Antibody variants and isoforms with reduced biological activity

Abstract

An antibody variant (Q-CDR-cleavage variant) in which a specific amino acid residue in a variable region is cleaved and deleted, and an antibody isoform in which an inter-heavy chain disulfide bond is hardly reduced under mild reduction conditions, are provided as antibody variants and isoforms having lower FVIII-like activity than eimeracib.

Description

[0001] This application is a divisional application of the application with an international application date of October 31, 2018, international application number PCT / JP2018 / 040436, which entered the Chinese national phase application number 201880080142.X, and invention name “Antibody variants and isoforms with reduced biological activity”. Technical Field

[0002] The present invention relates to antibody variants and isotypes with reduced biological activity. For example, the present invention relates to antibody variants and isotypes of Emicizumab, wherein the antibody variants and isotypes have reduced coagulation factor VIII (FVIII) simulation activity. The present invention further relates to pharmaceutical compositions comprising such antibody variants or isotypes at low content ratios. The present invention further relates to methods for detecting antibody variants and isotypes and methods for analyzing antibody variants and isotypes. Background Art

[0003] Since antibodies are highly stable in plasma and have few side effects, they have attracted attention as drugs. Among them, many IgG-type antibody drugs are available on the market, and many antibody drugs are currently under development (NPL 1, 2, and 3).

[0004] Hemophilia A is a bleeding abnormality caused by the hereditary reduction or lack of FVIII function. Hemophilia A patients usually use FVIII preparations for bleeding (administered as needed). In recent years, FVIII preparations have also been used prophylactically to prevent bleeding episodes (prophylactic administration; NPL 1 and 2). The half-life of FVIII preparations in blood is about 12 to 16 hours. Therefore, in order to prevent continuous prevention, FVIII preparations (NPL 3 and 4) are administered to patients three times a week. In administration as needed, FVIII preparations are additionally administered at certain intervals as needed to prevent rebleeding. In addition, the administration of FVIII preparations is intravenous administration. Therefore, it is strongly desired to have a lighter administration burden than FVIII preparations.

[0005] Occasionally, antibodies (inhibitors) against FVIII are produced in hemophiliacs. This inhibitor counteracts the effect of FVIII preparations. For bleeding in patients (inhibitor patients) who have produced inhibitors, bypass agents can be used. Their mechanism of action is independent of FVIII function, that is, the function of catalyzing the activation of coagulation factor X (FX) by activated coagulation factor IX (FIXa). Therefore, in some cases, bypass agents cannot fully stop bleeding. Therefore, there is a strong need for an agent that is not affected by the presence of inhibitors and functionally replaces FVIII.

[0006] As a means to solve this problem, bispecific antibodies and uses thereof that functionally replace FVIII have been reported (PTL 1, 2, 3 and 4). By placing the two factors close to each other to show FVIII mimetic activity, bispecific antibodies for FIXa and FX can functionally replace FVIII (NPL 5). It is reported that by optimizing the affinity to FIXa and FX, the FVIII mimetic activity of the antibody can be enhanced (NPL 6). It is reported that ACE910 (one of these antibodies) with high FVIII mimetic activity exerts a hemostatic effect in a monkey model of hemophilia (NPL 7 and 8); therefore, clinical trials are being conducted on patients with hemophilia A.

[0007] Reference list

[0008] [Patent Document]

[0009] [PTL 1] WO 2005 / 035754

[0010] [PTL 2] WO 2005 / 035756

[0011] [PTL 3] WO 2006 / 109592

[0012] [PTL 4] WO 2012 / 067176

[0013] [Non-patent literature]

[0014] [NPL 1] Blood 58, 1-13 (1981)

[0015] [NPL 2]Nature 312,330-337(1984)

[0016] [NPL 3]Nature 312,337-342(1984)

[0017] [NPL 4]Biochim.Biophys.Acta 871,268-278(1986)

[0018] [NPL 5]Nat Med. 2012 Oct;18(10):1570-4.

[0019] [NPL 6]PLoS One.2013;8(2):e57479.

[0020] [NPL 7] J Thromb Haemost. 2014 Feb;12(2):206-213.

[0021] [NPL 8] Blood. 2014 Nov 13;124(20):3165-71.

[0022] [NPL9]J.Appl.Cryst.13,577-584(1980)

[0023] [NPL 10]IUCrJ.2,9-18(2015) Summary of the invention

[0024] [Technical issues]

[0025] The present invention has been achieved in view of the above circumstances. An object of the present invention is to provide an antibody variant or isotype having reduced FVIII mimetic activity.

[0026] [Solution to the problem]

[0027] The present inventors have conducted special research to solve the above problems and have successfully identified antibody variants and isotypes contained in pharmaceutical compositions containing emicizumab as an active ingredient. The present inventors have also found that the FVIII mimetic activity of these antibody variants and isotypes is very low compared to the activity of emicizumab.

[0028] The present invention has been completed based on these findings and provides the following [1]-

[21] .

[0029] [1] An antibody variant comprising a variable region comprising the amino acid sequence SISPSGQSTYYRREVKG (SEQ ID NO: 2), wherein

[0030] (a) an amino acid residue R at the 12th position from the N-terminal side of the sequence (61st position from the N-terminal side of the Q chain of emicizumab: position 60 according to Kabat numbering) or

[0031] (b) amino acid residue YYR at positions 10 to 12 from the N-terminal side of the sequence (positions 59 to 61 from the N-terminal side of the Q chain of emicizumab: positions 58 to 60 according to Kabat numbering)

[0032] is deleted, and the variable region is cleaved at the deletion site.

[0033] [2] The antibody variant as described in [1], wherein the sequence is a CDR sequence.

[0034] [3] The antibody variant described in [1], wherein the sequence is a CDR2 sequence.

[0035] [4] The antibody variant according to [1], wherein the sequence is a sequence contained in the heavy chain.

[0036] [5] The antibody variant according to [1], which is a bispecific antibody variant.

[0037] [6] The antibody variant as described in [1], which is a variant of emicizumab.

[0038] [7] A method for detecting an antibody variant as described in any one of [1] to [6], comprising the step of separating a sample containing an 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, wherein the antibody comprises a variable region comprising the amino acid sequence SISPSGQSTYYRREVKG (SEQ ID NO: 2).

[0039] [8] The detection method as described in [7], which uses the antibody variant described in any one of [1] to [6] as a reference standard.

[0040] [8-2] The detection method of [8], comprising the step of performing one or more analyses, wherein the analyses are selected from the group consisting of quantitative analysis, qualitative analysis, and structural analysis.

[0041] [9] A pharmaceutical composition comprising the antibody variant according to any one of [1] to [6], wherein the percentage of the antibody variant in the total antibody molecules in the pharmaceutical composition is 5% or less.

[0042]

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

[0043]

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

[0044]

[12] A method for inhibiting the production of the antibody variant described in any one 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.

[0045] [12-2] The method according to

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

[0046]

[13] An isotype of a bispecific antibody comprising a first heavy chain (Q chain, SEQ ID NO: 10) and a second heavy chain (J chain, SEQ ID NO: 11), wherein

[0047] Disulfide bonds are formed in:

[0048] (1a) between the cysteine ​​at position 144 according to EU numbering (150th from the N-terminal side of SEQ ID NO: 10) of the first heavy chain and the cysteine ​​at position 200 according to EU numbering (202nd from the N-terminal side of SEQ ID NO: 11) of the second heavy chain; and

[0049] (1b) between the cysteine ​​at position 200 according to EU numbering (the 206th from the N-terminal side of SEQ ID NO: 10) of the first heavy chain and the cysteine ​​at position 144 according to EU numbering (the 146th from the N-terminal side of SEQ ID NO: 11) of the second heavy chain; or wherein

[0050] Disulfide bonds are formed in:

[0051] (2a) between the cysteine ​​at position 226 according to EU numbering (position 229 from the N-terminal side of SEQ ID NO: 10) of the first heavy chain and the cysteine ​​at position 229 according to EU numbering (position 228 from the N-terminal side of SEQ ID NO: 11) of the second heavy chain; and

[0052] (2b) between the cysteine ​​at position 229 according to EU numbering (position 232 from the N-terminus of SEQ ID NO: 10) of the first heavy chain and the cysteine ​​at position 226 according to EU numbering (position 225 from the N-terminus of SEQ ID NO: 11) of the second heavy chain.

[0053]

[14] The bispecific antibody isotype of

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

[0054]

[15] An isotype of a bispecific antibody 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 region closer to the basic side than the bispecific antibody.

[0055]

[16] The isotype of the bispecific antibody according to any one of

[13] to

[15] , which is an isotype of emicizumab.

[0056]

[17] A method for detecting the antibody isotype described in any one of

[13] to

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

[0057]

[18] The detection method of

[17] , which uses the isotype of the bispecific antibody described in any one of

[13] to

[16] as a reference standard.

[0058] [18-2] The detection method as described in

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

[0059]

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

[13] to

[16] , wherein in the pharmaceutical composition, the percentage of the antibody isotype in the total antibody molecules is 2% or less.

[0060]

[20] A method for reducing the percentage of isotype content of the bispecific antibody of any one of

[13] to

[16] , comprising a purification step by cation exchange chromatography.

[0061]

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

[13] or

[15] , wherein the biological activity of the antibody is significantly reduced.

[0062]

[22] An isotype of an antibody or a derivative thereof having two variable regions each recognizing a different epitope, wherein the isotype has an average Rg value that is 3% or more, preferably 4% or more, more preferably 5% or more, or even more preferably 6% or more smaller than the antibody or derivative thereof, and / or wherein the isotype has an average Dmax value that is 5% or more, preferably 6% or more, more preferably 7% or more, or even more preferably 7.5% or more smaller than the antibody or derivative thereof.

[0063]

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

[0064]

[24] The isoform of

[22] or

[23] , wherein the isoform has disulfide bonds that are different from the disulfide bonds in the antibody or its derivative.

[0065]

[25] The isotype of any one of

[22] -

[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 each forms a pair with the first heavy chain or the second heavy chain).

[0066]

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

[0067]

[27] An isoform as described in

[25] or

[26] , wherein the isoform has a disulfide bond between the cysteine ​​at position 144 according to EU numbering of the first heavy chain (position 150 from the N-terminal side of SEQ ID NO:10) and the cysteine ​​at position 200 according to EU numbering of the second heavy chain (position 202 from the N-terminal side of SEQ ID NO:11), and a disulfide bond between the cysteine ​​at position 200 according to EU numbering of the first heavy chain (position 206 from the N-terminal side of SEQ ID NO:10) and the cysteine ​​at position 144 according to EU numbering of the second heavy chain (position 146 from the N-terminal side of SEQ ID NO:11).

[0068]

[28] A pharmaceutical composition comprising emicizumab and the isoform described in any one of

[22] to

[27] , wherein in the pharmaceutical composition, the percentage of the isoform in the total antibody molecules is 2% or less.

[0069]

[29] An isoform of a bispecific antibody (Q499-z121 / J327-z119 / L404-k; emicizumab), the 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 L chain (SEQ ID NO: 12) each forming a pair with the first heavy chain or the second heavy chain, wherein the isoform has a molecular structure difference from emicizumab in the amino acid residues at the following positions: amino acids from position 146 according to EU numbering of the Q chain to position 174 according to EU numbering of the Q chain (152 to 180 from the N-terminal side of SEQ ID NO: 10), and amino acids from position 146 according to EU numbering of the J chain to position 174 according to EU numbering of the J chain (148 to 176 from the N-terminal side of SEQ ID NO: 11).

[0070]

[30] The isoforms of

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

[0071]

[31] A pharmaceutical composition comprising emicizumab and the isoform described in

[29] or

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

[0072] The present invention further provides the following [A1] to [A9].

[0073] [A1] The detection method as described in [7] or

[17] , which comprises the step of subjecting a sample containing the antibody and / or the antibody variant or isotype to a reduction reaction, a hydrolysis reaction (digestion reaction), a protein denaturation reaction or a combination thereof.

[0074] [A2] The detection method as described in [A1], wherein the reduction reaction is carried out under mild reducing conditions (for example, reduction with DTT in Tris buffer (pH 7.0) at 37°C).

[0075] [A3] The detection method as described in [A1], wherein the hydrolysis reaction is carried out using a site-specific cleavage enzyme (for example, a sequence-specific protease such as IdeS protease, Lys-C, papain, etc.).

[0076] [A4] A detection method as described in any one of [7],

[17] and [A1]-[A3], which comprises the step of separating a sample containing the antibody, the antibody variant or isotype, their reaction products 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.

[0077] [A5] The detection method as described in any one of [8],

[18] and [A1]-[A4], which comprises the step of analyzing by SE-HPLC analysis, dynamic light scattering (DLS) method, SAXS measurement, electron microscopy measurement, 3D modeling, SPR determination, HDX MS analysis or a combination thereof.

[0078] [A6] A method for quality control of a pharmaceutical composition comprising emicizumab, comprising the steps of [7], [8],

[17] ,

[18] or [A1] to [A5] or a combination of these methods.

[0079] [A7] A method for preparing a pharmaceutical composition comprising emicizumab, comprising the steps from the method of [A6].

[0080] [A8] A method for purifying a composition comprising emicizumab, characterized in that the method comprises a step of binding & elution mode of cation exchange chromatography (CEX).

[0081] [A9] A method for preparing a pharmaceutical composition comprising emicizumab, comprising the steps of the purification method from [A8].

[0082] Effects of the Invention

[0083] The inventors have successfully identified antibody variants and isoforms contained in pharmaceutical compositions containing emicizumab as an active ingredient. The inventors have also found that the FVIII mimetic activity of these antibody variants and isoforms is very low compared to the FVIII mimetic activity of emicizumab. Therefore, pharmaceutical compositions containing emicizumab and only such antibody variants and isoforms at low levels can be used as a means of treating hemophilia. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] [ Figure 1-1 ] Figure 1A shows the results of separation of emicizumab drug substance by CE-HPLC. The peaks marked with bold boxes represent Q-CDR-splicing variants.

[0085] [ Figure 1-21B is a diagram showing the molecular structure of the Q-CDR-splicing variant. The numbers in the figure and the letters directly below them represent the positions of the amino acid residues counted from the N-terminus of the Q chain of emicizumab and the amino acid residues at that position (single letter code), respectively.

[0086] [ Figure 2-1 ] Figure 2A shows the results of separation of emicizumab drug substance by CE-HPLC. The peak marked with a bold box represents the protected disulfide isoform.

[0087] [ Figure 2-2 ] FIG. 2B is a diagram showing the molecular structure of protected disulfide isoforms. The numbers in the figure and the letter C on the left thereof respectively represent the position of the amino acid residue counted from the N-terminus of the Q chain of Emicizumab and the cysteine ​​residue at that position. FIG. 2C shows the content ratio of protected disulfide isoforms in the drug substance of Emicizumab. The content ratio under various conditions (culture conditions of antibody-producing cells) is represented by the average value (mean) of the peak area percentage (area %) of the protected disulfide isoforms in the CE-HPLC separation results (shown in FIG. 2A) and their standard deviation (StdDev).

[0088] [ Figure 3 ] Figure 3 Shown are the results of separation of emicizumab and protected disulfide isoforms by reverse phase HPLC after IdeS digestion and reduction treatment. Figure 3 AD show the separation results of samples containing emicizumab (A and B) or protected disulfide isoforms (C and D) that were digested with IdeS and then reduced in the presence of denaturant (A and C: fully reduced conditions) or in the absence of denaturant (B and D: partially reduced conditions). For samples from fully reduced conditions ( Figure 3 A and C), for both emicizumab and the protected disulfide isoforms, 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: no difference in reduction pattern was detected between emicizumab and the protected disulfide isoforms. On the other hand, for samples from partially reduced conditions ( Figure 3 B and D), the same peaks representing Q chain Fd, J chain Fd, Q chain Fc, J chain Fc and L chain as under fully reduced conditions were detected only for the protected disulfide isoforms, and in addition to this, a unique peak indicating a heterodimer of Q chain Fd and J chain Fd (J-Fd-Q-Fd) bonded together by disulfides was detected.

[0089] [ Figure 4 ] Figure 4Shown are the results of separation of emicizumab and protected disulfide isoforms by reverse phase HPLC after IdeS digestion (and reduction treatment). Figure 4 A and B show the separation results of IdeS digested protected disulfide isoforms (A) or emicizumab (B). Figure 4 C and D show the separation results of the protected disulfide isoform (C) or emicizumab digested with IdeS and then denatured. Regardless of whether the denaturation treatment was performed, the F(ab')2 portion of the protected disulfide isoform (LC-J Fab-Q Fab-LC) was separated with a longer retention time than the main component of emicizumab.

[0090] [ Figure 5 ] Figure 5 The content ratio of Q-CDR-splicing variants in the culture supernatant of emicizumab-producing CHO cells cultured under various culture conditions is shown. The culture supernatant samples purified using protein A were used to measure the content ratio of Q-CDR-splicing variants. The vertical axis represents the Q-CDR-splicing variant content ratio (peak area %), and the horizontal axis represents various culture conditions.

[0091] [ Figure 6 ] Figure 6 The content ratio of Q-CDR-splicing variants in the culture supernatant of emicizumab-producing CHO cells cultured under various culture conditions is shown. The culture supernatant samples purified using protein A were used to measure the content ratio of Q-CDR-splicing variants. The vertical axis represents the Q-CDR-splicing variant content ratio (peak area %), and the horizontal axis represents various culture conditions.

[0092] [ Figure 7 ] Figure 7 The CE-HPLC analysis results of each fraction of the antibody Emicizumab solution containing Q-CDR-splicing variants are shown, and the fractions are obtained during the purification process including the steps in the cation exchange chromatography (CEX) binding & elution mode. The "loaded fraction" shows the CE-HPLC analysis results of the antibody solution loaded on the cation exchange column. The "washed fraction" represents the CE-HPLC analysis results of the column adsorption fraction obtained after passing through a pH7.2 phosphate buffer (washing) containing 25mmol / L sodium chloride. The "eluted fraction" is shown in the CE-HPLC analysis results of the column adsorption fraction obtained after passing through a pH6.5 phosphate buffer containing 100mmol / L sodium chloride. It was found that in the eluted fraction, the peak of the Q-CDR-splicing variant on the more acidic side than the peak of the antibody Emicizumab disappeared compared to the loaded fraction and the washed fraction.

[0093] [ Figure 8 ] Figure 8AD shows the results of analyzing the molecular structures of emicizumab (MAIN) and protected disulfide isoform (BiAb3) using a SAXS apparatus. "Pair-distance distribution function [p(r)]", "Rg(nm)" and "Dmax(nm)" represent the pair-distance distribution function, the radius of gyration and the maximum size, respectively.

[0094] [ Figure 9-1 ] FIG. 9A shows the residual graph of Emicizumab (the main component in cation exchange HPLC) and the protected disulfide isoform on the deuterium exchange rate (%D) in HDX-MS measurement (deuterium exchange time 30s, 60s, 120s, 240s, 480s, 960s, 1920s and 3840s). Each bar in the figure represents the sum of the result differences of each deuterium exchange time of Q chain, J chain and L chain. FIG. 9B shows the part of the molecular structure difference between the protected disulfide isoform and Emicizumab suggested by HDX-MS measurement. In the peptide comprising the amino acid residues from position 146 according to EU numbering of the Q chain to position 174 according to EU numbering of the Q chain (152 to 180 from the N-terminal side of SEQ ID NO: 10) and the peptide comprising the amino acid residues from position 146 according to EU numbering of the J chain to position 174 according to EU numbering of the J chain (148 to 176 from the N-terminal side of SEQ ID NO: 11), the difference in the molecular structure of the protected disulfide isoform and that of Emicizumab is significant. See the part indicated by an asterisk.

[0095] [ Figure 9-2 ] Figure 9-2 yes Figure 9-1 The continuation of. Specific implementation plan

[0096] One embodiment of the present invention relates to antibody variants and isotypes with reduced biological activity (e.g., reduced FVIII mimetic activity). During the inventors' analysis of the emicizumab drug substance, antibody variants and isotypes were identified as two types of structurally altered molecules (Q-CDR-splicing variants and protected disulfide isotypes). In the present application, "antibody variants" and "antibody isotypes" may also be referred to as mutants or isomers of antibody molecules.

[0097] Emicizumab is a bispecific humanized IgG4 antibody that exhibits the activity of functionally replacing FVIII as a cofactor and comprises anti-FIX(a) and anti-FX, and comprises two types of heavy chains (Q499 and J327) and a common L chain (L404), wherein each heavy chain recognizes FIX(a) and FX, respectively.

[0098] Specifically, emicizumab is a bispecific antibody, wherein the first polypeptide and the third polypeptide form a pair, and the second polypeptide and the fourth polypeptide form a pair; wherein the first polypeptide comprises an H chain, and the H chain comprises the amino acid sequences of H chain CDRs 1, 2 and 3 (H chain CDRs of Q499) of SEQ ID NOs: 1, 2 and 3, respectively; the second polypeptide comprises an H chain, and the H chain comprises the amino acid sequences of H chain CDRs 1, 2 and 3 (H chain CDRs of J327) of SEQ ID NOs: 4, 5 and 6, respectively; and the third polypeptide and the fourth polypeptide comprise a common L chain, and the common L chain comprises the amino acid sequences of L chain CDRs 1, 2 and 3 (L chain CDRs of L404) of SEQ ID NOs: 7, 8 and 9, respectively (Q499-z121 / J327-z119 / L404-k).

[0099] More specifically, Emicizumab is a bispecific antibody, wherein the first polypeptide and the third polypeptide form a pair, and the second polypeptide and the fourth polypeptide form a pair; wherein the first polypeptide comprises an H chain, the H chain comprises the amino acid sequence of the H chain variable region of SEQ ID NO: 13, the second polypeptide comprises an H chain, the H chain comprises the amino acid sequence of the H chain variable region of SEQ ID NO: 14, and the third polypeptide and the fourth polypeptide comprise a common L chain, the common L chain comprises the amino acid sequence of the L chain variable region of SEQ ID NO: 15.

[0100] Even more specifically, Emicizumab is a bispecific antibody, wherein the first polypeptide and the third polypeptide form a pair, and the second polypeptide and the fourth polypeptide form a pair; wherein the first polypeptide comprises an H chain, the H chain comprises the amino acid sequence of SEQ ID NO: 10, the second polypeptide comprises an H chain, the H chain comprises the amino acid sequence of SEQ ID NO: 11, and the third polypeptide and the fourth polypeptide comprise a common L chain of SEQ ID NO: 12 (Q499-z121 / J327-z119 / L404-k).

[0101] Such antibodies can be obtained by the methods described in WO2005 / 035756, WO2006 / 109592, WO2012 / 067176, and the like.

[0102] The antibodies used in the present invention are not particularly limited as long as they bind to the desired antigen, and they may be polyclonal antibodies or monoclonal antibodies. Monoclonal antibodies are preferred because homogeneous antibodies can be stably produced.

[0103] The amino acids contained in the amino acid sequences of the present invention may be post-translationally modified (for example, modification of N-terminal glutamine to pyroglutamic acid by pyroglutamylation is well known to those skilled in the art). Naturally, such post-translationally modified amino acids are included in the antibodies used in the present invention.

[0104] In the present invention, the biological activity of the antibody or antibody variant or antibody isotype is preferably FVIII mimetic activity. In the present invention, "FVIII mimetic activity" refers to the activity of functionally replacing FVIII (functionally replacing the activity of FVIII as a cofactor). In the present invention, the phrase "functionally replacing FVIII" refers to identifying FIX or FIXa and FX, and promoting FX activation by FIXa (promoting FXa production by FIXa). For example, a measurement system comprising FIXa, FX, a synthetic substrate S-2222 (a synthetic substrate of FXa) and a phospholipid can be used to evaluate FXa production promoting activity. This measurement system shows a correlation with disease severity and clinical symptoms in hemophilia A cases (Rosen S, Andersson M, M et al. Clinical applications of a chromogenic substrate method for determination of FVIIIactivity. ThrombHaemost 1985;54:811-23).

[0105] The FVIII mimetic activity of antibodies such as emicizumab and antibody variants and antibody isotypes can be evaluated according to the methods described in, for example, WO2005 / 035756, WO2006 / 109592, WO2012 / 067176, and the like.

[0106] In the present invention, when the biological activity of an antibody or antibody variant or isotype is reduced compared to the biological activity of a reference antibody, the antibody or antibody variant or isotype is referred to as "having reduced biological activity", and preferably, the reduction is statistically significant. In the present invention, when the biological activity of an antibody or antibody variant or antibody isotype is reduced by 10% or more, such as 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 reference antibody, the antibody or antibody variant or antibody isotype is referred to as "having significantly (or extremely) reduced biological activity".

[0107] In the present invention, the terms "H chain" and "J chain" refer to H chains (heavy chains) comprising variable regions capable of exhibiting binding abilities to FIX(a) and FX, respectively.

[0108] In the present invention, the term "common L chain" refers to an L chain that can form a pair with each of two or more different H chains and can exhibit binding ability to their respective antigens. Here, the term "different H chains" preferably refers to H chains of antibodies against different antigens, but is not limited thereto; it refers to H chains whose amino acid sequences are different from each other. The common L chain can be obtained, for example, according to the method described in WO2006 / 109592.

[0109] The term "antibody" is used in the broadest sense, including monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (such as bispecific antibodies), antibody derivatives and modified antibodies (Miller K et al. J Immunol. 2003, 170 (9), 4854-61), as long as they show the desired biological activity. The antibody can be a mouse antibody, a human antibody, a humanized antibody, a chimeric antibody or an antibody derived from another species, or an artificially synthesized antibody. The antibodies disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules. Immunoglobulins can be derived from any species (e.g., humans, mice or rabbits). The terms "antibody", "immunoglobulin" and "immunoglobulin" are used interchangeably in a broad sense.

[0110] "Bispecific antibodies" refer to antibodies with two variable regions, each of which recognizes a different epitope, wherein the variable regions are present in the same antibody molecule. Bispecific antibodies can be antibodies that recognize two or more different antigens, or antibodies that recognize two or more different epitopes on the same antigen. Bispecific antibodies can include not only complete antibodies, but also antibody derivatives.

[0111] Recombinant antibodies produced by using genetic engineering techniques can be used as antibodies. Recombinant antibodies can be obtained by cloning DNA encoding the antibody from a hybridoma or an antibody-producing cell (e.g., a sensitized lymphocyte producing the antibody); inserting it into a vector; and then introducing it into a host (host cell) to produce the antibody.

[0112] Bispecific antibodies are not limited to those of IgG type; for example, IgG type bispecific antibodies can be secreted from hybridized hybridomas (quadromas) produced by fusing two types of hybridomas producing IgG antibodies (Milstein C. et al., Nature 1983, 305: 537-540). They can also be secreted by introducing L chain and H chain genes constituting two IgGs of interest, i.e., a total of four genes, into cells to co-express these genes.

[0113] The antibodies of the present invention can be produced by methods known to those skilled in the art. Specifically, the DNA encoding the antibody of interest is inserted into an expression vector. Insertion into the expression vector is performed so that expression occurs under the control of expression regulatory regions such as enhancers and promoters. Then, the expression vector is used to transform host cells to express the antibody. In this case, a suitable combination of host and expression vector can be used.

[0114] The antibody of the present invention thus obtained can be separated from the host cell interior or cell exterior (culture medium etc.), and purified into substantially pure, homogeneous antibody.Can be separated and purified by the method for separating and purifying antibody conventionally, and the method is not limited in any way.For example, the method described in known WO2013 / 086448 is used to separate IgG2 disulfide isotypes.In order to separate and purify the antibody and antibody variant or antibody isotype in the present invention, for example, can separate and purify antibody and antibody variant or antibody isotype by suitable selection and combination column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization etc.For example, in the separation and purification using chromatographic column, various types of matrix can be used, including strong cation exchange matrix, weak cation exchange matrix, anti-human IgG affinity matrix and protein L matrix.

[0115] In one aspect, the invention relates to antibody variants (sometimes referred to herein as "Q-CDR-splicing variants") having the following features:

[0116] - Very low biological activity (FVIII mimetic activity) compared to emicizumab;

[0117] - The fragment on the N-terminal side of the deleted amino acid residue and the other fragment on the C-terminal side are linked together by a disulfide bond ( Figure 1B );

[0118] -The amount produced varies depending on the time, temperature and pH of culturing the antibody-producing cells.

[0119] In one embodiment, the Q-CDR-splicing variant is an antibody variant comprising a variable region comprising the amino acid sequence SISPSGQSTYYRREVKG (SEQ ID NO: 2), wherein

[0120] (a) an amino acid residue R at position 12 from the N-terminal side of the amino acid sequence of SEQ ID NO: 2 (position 61 from the N-terminal side of the Q chain of emicizumab: position 60 according to Kabat numbering); or

[0121] (b) amino acid residue YYR at positions 10 to 12 from the N-terminal side of the amino acid sequence of SEQ ID NO: 2 (positions 59 to 61 from the N-terminal side of the Q chain of emicizumab: positions 58 to 60 according to Kabat numbering),

[0122] is deleted, and the variable region is cleaved at the deletion site.

[0123] The Q-CDR-splicing variant is preferably a variant of a bispecific antibody, more preferably a variant of emicizumab.

[0124] In another aspect, the present invention also relates to a method for detecting Q-CDR-splicing variants and a method for analyzing Q-CDR-splicing variants. In one embodiment, the method for detecting Q-CDR-splicing variants comprises the step of separating a sample containing an 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, wherein the antibody comprises a variable region comprising the amino acid sequence SISPSGQSTYYRREVKG (SEQ ID NO: 2). In one embodiment, the method for analyzing Q-CDR-splicing variants comprises the step of performing one or more analyses using the Q-CDR-splicing variant as a reference standard, wherein the analyses are selected from the group consisting of quantitative analysis, qualitative analysis, and structural analysis.

[0125] In this detection method and analysis method, the presence or absence of the missing part in the 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 missing part can be detected, for example, by using the molecular weight change caused by the deletion in LCMS analysis as an indicator. In the Q-CDR-splicing variant, the fragment on the N-terminal side of the deleted amino acid residue and the other fragments on the C-terminal side are linked together by a disulfide bond; therefore, by analyzing the sample after the reaction of reducing the disulfide bond, various analytical techniques such as CE-HPLC, LCMS, LC-UV can be used to detect the difference in the reduction pattern caused by the presence or absence of the missing part. Structural analysis by NMR measurement and the like can also be used.

[0126] Alternatively, differences in resolution by ion exchange chromatography can be used as an indicator. For example, when separated by cation exchange chromatography, the Q-CDR-splice variants will separate in a more acidic region than the main peak of emicizumab.

[0127] In another aspect, in the present invention, the production and quality control of a pharmaceutical composition comprising Emicizumab can be implemented by performing one of the above-mentioned detection methods and analytical methods or any combination thereof. Therefore, the present invention relates to a method for quality control of a pharmaceutical composition comprising Emicizumab, comprising the step of performing the above-mentioned detection methods and analytical methods or the step of combining any of those methods. The present invention also relates to the preparation of a pharmaceutical composition comprising Emicizumab, comprising the step of performing such a method for quality control.

[0128] In another aspect, the present invention relates to a pharmaceutical composition comprising emicizumab and a Q-CDR splice variant, wherein in the pharmaceutical composition, the ratio of the Q-CDR splice variant in the total antibody molecule remains low. The pharmaceutical composition can be obtained by a purification method comprising purification by cation exchange chromatography (CEX). For example, an antibody solution containing emicizumab and a Q-CDR splice variant is adsorbed onto a cation exchange column, and then, only the acidic side variants including the Q-CDR splice variant can be selectively eluted and removed. The ratio of the Q-CDR-splicing variant in the total antibody molecule in the pharmaceutical composition can be evaluated by various methods, including the above-mentioned method for detecting / analyzing the Q-CDR-splicing variant, and can be represented by the ratio of the peak areas of the Q-CDR-splicing variants obtained by analyzing the pharmaceutical composition using, for example, cation exchange chromatography (CEX) or CE-HPLC (peak area ratio). The ratio of Q-CDR-splicing variants in the total antibody molecules in the pharmaceutical composition (e.g., CEX peak area ratio) 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.

[0129] The present invention further relates to a method for preparing a pharmaceutical composition in which the content ratio of Q-CDR splice variants is kept low, and a method for inhibiting the formation of Q-CDR splice variants. The amount of Q-CDR splice variants formed can be reduced by shortening the culture time of antibody-producing cells (e.g., shortening to 15 days or less, preferably 13 days or less), or by lowering the culture temperature of antibody-producing cells (e.g., lowering to 38° C. or less, preferably 37° C. or less, more preferably 36° C. or less), and / or by increasing the culture pH of antibody-producing cells (e.g., increasing to 6.7 or more, preferably 6.9 or more, more preferably 7.1 or more). Figure 4). Therefore, the above method is characterized in that the method includes the following steps: culturing the antibody (e.g., emicizumab) producing cells for a certain period of time (e.g., about 15 days or less) at a lower culture temperature (e.g., about 36°C or lower) and a higher pH (e.g., 7.1 or higher) than conventional. In one embodiment, the above method includes the step of culturing the antibody producing cells at a pH of 7.1 or higher and / or at a culture temperature of 36°C or lower. In certain embodiments, the above method is characterized in that, midway through the culture (e.g., on the second day of culture or later), the culture conditions of the antibody producing cells are changed to a pH of 7.1 or higher and / or a culture temperature of 36°C or lower.

[0130] In another aspect, the present invention relates to a method for purifying a composition comprising Emicizumab, the method being characterized by a step comprising a bind & elute mode of cation exchange chromatography (CEX). The present invention further relates to a method for preparing a pharmaceutical composition comprising Emicizumab, the method comprising the step of performing a purification method.

[0131] In another aspect, the invention relates to antibody isoforms (sometimes referred to herein as "protected disulfide isoforms") having the following characteristics:

[0132] - Very low biological activity (FVIII mimetic activity) compared to emicizumab;

[0133] -More hydrophobic than emicizumab;

[0134] - have inter-heavy chain disulfide bonds ( FIG. 2B ) that are less sensitive to reduction under mild conditions (partially reducing conditions) than emicizumab;

[0135] - Formed independently of conditions (production parameters) such as the dissolved oxygen concentration and initial pH of the culture medium for antibody-producing cells, and the culture time before the addition of MTX (methotrexate).

[0136] In one embodiment, the protected disulfide isoforms are characterized in that they are able to bind to the antigens FIX(a) and FX, but do not exhibit biological activity (FVIII mimetic activity).

[0137] In certain embodiments, a protected disulfide isoform is a structural isomer that has the same (normal) disulfide bonds as emicizumab, but is more hydrophobic than usual due to structural changes in the Fab portion and thus has inter-heavy chain disulfide bonds that are less sensitive to reduction than usual.

[0138] In another embodiment, the protected disulfide isoform is an isoform of a bispecific antibody comprising a first heavy chain (Q chain, SEQ ID NO: 10) and a second heavy chain (J chain, SEQ ID NO: 11), wherein, in the protected disulfide isoform,

[0139] Disulfide bonds are formed in:

[0140] (1a) between the cysteine ​​at position 144 according to EU numbering (150th from the N-terminal side of SEQ ID NO: 10) of the first heavy chain and the cysteine ​​at position 200 according to EU numbering (202nd from the N-terminal side of SEQ ID NO: 11) of the second heavy chain; and

[0141] (1b) between the cysteine ​​at position 200 according to EU numbering (the 206th from the N-terminal side of SEQ ID NO: 10) of the first heavy chain and the cysteine ​​at position 144 according to EU numbering (the 146th from the N-terminal side of SEQ ID NO: 11) of the second heavy chain; or wherein

[0142] Disulfide bonds are formed in:

[0143] (2a) between the cysteine ​​at position 226 according to EU numbering (position 229 from the N-terminal side of SEQ ID NO: 10) of the first heavy chain and the cysteine ​​at position 229 according to EU numbering (position 228 from the N-terminal side of SEQ ID NO: 11) of the second heavy chain; and

[0144] (2b) between the cysteine ​​at position 229 according to EU numbering (232nd from the N-terminal side of SEQ ID NO: 10) of the first heavy chain and the cysteine ​​at position 226 according to EU numbering (225th from the N-terminal side of SEQ ID NO: 11) of the second heavy chain.

[0145] In a specific embodiment, the protected disulfide isoform is an isoform of a bispecific antibody, and preferably an isoform of emicizumab, wherein, in the protected disulfide isoform,

[0146] Disulfide bonds are formed in:

[0147] (1a) between the cysteine ​​at position 144 according to EU numbering (150th from the N-terminal side of SEQ ID NO: 10) of the first heavy chain and the cysteine ​​at position 200 according to EU numbering (202nd from the N-terminal side of SEQ ID NO: 11) of the second heavy chain;

[0148] (1b) between the cysteine ​​at position 200 according to EU numbering (the 206th from the N-terminal side of SEQ ID NO: 10) of the first heavy chain and the cysteine ​​at position 144 according to EU numbering (the 146th from the N-terminal side of SEQ ID NO: 11) of the second heavy chain;

[0149] (1c) between the cysteine ​​at position 226 according to EU numbering (position 229 from the N-terminal side of SEQ ID NO: 10) of the first heavy chain and the cysteine ​​at position 226 according to EU numbering (position 225 from the N-terminal side of SEQ ID NO: 11) of the second heavy chain; and

[0150] (1d) between the cysteine ​​at position 229 according to EU numbering (position 232 from the N-terminal side of SEQ ID NO: 10) in the first heavy chain and the cysteine ​​at position 229 according to EU numbering (position 228 from the N-terminal side of SEQ ID NO: 11) in the second heavy chain; or wherein

[0151] Disulfide bonds are formed in:

[0152] (2a) between the cysteine ​​at position 226 according to EU numbering (229th from the N-terminal side of SEQ ID NO: 10) of the first heavy chain and the cysteine ​​at position 229 according to EU numbering (228th from the N-terminal side of SEQ ID NO: 11) of the second heavy chain;

[0153] (2b) between the cysteine ​​at position 229 according to EU numbering (position 232 from the N-terminal side of SEQ ID NO: 10) of the first heavy chain and the cysteine ​​at position 226 according to EU numbering (position 225 from the N-terminal side of SEQ ID NO: 11) of the second heavy chain;

[0154] (2c) between the cysteine ​​at position 144 according to EU numbering (the 150th from the N-terminal side of SEQ ID NO: 10) of the first heavy chain and the cysteine ​​at position 200 according to EU numbering (the 206th from the N-terminal side of SEQ ID NO: 10) of the first heavy chain; and

[0155] (2d) between the cysteine ​​at position 144 according to EU numbering (the 146th from the N-terminal side of SEQ ID NO: 11) of the second heavy chain and the cysteine ​​at position 200 according to EU numbering (the 202nd from the N-terminal side of SEQ ID NO: 11) of the second heavy chain.

[0156] In another aspect, the present invention relates to a method for detecting protected disulfide isoforms and a method for analyzing protected disulfide isoforms. In one embodiment, a method for detecting protected disulfide isoforms, comprising the 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. In one embodiment, a method for analyzing protected disulfide isoforms comprises the step of performing one or more analyses using protected disulfide isoforms as reference standards, wherein the analysis is selected from the group consisting of quantitative analysis, qualitative analysis, and structural analysis.

[0157] In such detection and analysis methods, the differences in the structure of the region forming the inter-heavy chain disulfide bonds and / or the Fab region between the protected disulfide isoforms and emicizumab can be used for analysis. The structural differences can be detected by various analytical methods (such as, for example, those shown below).

[0158] For example, peaks reflecting differences in three-dimensional structure or differences in hydrophobicity intensity between emicizumab and protected disulfide isoforms can be detected by analyzing the sample using a reverse phase column (e.g., a C4 column) after the sample has been treated under non-reducing conditions for an IdeS protease digestion reaction (which cleaves at a single site below the hinge region in IgG to yield F(ab')2 and Fc fragments).

[0159] Peaks reflecting the difference in sensitivity of disulfide bonds to reduction between emicizumab and protected disulfide isoforms can be detected by analyzing the samples using a reverse phase column after the samples have been treated for IdeS digestion and subsequently for a reduction reaction under mild reducing conditions (e.g., reduction with DTT in Tris buffer (pH 7.0) at 37°C).

[0160] When the reduction reaction is carried out under conditions where all disulfide bonds are reduced, no difference will be detected in the analytical results obtained using the reverse phase columns for emicizumab and the protected disulfide isoforms, whereas when the reduction reaction is carried out under the above-mentioned mild conditions, a difference in the reduction pattern will be detected in the analytical results obtained using the reverse phase columns for them. Without wishing to be bound by any particular theory, it is believed that under the above-mentioned mild reducing conditions, in the case of emicizumab, both the disulfide bonds between the heavy chain and the light chain and the disulfide bonds between the heavy chains are reduced, whereas in the case of the protected disulfide isoforms, only the disulfide bonds between the heavy chain and the light chain are reduced, while the disulfide bonds between the heavy chains are not reduced.

[0161] Peaks reflecting differences in Lys-C digestion patterns caused by differences in the three-dimensional structure between emicizumab and the protected disulfide isoforms can be detected by analyzing the sample using a reverse phase column (e.g., a C4 column) after the sample has been treated for limited Lys-C digestion (e.g., by stopping the Lys-C digestion reaction midway) under non-denaturing conditions (e.g., in Tris buffer). Without wishing to be bound by any particular theory, it is believed that Lys-C digestion patterns reflecting differences in the three-dimensional structure are detected as a result of preferential cleavage at positions that are accessible to Lys-C while maintaining the three-dimensional structure during Lys-C digestion under non-denaturing conditions.

[0162] Alternatively, peaks reflecting differences in Lys-C digestion patterns caused by differences in the three-dimensional structure between emicizumab and the protected disulfide isoforms can be detected by analyzing samples using a reverse phase column after a sample that has been pretreated for denaturation (e.g., 5 M guanidine at 37° C. for 30 minutes) but not reduced (i.e., retaining SS bonds) has been treated for limited Lys-C digestion. Without wishing to be bound by any particular theory, it is believed that when a denatured but not reduced sample is subjected to Lys-C digestion, cleavage is preferentially performed at a position that is easily accessible to Lys-C in a state where the three-dimensional structure is no longer retained but the SS bonds (disulfide bonds) are retained, so a Lys-C digestion pattern reflecting differences in SS bonds is detected.

[0163] In addition to the above-mentioned reduction reaction, IdeS digestion and limited Lys-C digestion under non-denaturing conditions or denaturing conditions, various other decomposition reactions such as papain digestion can be used. In addition to reverse phase chromatography using a C4 column, etc., various analytical techniques such as SE-HPLC analysis, dynamic light scattering (DLS), SAXS measurement, electron microscopy measurement, 3D modeling, SPR determination, HDX MS analysis can be used.

[0164] In another aspect, in the present invention, the production and quality control of a pharmaceutical composition comprising Emicizumab can be implemented by performing one of the above-mentioned detection methods and analytical methods or any combination thereof. Therefore, the present invention relates to a method for quality control of a pharmaceutical composition comprising Emicizumab, comprising the step of performing the above-mentioned detection methods and analytical methods or the step of combining any of those methods. The present invention also relates to the preparation of a pharmaceutical composition comprising Emicizumab, comprising the step of performing such a method for quality control.

[0165] In another aspect, the present invention relates to a pharmaceutical composition comprising emicizumab and a protected disulfide isoform, wherein in the pharmaceutical composition, the ratio of the protected disulfide isoform in the total antibody molecule remains low. The ratio of the protected disulfide isoform in the total antibody molecule in the pharmaceutical composition can be evaluated by various methods, including the above-mentioned method for detecting / analyzing the protected disulfide isoform, and can be represented by the ratio of the peak area of ​​the protected disulfide isoform obtained by analyzing the pharmaceutical composition, for example, using cation exchange chromatography (CEX) or CE-HPLC (peak area ratio). The ratio of the protected disulfide isoform in the total antibody molecule 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.

[0166] In another aspect, the present invention relates to a method for purifying a composition comprising Emicizumab, the method being characterized by a step comprising a bind & elute mode of cation exchange chromatography (CEX). The present invention further relates to a method for preparing a pharmaceutical composition comprising Emicizumab, the method comprising the step of performing a purification method.

[0167] In another aspect, the present invention discloses an isoform of Emicizumab (protected disulfide isoform), which has the same heavy chain and light chain amino acid sequences as Emicizumab, but has a molecular structure with a smaller Rg (nm) value and / or Dmax (nm) value than Emicizumab. This isoform has a molecular structure in which the distance between the N-termini of the J chain / Q chain is shorter than that of Emicizumab, and in particular has an average Rg value of 3% or more, preferably 4% or more, more preferably 5% or more, or even more preferably 6% or more, less than Emicizumab measured by a SAXS device, and / or has an average Dmax value of 5% or more, preferably 6% or more, more preferably 7% or more, or even more preferably 7.5% or more, less than Emicizumab measured by a SAXS device. The isoforms may have an average Rg value that is 0.15 nm or more, preferably 0.2 nm or more, more preferably 0.25 nm or more, or even more preferably 0.3 nm or more less than emicizumab, and / or may have an average Dmax value that is 0.5 nm or more, preferably 1.0 nm or more, more preferably 1.2 nm or more, or even more preferably 1.4 nm or more less than emicizumab. These isoforms may have an average Rg value of 4.9 nm or less, or preferably 4.8 nm or less, and / or have an average Dmax value of 17.0 nm or less, or preferably 16.5 nm or less.

[0168] Rg and Dmax values ​​can be determined under the following identified conditions:

[0169] (1) Antibody concentration: The antibody concentration is 7.54 mg / mL;

[0170] (2) Solvent conditions: 150 mmol / L arginine, 20 mmol / L histidine-aspartic acid, pH 6.0; and

[0171] (3) Temperature: 25°C.

[0172] Here, the average Rg value can be obtained by calculating the Rg of each measurement from the Guinier plot and averaging them. The average Dmax value can be obtained by calculating the Dmax of each measurement from the x intercept of p(r) and averaging them. For the method of analyzing the Guinier plot and p(r), see Example 9 described below.

[0173] In another aspect, the present invention discloses an isoform of Emicizumab (protected disulfide isoform) having the same heavy chain and light chain amino acid sequences as Emicizumab, but having a different molecular structure from Emicizumab in the following amino acid residues: amino acid residues from position 146 according to EU numbering of the Q chain to position 174 according to EU numbering of the Q chain (152 to 180 from the N-terminal side of SEQ ID NO: 10), and amino acid residues from position 146 according to EU numbering of the J chain to position 174 according to EU numbering of the J chain (148 to 176 from the N-terminal side of SEQ ID NO: 11). The difference in molecular structure can be measured as a difference in deuterium exchange rate (%D) in HDX-MS measurement, and can be confirmed as a difference in deuterium exchange time of peptides containing amino acid residues in these regions, as specifically shown in FIG. 9A .

[0174] In another aspect, the present invention discloses a pharmaceutical composition comprising emicizumab and an isotype, wherein the percentage of the isotype among the total antibody molecules in the pharmaceutical composition is 2% or less.

[0175] In the above-mentioned antibody molecules, antibody variants, antibody isotypes, pharmaceutical compositions comprising antibody variants or isotypes, methods for analyzing antibody variants or isotypes, or methods for inhibiting the formation of antibody variants or isotypes, the antibody is preferably a bispecific antibody, more preferably emicizumab (ACE910).

[0176] As used herein, aspects referred to by the expression "comprising" include those referred to by the expression "consisting essentially of" and those referred to by the expression "consisting of.

[0177] The numerical values ​​described herein may vary within a certain range, for example, depending on instruments or equipment, measurement conditions and procedures used by those skilled in the art, and as long as they are within the range allowing the purpose of the present invention to be achieved, they may include a deviation of, for example, about 10%.

[0178] All patents and literature references explicitly cited herein are incorporated by reference into this specification in their entirety.

[0179] The present invention is further illustrated by the following examples, but should not be construed as being limited thereto.

[0180] Specific implementation plan [Example 1] Genetically engineered humanized bispecific monoclonal antibody (antibody Emycel Preparation of β-cell monoclonal antibody

[0181] For the structural analysis of emicizumab isomers (antibody variants and isotypes), emicizumab antibodies were prepared in large quantities by the following method. CHO cells into which the gene encoding emicizumab was introduced were cultured as emicizumab producing cells in a commercially available basal medium (basal medium for culturing animal cells). Cultivation was performed under an environment that was generally suitable for culturing CHO cells.

[0182] The expressed antibodies are purified by a combination of standard column chromatography (such as affinity chromatography, ion exchange chromatography, hydrophobic chromatography, etc.).

[0183] [Example 2] Separation of Q-CDR-Splice Variants and Protected Disulfide Isoforms (Cation Exchange High Efficiency Liquid chromatography

[0184] The sample solution prepared by diluting the sample for analysis with mobile phase solution A (whose composition is described below) was injected into a cation exchange column (ProPac WCX-10: particle size 10 μm; inner diameter 4.0 mm, length 250 mm). Then, separation was performed by liquid chromatography (column temperature: 30+ / -5°C, measurement wavelength: 280 nm, flow rate: 1.0 mL / min) using an acidic mobile phase (mobile phase solution A, containing 9.6 mmol / L Tris, 6.0 mmol / L piperazine, 11.0 mmol / L imidazole buffer (pH 6.0)) and an alkaline mobile phase (mobile phase solution 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)). As a result, it was confirmed that the Q-CDR-splicing variant and the protected disulfide isoform were separated in more acidic and more basic regions, respectively, compared to the main peak corresponding to emicizumab ( FIG. 1A and FIG. 2A ).

[0185] [Example 3] Separation of protected disulfide isoforms (IdeS digestion, partial reduction, reversed-phase HPLC Chromatography

[0186] The sample was diluted with phosphate buffer, digested with IdeS protease and PNGase-F, and then partially reduced with DTT in Tris buffer without denaturant. The sample diluted with TFA solution was poured into a reversed-phase HPLC column and separated. It was found that the main component of emicizumab was separated on the chromatograph in a state where the disulfide bonds between the heavy chain and the light chain were reduced, while the protected disulfide isoform was detected with a unique peak representing a state where the disulfide bonds between the heavy chains remained unreduced ( Figure 3 B and Figure 3 D).

[0187] [Example 4] Separation of protected disulfide isoforms (IdeS digestion, denaturation, reversed-phase HPLC)

[0188] The sample was diluted with buffer, digested with IdeS protease and PNGase-F, and then the protein was denatured using denaturing buffer. The sample diluted with TFA solution was then poured into a reversed-phase HPLC column and separated. It was found that the F(ab')2 part of the protected disulfide isoform was separated after a longer retention time compared to the main component of emicizumab ( Figure 4 C and Figure 4 D). [Example 5] Separation of protected disulfide isoforms (IdeS digestion, reverse phase high efficiency Liquid chromatography

[0189] The samples were diluted with buffer and digested with IdeS protease and PNGase-F. The samples diluted with TFA solution were separated by reverse phase HPLC. It was found that the F(ab')2 part of the protected disulfide isoform was separated after a longer retention time compared to the main component of emicizumab ( Figure 4 A and Figure 4 B).

[0190] [Example 6] Evaluation of the biological activity of Q-CDR-splicing variants and protected disulfide isoforms

[0191] Chromogenic assay

[0192] Use specific chromogenic substrate, quantitative determination activated coagulation factor X (FXa) amount, described activated coagulation factor X (FXa) is in the reaction field that phospholipid is provided, in the presence of FIXa and FX, by the reaction generation of imipenem.Specifically, by adding the solution (TBSB) containing tris-hydroxymethylaminomethane, sodium chloride and BSA in sample, prepare the imipenem solution diluted with various concentrations.Each kind of dilution solution is added in the respective hole of 96-well microplates, to which the coagulation factor solution containing FIXa, FX, calcium chloride, magnesium chloride, phospholipid and TBSB is added, after shaking, plate is placed 30 minutes.Ethylenediaminetetraacetic acid solution is added in each hole, shakes flat board, and chromogenic substrate solution is added in each hole (N-benzoyl-L-isoleucyl-L-glutamyl-glycyl-L-arginine-p-nitroaniline hydrochloride and methyl ester thereof).After shaking, plate is placed 35 minutes. After shaking for one to two minutes, the absorbance (Abs) at 405nm of each well was measured using a microplate reader. Based on the absorbance values ​​obtained from the standard solution and the sample solutions of various concentrations, the specific activity of the sample relative to the standard solution was determined from the regression curve generated using a 4-parameter parallel line logarithmic analysis program. As a result, relative to the emicizumab standard solution, the Q-CDR-splicing variant showed 18+ / -1% of biological activity, and the protected disulfide isoform showed 8+ / -0% of biological activity.

[0193] Coagulation assay

[0194] In this assay, in a system using a reconstructed endogenous coagulation activation mechanism of human plasma deficient in factor VIII, the time until coagulation due to fibrin formation is measured based on the change in turbidity as an indicator. Specifically, a solution containing tris-hydroxymethylaminomethane, sodium chloride and BSA is added to the sample to prepare an emicizumab solution diluted in various concentrations. Using an automatic coagulation measuring device, factor VIII-deficient plasma is added to the dilute solution and incubated, then the APTT reagent is added and incubated, and finally a calcium chloride solution is added and measured to determine the coagulation time. The specific activity of the sample relative to the standard substance is calculated by parallel line determination. As a result, relative to the emicizumab standard solution, the Q-CDR-splicing variant showed 18+ / -1% of biological activity, and the protected disulfide isoform showed 16+ / -1% of biological activity.

[0195] [Example 7] Evaluation of the effects of culture parameters on the ratio of Q-CDR splice variants

[0196] [Initial culture medium]

[0197] Plant-derived hydrolyzates, amino acids, etc. are added and dissolved in a commercially available basal medium, and the mixture is then sterilized by filtration.

[0198] [Feed medium]

[0199] Glucose, amino acids, etc. are added and dissolved in a commercially available basal medium. The mixture is then sterilized by filtration.

[0200] [cell[

[0201] Emicizumab was used to produce CHO cells (strain DXB-11) containing a gene encoding emicizumab incorporated therein.

[0202] [Cultivation method]

[0203] The production medium (to + / -10% of the standard concentration) was poured into a 1L scale cell culture apparatus and the above CHO cell lines were inoculated therein to obtain 2 to 6 × 10 5 Cells / mL. Cell culture was started under the following conditions: temperature 36-38°C, dissolved oxygen concentration 40%, initial pH 7.20. From day 1 to day 3 of culture, feed medium was added at a constant flow rate (to + / -10% of the standard concentration), and on day 3 of culture, the pH was shifted to 6.70-7.10. Culture was continued for 13-15 days.

[0204] According to the experimental plan designed based on a central composite design including 12 central points (6 factors: concentration of production medium, concentration of feed medium, initial cell density, temperature, time of starting to add feed medium, pH after conversion), cultivation was performed under a total of 56 conditions. For all conditions, the culture medium was sampled on the 13th, 14th and 15th day of cultivation (a total of 168 samples) and centrifuged (centrifugation at 3000 rpm for 5 minutes). The supernatant was protein A purified and then used to measure the ratio of Q-CDR-splicing variants.

[0205] [Analysis method]

[0206] The number of live cells and the ratio of live cells were measured by trypan blue staining. The Q-CDR splice variants were detected as peaks by cation exchange HPLC using a cation column (ProPac WCX-10).

[0207] [result]

[0208] The results are shown in Figure 5 and Figure 6The ratio of Q-CDR-splicing variants is affected by temperature and post-conversion pH. In the range tested (temperature 36-38°C, post-conversion pH 6.70-7.10), the ratio of Q-CDR-splicing variants decreased more in culture at 36°C and in culture at a conversion pH of 7.10. There is an interaction between temperature and post-conversion pH (conversion pH). It has been found that when the conversion pH is as low as 6.70, the ratio of Q-CDR splicing variants can be reduced even under 38°C culture conditions.

[0209] The Q-CDR-splicing variants were successfully controlled to 4% or less by controlling the culture temperature at 36-38°C and the pH at 6.70-7.10 on the third day of culture or thereafter.

[0210] [Example 8] Removal of Q-CDR splice variants by cation exchange chromatography

[0211] A method for separating Q-CDR splice variants and the antibody emicizumab was developed using differences in their electrostatic properties. Examples are described below.

[0212] The column was filled with Capto SPImpRes (GE) or its permissible substitute as a cation exchange resin and balanced. The column was loaded with a solution of the antibody Emicizumab containing the Q-CDR shear variant so that the two were adsorbed. After loading, the column was washed with a phosphate buffer containing sodium chloride, and then, only the variants on the acidic side of the Q-CDR- shear variant were specifically eluted to separate and remove them from the antibody Emicizumab. In order to show how the removal effect is, the washing fraction, the eluted fraction and the loading fraction before separation were analyzed by CE-HPLC ( Figure 7 ).

[0213] The conditions for loading, washing, and elution are shown below.

[0214] Loading: 33 g of antibody Emicizumab per 1 L of resin was loaded with Tris-HCl buffer adjusted to pH 5.0 containing antibody Emicizumab.

[0215] 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.

[0216] Elution: Phosphate buffer adjusted to pH 6.5 and containing 100 mmol / L sodium chloride was passed through the column at room temperature for 6.5 CV.

[0217] [Example 9] Molecular structure analysis of protected disulfide isoforms

[0218] Preparations of antibodies of emicizumab and protected disulfide isoforms (antibody concentration of 7.54 mg / mL, 150 mmol / L arginine, 20 mmol / L histidine-aspartic acid, pH 6.0) were prepared. SAXS measurements were performed using a linear collimated X-ray beam (Cu Kα, λ=0.1542 nm) generated by a SAXSess mc2 system (Anton Paar, Graz, Austria). The measurement temperature was set to 25°C and a two-dimensional imaging plate was used for detection. The exposure time of the X-ray beam was set to 30 minutes. The two-dimensional scattering intensity was converted to a one-dimensional scattering intensity I (q) on the SAXSQuant software (Anton Paar). Here, q is a scattering vector, defined as q=(4π / λ)sin(θ / 2) (θ is the scattering angle). For the light beam transmitted through the beam stopper, the scattering curve was normalized for the scattering intensity at q=0, and then blank correction (with buffer and capillary) and optical system (decontamination) correction were performed. Under the condition that q xRg<1.3, Guinier plotting is performed on the corrected scattering curve to obtain the radius of gyration, Rg (nm); however, when there is a drop in scattering intensity on the small angle side, the data of the q range corresponding thereto is excluded when performing the Guinier plotting to avoid the influence of interparticle repulsion. In a system assuming no interparticle interaction (structure factor S(q) = 1), the scattering intensity I(q) is given as the Fourier transform of the distance distribution function p(r). The p(r) of the particle is obtained by applying the indirect Fourier transform method to the corrected scattering curve (NPL9). The maximum size Dmax (nm) is obtained from the x-intercept of p(r). Three measurements were performed on each of the emicizumab antibody preparation (MAIN) and the protected disulfide isotype antibody preparation (BiAb3).

[0219] As a result, the protected disulfide isotype had an average Rg value of 4.8 nm or less (a value 0.3 nm or more smaller than that of Emicizumab) and an average Dmax value of 16.5 nm or less (a value 1.4 nm or more smaller than that of Emicizumab), and exhibited an average Rg value of 6% or more smaller than that of Emcizumab and an average Dmax value of 7.5% or more smaller than that of Emcizumab. It is reported that the Dmax value of an IgG4 antibody molecule (which is the same antibody subclass as Emicizumab) corresponds to the distance between the tips of the two Fab domains (NPL 10); therefore, it is believed that the distance between the tips of the two Fab domains of the protected disulfide isotype is shortened, thereby giving a smaller Rg value, which represents the distance from the center of mass of the molecule. Based on the above, it was confirmed that the protected disulfide isotype has a molecular structure that has a shorter distance between the N-termini of the J chain / Q chain compared to Emicizumab ( Figure 8 ). For bispecific antibodies that mediate interactions between two types of antigens, the distance between the Fab domains should be crucial in determining the interactions between the antigens, given the three-dimensional structure. Therefore, how to control the percentage of such isotypes in a pharmaceutical composition is an important task, not only for Emicizumab, but also for antibody drugs containing bispecific antibodies in general.

[0220] [Example 10] Molecular structure analysis by HDX-MS (hydrogen deuterium exchange mass spectrometry)

[0221] Antibody preparations of emicizumab and each protected disulfide isoform were prepared (antibody concentration was 1 mg / mL, 150 mmol / L arginine, 20 mmol / L histidine-aspartic acid, pH 6.0), and HDX-MS measurements were performed using an HDX-MS apparatus (Orbitrap Fusion Lumos (Thermo Fisher Scientific) with HDX-PAL (LEAP Technologies), UltiMate30000RSLCnano (Thermo Fisher Scientific)) (measured at deuterium exchange times of 30 s, 60 s, 120 s, 240 s, 480 s, 960 s, 1920 s, and 3840 s).

[0222] As a result, a significant difference in the deuterium exchange rate (%D) in the HDX-MS measurement was observed in a peptide comprising amino acid residues from position 146 according to EU numbering of the Q chain to position 174 according to EU numbering of the Q chain (152 to 180 from the N-terminal side of SEQ ID NO: 10) and a peptide comprising amino acid residues from position 146 according to EU numbering of the J chain to position 174 according to EU numbering of the J chain (148 to 176 from the N-terminal side of SEQ ID NO: 11). Based on this result, it was confirmed that the protected disulfide isoform has a different structure in these regions compared to emicizumab ( FIG. 9 ).

[0223] Industrial Applicability

[0224] Compared to Emicizumab, the antibody variants and isoforms of the present invention have extremely reduced FVIII mimetic activity; therefore, the pharmaceutical composition of the present invention comprising Emicizumab and having a reduced content ratio of such antibody variants and isoforms can be used as a means of treating hemophilia. The method of the present invention for analyzing antibody variants and isoforms is useful for evaluating the quality of Emicizumab preparations, and is also useful for developing Emicizumab preparations with reduced content ratios of antibody variants and isoforms or developing methods for inhibiting the formation of antibody variants and isoforms.

Claims

1. An isotype of a bispecific antibody comprising a first heavy chain and a second heavy chain, wherein Disulfide bonds are formed in: (1a) between the cysteine ​​at position 144 according to EU numbering of the first heavy chain and the cysteine ​​at position 200 according to EU numbering of the second heavy chain; as well as (1b) between the cysteine ​​at position 200 according to EU numbering of the first heavy chain and the cysteine ​​at position 144 according to EU numbering of the second heavy chain; or wherein Disulfide bonds are formed in: (2a) between the cysteine ​​at position 226 according to EU numbering of the first heavy chain and the cysteine ​​at position 229 according to EU numbering of the second heavy chain; as well as (2b) between the cysteine ​​at position 229 according to EU numbering of the first heavy chain and the cysteine ​​at position 226 according to EU numbering of the second heavy chain.

2. The bispecific antibody isoform of claim 1, wherein a disulfide bond is formed in (1a) and (1b).

3. The bispecific antibody isotype of claim 1, comprising a first heavy chain and a second heavy chain, characterized in that When separated using cation exchange chromatography, it elutes in a region closer to the basic side than the bispecific antibody.

4. The bispecific antibody isoform of any one of claims 1 to 3, which is an isoform of emicizumab.

5. The method for detecting the antibody isotype of any one of claims 1 to 4, comprising the step of separating a sample containing the 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.

6. The detection method according to claim 5, which uses the antibody isotype according to any one of claims 1 to 4 as a reference standard.

7. A pharmaceutical composition comprising the bispecific antibody isotype of any one of claims 1 to 4, wherein in the pharmaceutical composition, the percentage of the antibody isotype in the total antibody molecules is 2% or less.

8. A method for reducing the percentage of isotypes of the bispecific antibody of any one of claims 1 to 4, comprising a purification step by cation exchange chromatography.

9. The antibody isoform of any one of claims 1 to 4, wherein the biological activity of the antibody is significantly reduced.

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