q-CDR clipped variant ratio. An interaction between temperature and post-shift pH was also observed; even under 38°C incubation conditions, lowering the post-shift pH to 6.70 reduced the Q-CDR clipped variant ratio. By controlling the culture temperature at 36-38°C and the pH at 6.70-7.10 from the third day after the start of culture, the Q-CDR clipped variants could be controlled to 4% or less.

[0058] [Example 8] Removal of Q-CDR Clipped Variants by Cation Exchange Chromatography We established a method for separating Q-CDR clipped variants and emicizumab antibodies by utilizing the difference in electrostatic properties between the two. An example is shown below. A column was packed with GE Capto SP ImpRes or an equivalent cation exchange resin. After equilibration, the emicizumab antibody solution containing the Q-CDR clipped variant was loaded, and both were initially adsorbed. After loading, the column was washed with a phosphate buffer containing sodium chloride, and only the acidic variants, including the Q-CDR clipped variant, were specifically eluted, separated from the emicizumab antibody, and removed. To demonstrate the removal status, the wash fraction, elution fraction, and the load fraction before separation were analyzed by CE-HPLC (Figure 7). The loading, washing, and elution conditions were as follows: Loading: Tris-HCl buffer containing emicizumab antibody, adjusted to pH 5.0, was loaded at 33 g of emicizumab antibody per 1 L of resin. Washing: Phosphate buffer containing 25 mmol / L sodium chloride, adjusted to pH 7.2, was passed through the column for 3.5 CV at room temperature. Elution: Phosphate buffer containing 100 mmol / L sodium chloride, adjusted to pH 6.5, was passed through the column for 6.5 CV at room temperature.

[0059] [Example 9] Analysis of the molecular structure of protected disulfide isoforms Antibody formulations (antibody concentration 7.54 mg / mL, 150 mmol / L arginine, 20 mmol / L histidine-aspartic acid, pH 6.0) were prepared for emicizumab and the protected disulfide isoform, respectively. SAXS measurements were performed using a line-collimated X-ray beam (Cu Kα, λ = 0.1542 nm) on a SAXSess mc2 system (Anton Paar, Graz, Austria). The measurement temperature was 25°C. A two-dimensional imaging plate was used for detection. The X-ray exposure time was 30 minutes. The two-dimensional scattering intensity was converted to one-dimensional scattering intensity I(q) using SAXSQuant software (Anton Paar). Here, q is the scattering vector, defined as q = (4π / λ) sin(θ / 2), and θ is the scattering angle. The scattering curve was normalized by the scattering intensity at q = 0 after passing through the beam stopper, and then blank (capillary and buffer) correction and optical system (Desmear) correction were performed. A Guinier plot was performed on the corrected scattering curve under the condition of q × Rg < 1.3 to determine the radius of gyration Rg (nm). However, if a decrease in scattering intensity was observed at small angles, the Guinier plot was performed excluding such q range to avoid the contribution of interparticle repulsion. In a system assuming no interparticle interactions (structure factor S(q) = 1), the scattering intensity I(q) is given as the Fourier transform of the pair distance distribution function p(r). The indirect Fourier transform method (Non-Patent Document 9) was applied to the corrected scattering curve to obtain the particle p(r). The maximum length Dmax (nm) was obtained from the x-axis intercept of p(r). Measurements were performed three times for each of the emicizumab antibody formulation (Main) and the protected disulfide isoform antibody formulation (BiAb3). As a result, the average Rg value of the protected disulfide isoform was 4.8 nm or less (more than 0.3 nm smaller than emicizumab), and the average Dmax value was 16.5 nm or less (more than 1.4 nm smaller than emicizumab). Compared to emicizumab, the average Rg was more than 6% smaller and the average Dmax was more than 7.5% smaller. It has been reported that the Dmax of IgG4 antibody molecules of the same subclass as emicizumab corresponds to the distance between the tips of the two Fab domains (Non-Patent Document 10). Therefore, in the protected disulfide isoform, the distance between the tips of the two Fab domains is shorter, which is thought to explain the smaller Rg value, which indicates the spread from the center of gravity of the molecule. These results confirm that the protected disulfide isoform has a molecular structure in which the distance between the N-terminus of the J chain and the Q chain is shorter than that of emicizumab (Figure 8). In bispecific antibodies that mediate the interaction between two antigens, the distance between the Fab domains is thought to play a crucial role in the three-dimensional structure of the antibody. Therefore, controlling the ratio of the isoforms in a pharmaceutical composition is important not only for emicizumab but also for bispecific antibody pharmaceuticals in general.

[0060] [Example 10] Analysis of molecular structure by HDX-MS (Hydrogen-Deuterium exchange mass spectrometry) Emicizumab and its protected disulfide isoform were prepared as antibody formulations (antibody concentration 1 mg / mL, 150 mmol / L arginine, 20 mmol / L histidine-aspartic acid, pH 6.0) and subjected to HDX-MS analysis using an HDX-MS instrument (Orbitrap Fusion Lumos (Thermo Fisher Scientific), UltiMate30000RSLCnano (Thermo Fisher Scientific) with HDX-PAL (LEAP Technologies)) with deuterium exchange times of 30 s, 60 s, 120 s, 240 s, 480 s, 960 s, 1920 s, and 3840 s). As a result, a significant difference in the heavy water exchange rate (D%) in HDX-MS measurements was observed for peptides containing amino acid residues at positions 146 to 174 (EU numbering) in the Q chain (positions 152 to 180 from the N-terminus of SEQ ID NO: 10) and amino acid residues at positions 146 to 174 (EU numbering) in the J chain (positions 148 to 176 from the N-terminus of SEQ ID NO: 11). These results confirmed that the structure of the protected disulfide isoform in this region was altered compared to emicizumab (Figure 9). [Industrial Applicability]

[0061] The antibody variants and isoforms of the present invention have extremely low FVIII-like activity compared to emicizumab, and pharmaceutical compositions of the present invention containing emicizumab and having reduced contents of these antibody variants and isoforms are useful as a means for treating hemophilia. Furthermore, the analytical methods for antibody variants and isoforms of the present invention are useful not only for evaluating the quality of emicizumab preparations, but also for developing emicizumab preparations having reduced contents of antibody variants and isoforms, and for developing methods for suppressing the production of antibody variants and isoforms.

Claims

1. A variant of an antibody having a variable region comprising the amino acid sequence SISPSGQSTYYRREVKG (SEQ ID NO: 2), (a) an amino acid residue R at the 12th position from the N-terminus of said sequence; or (b) amino acid residues YYR at positions 10 to 12 from the N-terminus of the sequence; and the variable region is truncated at the deletion site.

2. The antibody variant of claim 1 , wherein the sequence is a CDR sequence.

3. The antibody variant of claim 1, wherein the sequence is a CDR2 sequence.

4. The antibody variant of claim 1 , wherein the sequence is a sequence contained in the heavy chain.

5. The antibody variant of claim 1, which is a bispecific antibody variant.

6. The antibody variant of claim 1, which is a variant of emicizumab.

7. A method for detecting an antibody variant according to any one of claims 1 to 6, comprising a step of separating a sample containing an antibody having a variable region comprising the amino acid sequence SISPSGQSTYYRREVKG (SEQ ID NO: 2) 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.

8. The detection method according to claim 7, wherein the antibody variant according to any one of claims 1 to 6 is used as a standard.

9. A pharmaceutical composition comprising the antibody variant of any one of claims 1 to 6, wherein the proportion of the antibody variant in all antibody molecules in the pharmaceutical composition is 5% or less.

10. The pharmaceutical composition of claim 9, wherein the antibody is emicizumab.

11. 10. The pharmaceutical composition of claim 9, obtained by a purification process comprising purification by cation exchange chromatography (CEX).

12. A method for inhibiting the production of an antibody variant described in any one of claims 1 to 6, comprising a step of culturing antibody-producing cells at a pH of 7.1 or higher and / or a culture temperature of 36°C or lower.

13. An isoform of a bispecific antibody comprising a first heavy chain and a second heavy chain, (1a) between the cysteine ​​at position 144 (EU numbering) in the first heavy chain and the cysteine ​​at position 200 (EU numbering) in the second heavy chain; and (1b) between the cysteine ​​at position 200 (EU numbering) in the first heavy chain and the cysteine ​​at position 144 (EU numbering) in the second heavy chain; forming a disulfide bond in (2a) between the cysteine ​​at position 226 (EU numbering) in the first heavy chain and the cysteine ​​at position 229 (EU numbering) in the second heavy chain; and (2b) between the cysteine ​​at position 229 (EU numbering) in the first heavy chain and the cysteine ​​at position 226 (EU numbering) in the second heavy chain. A bispecific antibody isoform that forms a disulfide bond in the

14. The bispecific antibody isoform according to claim 13, wherein (1a) and (1b) form a disulfide bond.

15. A bispecific antibody isoform comprising a first heavy chain and a second heavy chain, characterized in that when separated using cation exchange chromatography, the bispecific antibody isoform is eluted in a more alkaline region than the bispecific antibody.

16. The bispecific antibody isoform of any one of claims 13 to 15, which is an isoform of emicizumab.

17. The method for detecting antibody isoforms according to any one of claims 13 to 16, comprising a step of separating a sample containing a bispecific 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.

18. The detection method according to claim 17, wherein the antibody isoform according to any one of claims 13 to 16 is used as a standard.

19. A pharmaceutical composition comprising the bispecific antibody isoform of any one of claims 13 to 16, wherein the proportion of the antibody isoform in all antibody molecules in the pharmaceutical composition is 2% or less.

20. A method for reducing the content of a bispecific antibody isoform according to any one of claims 13 to 16, comprising a step of purifying by cation exchange chromatography.

21. 16. The antibody isoform or variant of claim 1, 13, or 15, wherein the biological activity of the antibody is significantly reduced.

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