HK40031063A - Antibody variant and isoform with lowered biological activity - Google Patents

Description

Antibody variants and isotypes with reduced biological activity

Technical Field

The present invention relates to antibody variants and isotypes having reduced biological activity. For example, the present invention relates to antibody variants and isotypes of eimeria-lizumab (emilizumab) that have reduced coagulation factor viii (fviii) mimetic activity. The invention also relates to pharmaceutical compositions comprising such antibody variants or isotypes at low content rates. The invention further relates to methods for detecting and methods for analyzing antibody variants and isotypes.

Background

Among them, many IgG-type antibody drugs are commercially available and many are currently under development (NP L1, 2 and 3).

In recent years, FVIII preparations have also been administered prophylactically to prevent bleeding events (prophylactic administration; NP L1 and 2). FVIII preparations have a half-life in blood of about 12 to 16 hours.

Occasionally, antibodies (inhibitors) against FVIII are produced in hemophiliacs. This inhibitor counteracts the action of FVIII formulations. For bleeding in patients who have developed inhibitors (inhibitor patients), a bypass agent may be administered. Their mechanism of action is independent of FVIII function, i.e. the function of catalyzing the activation of factor x (fx) by activated factor ix (fixa). Thus, in some cases, the bypass agent does not sufficiently stop bleeding. Therefore, there is a strong need for agents that are not affected by the presence of inhibitors and that functionally replace FVIII.

As a means for solving this problem, bispecific antibodies functionally replacing FVIII and their use have been reported (PT L1, 2, 3 and 4): bispecific antibodies against FIXa and FX can functionally replace FVIII (NP L5) by placing two factors close to each other to exhibit FVIII mimetic activity, FVIII mimetic activity of antibodies can be reported to be enhanced by optimizing affinity for FIXa and FX (NP L6) and eimeislizumab (ACE910), which is one of these antibodies, having high FVIII mimetic activity is reported to exert hemostatic effects in a monkey model of hemophilia (NP L7 and 8), and thus clinical trials are being conducted on hemophilia a patients.

List of documents

[ patent document ]

[PTL 1]WO 2005/035754

[PTL 2]WO 2005/035756

[PTL 3]WO 2006/109592

[PTL 4]WO 2012/067176

[ non-patent document ]

[NPL 1]Blood 58，1-13(1981)

[NPL 2]Nature 312，330-337(1984)

[NPL3]Nature 312，337-342(1984)

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

[ NP L5 ] Nat Med.2012 for 10 months, 18 (10): 1570-4.

[NPL 6]PLoS One.2013；8(2)：e57479.

[ NP L7 ] J ThrombHaemost.2014 2 months, 12 (2): 206-.

[ NP L8 ] blood.2014, 11 and 13 days; 124 (20): 3165-71.

[NPL9]J.Appl.Cryst.13，577-584(1980)

[NPL 10]IUCrJ.2，9-18(2015)

Disclosure of Invention

[ problem ] to

The present invention has been achieved in view of the above circumstances. It is an object of the present invention to provide antibody variants or isotypes with reduced FVIII mimicking activity.

[ means for solving the problems ]

The present inventors have conducted a dedicated study to solve the above-mentioned problems and succeeded in identifying antibody variants and isotypes contained in a pharmaceutical composition containing eimeria-zumab as an active ingredient. The inventors have also found that FVIII mimicking activity of these antibody variants and isotypes is very low compared to the activity of eimeria-vizumab.

The present invention has been completed based on these findings, and the following [1] to [21] are provided.

[1] Antibody variants comprising a variable region comprising amino acid sequence SISPSGQSTYYRREVKG (SEQ ID NO: 2), wherein

(a) An amino acid residue R at position 12 from the N-terminal side of the sequence (position 61 from the N-terminal side of the Eimelizumab Q chain: position 60 according to Kabat numbering)

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

Is deleted and the variable region is cleaved at the site of deletion.

[2] The antibody variant according to [1], wherein the sequence is a CDR sequence.

[3] The antibody variant according to [1], wherein the sequence is a CDR2 sequence.

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

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

[6] The antibody variant according to [1], which is a variant of eimeiszumab.

[7] [1] to [6], which comprises a step of separating a sample containing an antibody comprising a variable region comprising amino acid sequence SISPSGQSTYYRREVKG (SEQ ID NO: 2) by affinity chromatography, ion exchange chromatography, normal phase chromatography, reverse phase chromatography, hydrophilic interaction chromatography (HI L IC), 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], which uses the antibody variant according to any one of [1] to [6] as a reference standard.

[8-2] the detection method according to [8], which comprises a step of performing one or more kinds of analysis 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 percentage of the antibody variant in the total antibody molecules in the pharmaceutical composition is 5% or less.

[10] The pharmaceutical composition of [9], wherein the antibody is eimeria-lizumab.

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

[12] A method for inhibiting the production of the antibody variant of any one of [1] to [6], which comprises the step of culturing an antibody-producing cell at a pH of 7.1 or more and/or at a culture temperature of 36 ℃ or less.

[12-2] the method according to [12], wherein, during the culture, the conditions for culturing the antibody-producing cells are changed to a pH of 7.1 or more and/or a culture temperature of 36 ℃ or less.

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

Disulfide bonds are formed in:

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

(1b) between cysteine at position 200 (position 206 from the N-terminal side of SEQ ID NO: 10) according to EU numbering of the first heavy chain and cysteine at position 144 (position 146 from the N-terminal side of SEQ ID NO: 11) according to EU numbering of the second heavy chain; or wherein disulfide bonds are formed in:

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

(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.

[14] The bispecific antibody isotype of [13], wherein disulfide bonds are formed in (1a) and (1 b).

[15] 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), characterized in that it elutes in a region closer to the basic side than the bispecific antibody when separated using cation exchange chromatography.

[16] An isotype of the bispecific antibody of any one of [13] to [15], which is an isotype of eimeislizumab.

[17] A detection method for detecting an antibody isotype of any one of [13] - [16], which comprises 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 (HI L IC), Hydrophobic Interaction Chromatography (HIC), charge-based separation, Size Exclusion Chromatography (SEC), Gel Permeation Chromatography (GPC), or a combination thereof.

[18] The detection method as described in [17], which uses an isotype of the bispecific antibody as described in any one of [13] to [16] as a reference standard.

[18-2] the detection method according to [18], which comprises a step of performing one or more kinds of analysis selected from the group consisting of quantitative analysis, qualitative analysis and structural analysis.

[19] A pharmaceutical composition comprising the bispecific antibody isotype of any one of [13] - [16], wherein in the pharmaceutical composition, the percentage of the antibody isotype in the total antibody molecules is 2% or less.

[20] A method for reducing the percentage content of a bispecific antibody isotype of any of [13] - [16], comprising a step of purification by cation exchange chromatography.

[21] The antibody isoform or variant according to [1], [13] or [15], wherein the biological activity of said antibody is significantly reduced.

[22] An isotype of an antibody or derivative thereof having two variable regions each recognizing a different epitope, wherein said 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, relative to said antibody or derivative thereof, and/or wherein said 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, relative to said antibody or derivative thereof.

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

[24] The isotype of [22] or [23], wherein the isotype has a disulfide bond different from a disulfide bond in the antibody or derivative thereof.

[25] The isotype of any of [22] - [24], wherein the antibody or derivative thereof is eimeria-lizumab (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) each forming a pairing with either the first heavy chain or the second heavy chain).

[26] Isoforms of eimeislizumab, wherein the isoforms have an average Rg value of 4.9nm or less, or preferably 4.8nm or less, and/or wherein the isoforms have an average Dmax value of 17.0nm or less, or preferably 16.5nm or less.

[27] The isoform according to [25] or [26], wherein the isoform has a disulfide bond between a 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 a 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 a 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 a 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).

[28] A pharmaceutical composition comprising eimeria-lizumab and an isoform as defined in any one of [22] to [27], wherein in said pharmaceutical composition the percentage of said isoform in the total antibody molecules is 2% or less.

[29] An isotype of a bispecific antibody (Q499-z121/J327-z 119/L404-k; eimeishu mab) 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 isotype and the eimeishu mab have a difference in molecular structure in 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 (from position 152 to position 180 from the N-terminal side of SEQ ID NO: 10), and from position 146 according to EU numbering of the J chain to position 174 according to EU numbering of the J chain (from position 148 to position 176 from the N-terminal side of SEQ ID NO: 11).

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

[31] A pharmaceutical composition comprising eimeria-lizumab and an isoform according to [29] or [30], wherein in said pharmaceutical composition the percentage of said isoform in the total antibody molecules is 2% or less.

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

[A1] The detection method according to [7] or [17], which comprises a step of subjecting a sample containing the antibody and/or the 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 according to [ A1], wherein the reduction reaction is carried out under mild reduction conditions (for example, reduction with DTT in Tris buffer (pH7.0) at 37 ℃).

[A3] The detection method as described in [ A1], wherein the hydrolysis reaction is carried out using a site-specific cleaving enzyme (e.g., a sequence-specific protease such as IdeS protease, L ys-C, papain, etc.).

[A4] The detection method of any one of [7], [17] and [ A1] - [ A3], comprising the step of separating a sample containing the antibody, the 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 (HI L IC), Hydrophobic Interaction Chromatography (HIC), charge-based separation, Size Exclusion Chromatography (SEC), Gel Permeation Chromatography (GPC), or a combination thereof.

[A5] The detection method of any one of [8], [18] and [ A1] - [ A4], comprising the step of analyzing by SE-HP L C analysis, dynamic light scattering (D L S) method, SAXS measurement, electron microscopy measurement, 3D modeling, SPR determination, HDX MS analysis, or a combination thereof.

[A6] A method for quality control of a pharmaceutical composition comprising eimeria-zumab, comprising the steps of [7], [8], [17], [18] or [ a1] - [ a5] or a step of combining these methods.

[A7] A method of preparing a pharmaceutical composition comprising eimeria-lizumab comprising the steps of the method from [ a6 ].

[A8] Method for the purification of a composition comprising eimeria-zumab, characterized in that it comprises a step of a binding & elution mode of cation exchange Chromatography (CEX).

[A9] A process for the preparation of a pharmaceutical composition comprising eimeria-lizumab, comprising the steps from the purification process of [ A8 ].

Effects of the invention

The present inventors have succeeded in identifying antibody variants and isotypes contained in a pharmaceutical composition containing eimeria-lizumab as an active ingredient. The inventors have also found that FVIII mimetic activity of these antibody variants and isotypes is very low compared to that of eimeria-zumab. Thus, pharmaceutical compositions comprising eimeria-lizumab and such antibody variants and isotypes only at low levels may be used as a means of treating hemophilia.

Drawings

FIG. 1-1 FIG. 1A shows the results of isolating the eimeria-lizumab drug substance by CE-HP L C the peaks marked with bold boxes represent Q-CDR-cleaved variants.

FIGS. 1-2 FIG. 1B is a diagram showing the molecular structure of a Q-CDR-spliced variant. The number in the figure and the letter immediately below thereof indicate the position of the amino acid residue counted from the N-terminal of the eimeria-lizumab Q chain and the amino acid residue at that position (one-letter code), respectively.

FIG. 2-1 FIG. 2A shows the results of the isolation of the eimeria-lizumab drug substance by CE-HP L C the peaks marked with bold boxes represent the protected disulfide isoforms.

[ FIGS. 2-2] FIG. 2B is a graph showing the molecular structure of protected disulfide isoforms, the numbers in the graph and the letter C on the left thereof indicate the position of an amino acid residue counted from the N-terminus of the Eimelizumab Q chain and a cysteine residue at that position, respectively, FIG. 2C shows the content ratio of the protected disulfide isoforms in the Eimelizumab drug substance, the content ratio under various conditions (culture conditions of antibody-producing cells) is represented by the average (mean) of the peak area percentages (area%) of the protected disulfide isoforms in the CE-HP L C isolation results (shown in FIG. 2A) and their standard deviations (StdDev).

FIG. 3 shows the results of the separation of eimeria mab and protected disulfide isoforms by reversed phase high performance liquid chromatography after IdeS digestion and reduction treatment FIGS. 3A-D show the results of the separation of samples containing eimeria mab (A and B) or protected disulfide isoforms (C and D), which were digested with IdeS and then reduced in the presence (A and C: fully reduced conditions) or in the absence (B and D: partially reduced conditions) of a denaturing agent for samples from fully reduced conditions (FIGS. 3A and C), for both eimeria mab and protected isoform disulfides, peaks representing the Q chain Fd (Q-Fd), the J chain Fd (J-Fd), the Q chain Fc (Q-Fc), the J chain Fc (J-Fc), and the L chain (L C) were detected, the difference between the modes of the former, the latter, and the latter, being detected for samples from the same conditions, the peaks of the same disulfide isoforms (Q-Fd, J-Fc) and for the same isoforms (J-Fd), the peaks of the J chain Fc (J-Fc) and the former, the latter being detected for the same sample.

FIG. 4 shows the results of separation of eimeria mab and protected disulfide isoforms by reversed phase high performance liquid chromatography after IdeS digestion (and reduction treatment). FIGS. 4A and B show the results of separation of protected disulfide isoform (A) or eimeria mab (B) digested by IdeS.FIGS. 4C and D show the results of separation of protected disulfide isoform (C) or eimeria mab digested by IdeS and then denatured.

FIG. 5 shows the content ratio of Q-CDR-cleaved variants in the culture supernatants of eimeria-mab producing CHO cells cultured under various culture conditions. Protein A purified culture supernatant samples were used for measuring the content ratio of Q-CDR-cleaved variants. The vertical axis represents the Q-CDR-cut variant content ratio (% peak area), and the horizontal axis represents various culture conditions.

FIG. 6 shows the content ratio of Q-CDR-cleaved variants in the culture supernatants of eimeria-mab producing CHO cells cultured under various culture conditions. Protein A purified culture supernatant samples were used for measuring the content ratio of Q-CDR-cleaved variants. The vertical axis represents the Q-CDR-cut variant content ratio (% peak area), and the horizontal axis represents various culture conditions.

FIG. 7 is a graph showing the results of CE-HP L C analysis of each fraction of an antibody eimeria mab solution containing a Q-CDR-cleaved variant, which is obtained during purification including steps in a cation exchange Chromatography (CEX) bind & elute mode, "load fraction" shows the results of CE-HP L C analysis of the antibody solution loaded onto a cation exchange column, "wash fraction" shows the results of CE-HP L C analysis of the column adsorption fraction obtained after passing through a pH7.2 phosphate buffer (wash) containing 25 mmol/L sodium chloride, "elute fraction" shows the results of CE-HP L C analysis of the column adsorption fraction obtained after passing through a pH6.5 phosphate buffer containing 100 mmol/L sodium chloride.

FIG. 8 FIGS. 8A-D show the results of analyzing the molecular structures of eimeria-lizumab (MAIN) and protected disulfide isoform (BiAb3) using a SAXS apparatus. "pair-distance distribution function [ p (r) ]", "Rg (nm)" and "Dmax (nm)" respectively represent a pair-distance distribution function, a radius of gyration, and a maximum dimension.

FIG. 9-1 FIG. 9A shows the residual profiles on deuterium exchange rate (% D) of eimercetin (the main component in cation exchange high performance liquid chromatography) and protected disulfide isoforms (deuterium exchange times 30s, 60s, 120s, 240s, 480s, 960s, 1920s and 3840s) in HDX-MS measurement, each bar in the graph representing the sum of the resultant differences of each deuterium exchange time of the Q chain, the J chain and the L chain, FIG. 9B shows a portion indicating the difference in molecular structure of the protected disulfide isoform and eimercetin by HDX-MS measurement, a peptide comprising an amino acid residue from position 146 according to EU numbering of the Q chain to position 174 according to EU numbering of the Q chain (from position 152 to position 180 from the N-terminal side of SEQ ID NO: 10) and a peptide comprising an amino acid residue from position 146 according to EU numbering of the J chain to position 174 according to EU numbering (from position 152 to position 148 from the N-terminal side of SEQ ID NO: 11) are shown as a single disulfide molecule represented by a single disulfide isoform.

Fig. 9-2 is a continuation of fig. 9-1.

Detailed description of the preferred embodiments

One embodiment of the invention relates to antibody variants and isotypes having reduced biological activity (e.g., reduced FVIII mimicking activity). During the present inventors' analysis of eimeria-zumab drug substances, antibody variants and isotypes were identified as two types of structurally altered molecules (Q-CDR-cleaved variants and protected disulfide isotypes). In the present application, "antibody variants" and "antibody isotypes" may also be referred to as mutants or isomers of the antibody molecule.

Eimeria zumab is a bispecific humanized IgG4 antibody that exhibits functional replacement FVIII activity as a cofactor and comprises anti-fix (a) and anti-FX, and comprises two types of heavy chains (Q499 and J327) each recognizing fix (a) and FX, respectively, and a common L chain (L404).

Specifically, eimeria-vizumab 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 comprises an H chain comprising the amino acid sequences of H chain CDRs 1, 2 and 3 of SEQ ID NOS: 1, 2 and 3, respectively (the H chain CDRs of Q499), the second polypeptide comprises an H chain comprising the amino acid sequences of H chain CDRs 1, 2 and 3 of SEQ ID NOS: 4, 5 and 6, respectively (the H chain CDRs of J327), and the third and fourth polypeptides comprise a common L chain comprising the amino acid sequences of L chain CDRs 1, 2 and 3 of SEQ ID NOS: 7, 8 and 9, respectively (the L chain CDRs of L) (Q499-121 z/J327-119/L z/36404-404 k).

More specifically, eimeria zumab is a bispecific antibody, wherein 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 comprises an H chain comprising the amino acid sequence of the H chain variable region of SEQ ID NO: 13, the second polypeptide comprises an H chain comprising 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 comprising the amino acid sequence of the L chain variable region of SEQ ID NO: 15.

Still more specifically, eimeria-zumab is a bispecific antibody, wherein 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 comprises an H chain comprising the amino acid sequence of SEQ ID NO: 10, the second polypeptide comprises an H chain comprising the amino acid sequence of SEQ ID NO: 11, and the third polypeptide and the fourth polypeptide comprise the common L chain of SEQ ID NO: 12 (Q499-z121/J327-z 119/L404-k).

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

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

The amino acids comprised in the amino acid sequence of the present invention may be post-translationally modified (e.g., modification of the N-terminal glutamine to pyroglutamic acid by pyroglutamate acylation 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.

In the present invention, the biological activity of an antibody or antibody variant or antibody isotypePreferably, the FVIII mimicking activity. In the present invention, "FVIII mimicking activity" refers to activity of functionally substituted FVIII (activity of functionally substituted FVIII as a cofactor). In the present invention, the phrase "functionally substituted for FVIII" means that FIX or FIXa and FX are recognized and FX activation is promoted by FIXa (FXa production is promoted by FIXa). The FXa production promoting activity can be evaluated using, for example, a measurement system comprising FIXa, FX, synthetic substrate S-2222 (synthetic substrate for FXa) and phospholipid. This measurement system shows a correlation with disease severity and clinical symptoms in hemophilia a cases (Rosen S, Andersson M,m. clinical applications of a colorimetric substrate method for determination of FVIIIactivity.ThrombHaemost 1985; 54: 811-23).

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

In the present invention, an antibody or antibody variant or isotype is said to have reduced biological activity when the biological activity of the antibody or antibody variant or isotype is reduced compared to the biological activity of a reference antibody, and preferably, the reduction is statistically significant. In the present invention, an antibody or antibody variant or antibody isotype is said to have "significantly (or extremely) reduced biological activity" when the biological activity of the 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.

In the present invention, the terms "Q chain" and "J chain" refer to an H chain (heavy chain) comprising a variable region capable of exhibiting binding ability to fix (a) and FX, respectively.

In the present invention, the term "common L chain" refers to a L chain capable of forming a pair with each of two or more different H chains and capable of exhibiting binding ability to their respective antigens the term "different H chains" herein preferably refers to H chains of antibodies directed to 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 WO 2006/109592.

The term "antibody" is used in the broadest sense and includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (such as bispecific antibodies), antibody derivatives, and modified antibodies (Miller K et al JImmunol.2003, 170(9), 4854-61) so long as they exhibit the desired biological activity. The antibody may 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 of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule. The immunoglobulin may be derived from any species (e.g., human, mouse, or rabbit). The terms "antibody," "immunoglobulin," and "immunoglobulin" are used interchangeably in a broad sense.

"bispecific antibody" refers to an antibody having two variable regions, each variable region recognizing 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 intact antibodies, but also antibody derivatives.

Recombinant antibodies produced by using genetic engineering techniques can be used as antibodies. Recombinant antibodies can be obtained by: cloning of DNA encoding the antibody from the hybridoma or antibody-producing cell (e.g., sensitized lymphocytes that produce the antibody); inserting it into a vector; and then introduced into a host (host cell) to produce the antibody.

Bispecific antibodies are not limited to those of the IgG type, for example, IgG type bispecific antibodies can be secreted from a hybrid hybridoma (quadroma) produced by fusing two types of IgG antibody-producing hybridomas (Milstein C. et al, Nature1983, 305: 537-540.) they can also be secreted by introducing L chain and H chain genes constituting two types of IgG of interest, i.e., a total of four genes, into a cell to coexpress these genes.

The antibodies of the invention can be produced by methods known to those skilled in the art. Specifically, a DNA encoding the antibody of interest is inserted into an expression vector. Insertion into an expression vector is performed such that expression occurs under the control of expression regulatory regions such as enhancers and promoters. Subsequently, the host cell is transformed with the expression vector to express the antibody. In this case, an appropriate combination of the host and the expression vector may be used.

The antibodies of the present invention thus obtained can be isolated and purified from the inside or outside of the host cell (culture medium, etc.) and purified as substantially pure, homogeneous antibodies, the antibodies can be isolated and purified by methods generally used for isolating and purifying antibodies, and the methods are not limited in any way, for example, the method described in WO2013/086448 is known for isolating IgG2 disulfide isotype, for isolating and purifying the antibodies and antibody variants or antibody isotypes in the present invention, for example, the antibodies and antibody variants or antibody isotypes can be isolated and purified by appropriately selecting and combining column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, and the like.

In one aspect, the invention relates to antibody variants (sometimes referred to herein as "Q-CDR-scissioned variants") having the following characteristics:

-very low biological activity (FVIII mimicking activity) compared to eimeria-vizumab;

-the N-terminal side fragment and the C-terminal side other fragment of the deleted amino acid residue are linked together by a disulfide bond (fig. 1B);

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

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

(a) From SEQ ID NO: 2 (position 61 from the N-terminal side of the eimeria-lizumab Q chain: position 60 according to Kabat numbering); or

(b) From SEQ ID NO: 2 (positions 10 to 12 from the N-terminal side of the amino acid sequence of eimeria-lizumab Q chain (positions 59 to 61 from the N-terminal side of the chain: positions 58 to 60 according to Kabat numbering),

is deleted and the variable region is cleaved at the site of deletion.

The Q-CDR-spliced variant is preferably a variant of a bispecific antibody, more preferably a variant of eimeria-seuzumab.

In another aspect, the present invention also relates to a method for detecting a Q-CDR-cleaved variant and a method for analyzing a Q-CDR-cleaved variant in one embodiment, the method for detecting a Q-CDR-cleaved variant comprises the step of isolating a sample comprising an antibody comprising a variable region comprising amino acid sequence SISPSGQSTYYRREVKG (SEQ ID NO: 2) by affinity chromatography, ion exchange chromatography, normal phase chromatography, reverse phase chromatography, hydrophilic interaction chromatography (HI L IC), Hydrophobic Interaction Chromatography (HIC), charge-based separation, Size Exclusion Chromatography (SEC), Gel Permeation Chromatography (GPC), or a combination thereof.

In this detection method and analysis method, the presence or absence of a deleted portion in Fab (Q chain Fab) having an amino acid sequence of SEQ ID NO: 2 can be used as an index for detection and analysis.the deleted portion can be detected, for example, by using a change in molecular weight caused by deletion in L CMS analysis as an index.in the Q-CDR-cleaved variant, a fragment on the N-terminal side of the deleted amino acid residue and other fragments on the C-terminal side are linked together by a disulfide bond.accordingly, by analyzing a sample after carrying out a reaction to reduce the disulfide bond, a difference in the reduction pattern based on the presence or absence of the deleted portion can be detected using various analysis techniques such as CE-HP L C, L CMS, L C-UV.

Alternatively, the difference in resolution by ion exchange chromatography may be used as an index. For example, when isolated by cation exchange chromatography, the Q-CDR-cleaved variants will be isolated in regions that are more acidic than the main peak of eimeria-lizumab.

In another aspect, in the present invention, the production and quality control of a pharmaceutical composition comprising eimeria-lizumab may be carried out by performing one or any combination of the above-described detection methods and analysis methods. Accordingly, the present invention relates to a method for quality control of a pharmaceutical composition comprising eimeria-lizumab, comprising the step of performing the above-described detection methods and analytical methods or the step of combining any of those methods. The invention also relates to the preparation of a pharmaceutical composition comprising eimeria-lizumab, comprising the steps of carrying out such a method for quality control.

In another aspect, the present invention relates to a pharmaceutical composition comprising eimeria mab and a Q-CDR cleaved variant, wherein in the pharmaceutical composition the ratio of the Q-CDR cleaved variant in the total antibody molecule remains low the pharmaceutical composition is obtainable by a purification method comprising purification by cation exchange Chromatography (CEX) for example, an antibody solution containing eimeria mab and a Q-CDR cleaved variant is adsorbed onto a cation exchange column, then only acidic side variants including the Q-CDR cleaved variant can be selectively eluted and removed the ratio of the Q-CDR-cleaved variant in the total antibody molecule in the pharmaceutical composition can be evaluated by various methods including the above-described method for detecting/analyzing the Q-CDR-cleaved variant, and the ratio of peak areas of the Q-CDR-cleaved variant that can be obtained by analyzing the pharmaceutical composition, for example, using cation exchange Chromatography (CEX) or CE-HP L C (peak area ratio) means the ratio of the Q-CDR-cleaved variant in the total antibody molecule in the pharmaceutical composition (e.g., the CEX ratio) is preferably 5% or less, such as 5.0.0% or less, 0.0% or less, or 0.0% or less.

The invention further relates to a method for the preparation of a pharmaceutical composition wherein the content ratio of Q-CDR-spliced variants is kept low, and to a method for inhibiting the formation of Q-CDR-spliced variants. The amount of formation of the Q-CDR cleavage variant can be reduced by shortening the culture time of the antibody-producing cell (e.g., to 15 days or less, preferably 13 days or less), or by lowering the culture temperature of the antibody-producing cell (e.g., to 38 ℃ or less, preferably 37 ℃ or less, more preferably 36 ℃ or less), and/or by increasing the culture pH of the antibody-producing cell (e.g., to 6.7 or more, preferably 6.9 or more, more preferably 7.1 or more) (fig. 4). The above method is therefore characterized in that it comprises the following steps: antibody-producing cells (e.g., eimeria mab) are cultured for a time (e.g., about 15 days or less) at a lower culture temperature (e.g., about 36 ℃ or less) and at a higher pH (e.g., 7.1 or more) than conventional. In one embodiment, the above method comprises the step of culturing the antibody-producing cell at a pH of 7.1 or more and/or at a culture temperature of 36 ℃ or less. In certain embodiments, the above-described method is characterized in that the culture conditions of the antibody-producing cells are changed to a pH of 7.1 or more and/or a culture temperature of 36 ℃ or less in the middle of the culture (for example, on day 2 or later of the culture).

In another aspect, the invention relates to a method for purifying a composition comprising eimeria-zumab, the method characterized by a step comprising a bind & elute mode of cation exchange Chromatography (CEX). The present invention further relates to a process for the preparation of a pharmaceutical composition comprising eimeria-lizumab, said process comprising the step of performing a purification process.

In another aspect, the invention relates to antibody isotypes (sometimes referred to herein as "protected disulfide isotypes") that have the following characteristics:

-very low biological activity (FVIII mimicking activity) compared to eimeria-vizumab;

-more hydrophobic than eimeria-mab;

-has an inter-heavy chain disulfide bond (fig. 2B) that is less susceptible to reduction under mild conditions (partially reducing conditions) than eimeutralizumab;

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

In one embodiment, the protected disulfide isoforms are characterized in that they are capable of binding antigens fix (a) and FX, but do not exhibit biological activity (FVIII mimicking activity).

In certain embodiments, the protected disulfide isoforms are structural isomers that have the same (normal) disulfide bond as eimeria-lizumab, but have a stronger hydrophobicity than usual due to structural changes in the Fab portion, and thus have an inter-heavy chain disulfide bond that is less sensitive to reduction than usual.

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,

disulfide bonds are formed in:

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

(1b) between cysteine at position 200 (position 206 from the N-terminal side of SEQ ID NO: 10) according to EU numbering of the first heavy chain and cysteine at position 144 (position 146 from the N-terminal side of SEQ ID NO: 11) according to EU numbering of the second heavy chain; or wherein disulfide bonds are formed in:

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

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

In a particular embodiment, the protected disulfide isoform is an isoform of a bispecific antibody, and preferably an isoform of eimeisubzumab, wherein, in the protected disulfide isoform,

disulfide bonds are formed in:

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

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

(1c) between 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 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

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

Disulfide bonds are formed in:

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

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

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

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

In another aspect, the 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 isolating a bispecific antibody containing sample by affinity chromatography, ion exchange chromatography, normal phase chromatography, reverse phase chromatography, hydrophilic interaction chromatography (HI L IC), Hydrophobic Interaction Chromatography (HIC), charge-based separation, Size Exclusion Chromatography (SEC), Gel Permeation Chromatography (GPC), or a combination thereof.

In such detection and analysis methods, the analysis can be performed using the difference in the structure of the region forming the inter-heavy chain disulfide bond and/or the Fab region between the protected disulfide isoform and eimeria-lizumab. Structural differences can be detected by various analytical methods such as, for example, those shown below.

For example, peaks reflecting three-dimensional structural differences or differences in hydrophobic strength between eimeria-vizumab 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 processed under non-reducing conditions for IdeS protease digestion reactions (cleavage at a single site below the hinge region in IgG to yield F (ab') 2 and Fc fragments).

The peak reflecting the difference in disulfide bond sensitivity to reduction between eimeria-mab and the protected disulfide isoform can be detected by analyzing the sample using a reverse phase column after the sample has been treated for IdeS digestion and subsequently used for reduction reactions under mild reduction conditions (e.g., reduction with DTT in Tris buffer (ph7.0) at 37 ℃).

When the reduction reaction was performed under the condition that all disulfide bonds were reduced, no difference was detected in the analysis results obtained using the reverse phase column for eimeria mab and protected disulfide isoforms, whereas when the reduction reaction was performed under the above-mentioned mild conditions, a difference in the reduction pattern was detected in the analysis results obtained using the reverse phase column for them. Without wishing to be bound by any particular theory, it is believed that under the mild reducing conditions described above, in the case of eimeria-mab, both the disulfide bonds between the heavy and light chains 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 and light chains are reduced, while the disulfide bonds between the heavy chains are not reduced.

Without wishing to be bound by any particular theory, it is believed that a L ys-C digestion pattern reflecting three dimensional structural differences is detected with the result that the cleavage is performed at a state where the three dimensional structure is maintained during L ys-C digestion under non-denaturing conditions, preferably at a position where L ys-C is readily accessible, after the sample has been treated under non-denaturing conditions (e.g., in Tris buffer) for limited L ys-C digestion (e.g., by stopping L ys-C digestion reactions halfway).

Alternatively, peaks reflecting L ys-C digestion pattern differences arising from three-dimensional structural differences between eimeria-zumab and the protected disulfide isoforms can be detected by analyzing the sample using a reverse phase column after a sample that has been pretreated for denaturation (e.g., treated with 5M guanidine for 30 minutes at 37 ℃) but not reduced (i.e., retains SS bonds) has been treated for limited L ys-C digestion.

In addition to the above reduction reactions, IdeS digestion, and limited L ys-C digestion under non-denaturing or denaturing conditions, various other decomposition reactions such as papain digestion can be used, in addition to reverse phase chromatography using C4 columns and the like, various analytical techniques can be used, such as SE-HP L C analysis, dynamic light scattering (D L S), SAXS measurement, electron microscopy measurement, 3D modeling, SPR determination, HDX MS analysis.

In another aspect, in the present invention, the production and quality control of a pharmaceutical composition comprising eimeria-lizumab may be carried out by performing one or any combination of the above-described detection methods and analysis methods. Accordingly, the present invention relates to a method for quality control of a pharmaceutical composition comprising eimeria-lizumab, comprising the step of performing the above-described detection methods and analytical methods or the step of combining any of those methods. The invention also relates to the preparation of a pharmaceutical composition comprising eimeria-lizumab, comprising the steps of carrying out such a method for quality control.

In another aspect, the present invention relates to a pharmaceutical composition comprising eimeria-lizumab 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 methods described above for detecting/analyzing the protected disulfide isoform, and can be represented by the ratio of the peak areas (peak area ratio) of the protected disulfide isoform obtained by analyzing the pharmaceutical composition, for example, using cation exchange Chromatography (CEX) or CE-HP L C the ratio of the protected disulfide isoform in the total antibody molecule (e.g., CEX peak area ratio) in the pharmaceutical composition 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 invention relates to a method for purifying a composition comprising eimeria-zumab, the method characterized by a step comprising a bind & elute mode of cation exchange Chromatography (CEX). The present invention further relates to a process for the preparation of a pharmaceutical composition comprising eimeria-lizumab, said process comprising the step of performing a purification process.

In another aspect, the invention discloses isoforms of eimeislizumab (protected disulfide isoforms) having the same heavy and light chain amino acid sequences as eimeislizumab, but having a molecular structure with a smaller rg (nm) value and/or dmax (nm) value than eimeislizumab. Such isoforms have a molecular structure that is shorter than eimeria-lizumab in the distance between the J chain/Q chain N-termini, and in particular have 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 less than eimeria-lizumab measured with an SAXS device, and/or have 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 less than eimeria-lizumab measured with an SAXS device. The isoforms may have an average Rg value that is 0.15nm or more, preferably 0.2nm or more, more preferably 0.25nm or more, or even more preferably 0.3nm or more smaller relative to eimeiszumab, and/or may have an average Dmax value that is 0.5nm or more, preferably 1.0nm or more, more preferably 1.2nm or more, or even more preferably 1.4nm or more smaller relative to eimeiszumab. These isoforms may have an average Rg value of 4.9nm or less, or preferably 4.8nm or less, and/or an average Dmax value of 17.0nm or less, or preferably 16.5nm or less.

Rg and Dmax values can be determined under the conditions identified below:

(1) the antibody concentration is 7.54mg/m L;

(2) solvent conditions of 150 mmol/L arginine, 20 mmol/L histidine-aspartic acid, pH6.0, and

(3) temperature: at 25 ℃.

Here, the average Rg value can be obtained by calculating Rg of each measurement from the genie graph and averaging them. The average Dmax value can be obtained by calculating the Dmax for each measurement from the x-intercept of p (r) and averaging them. See example 9 described below for methods of analyzing the ginier plots and p (r).

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

In another aspect, the invention discloses a pharmaceutical composition comprising eimeria-lizumab and an isoform, wherein the percentage of isoform in the total antibody molecules in the pharmaceutical composition is 2% or less.

In the above-described antibody molecule, antibody variant, antibody isotype, pharmaceutical composition comprising an antibody variant or isotype, method for analyzing an antibody variant or isotype, or method for inhibiting formation of an antibody variant or isotype, the antibody is preferably a bispecific antibody, more preferably eimeria mab (ACE 910).

As used herein, aspects to which the expression "comprises" refers include those to which the expression "consists essentially of.

The numerical values set forth herein may vary within certain limits, for example, depending on the instrumentation or equipment, the measurement conditions and procedures used by those skilled in the art, and they may cover deviations of, for example, about 10%, provided they are within the ranges that allow the objectives of the present invention to be achieved.

All patents and references explicitly cited herein are incorporated by reference in their entirety.

The invention is further illustrated by the following examples, which should not be construed as limiting.

Detailed description of the preferred embodiments

[ example 1]Preparation of genetically engineered humanized bispecific monoclonal antibody (antibody eimeria mab)

For structural analysis of eimeutlizumab isomers (antibody variants and isotypes), the eimeutlizumab antibodies were prepared in large quantities by the following method. The CHO cells into which the gene encoding eimeria mab was introduced were cultured in a commercially available basal medium (basal medium for culturing animal cells) as eimeria mab-producing cells. The culture is performed under conditions that render it generally suitable for culturing CHO cells.

The expressed antibody is purified by a combination of standard column chromatography (such as affinity chromatography, ion exchange chromatography, hydrophobic chromatography, etc.).

[ example 2]Isolation of Q-CDR-splicing variants and protected disulfide isoforms (cation exchange high Performance) Liquid chromatography)

A sample solution prepared by diluting a sample for analysis with mobile phase solution A (composition described below) was injected into a cation exchange column (ProPac WCX-10: particle size 10 μm; inner diameter 4.0mm, length 250 mm.) then, separation was performed by liquid chromatography (column temperature: 30+/-5 ℃, measurement wavelength: 280nm, flow rate: 1.0m L/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 a basic 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 (pH9.9)), the results confirmed that the Q-CDR-sheared variant and the protected isoform are separated in more acidic and more basic disulfide regions, respectively, as compared to the main peak corresponding to Eimeria beads (FIG. 1A +/-5 ℃ and FIG. 2).

[ example 3]Isolation of protected disulfide isoforms (IdeS digestion, partial reduction, reverse phase high performance liquid phase) Chromatography)

The samples were diluted with phosphate buffer, digested with IdeS protease and PNGase-F, and partially reduced with DTT in Tris buffer without denaturant. The sample diluted with TFA solution was poured onto a reverse phase high performance liquid chromatography column and separated. As a result, it was found that the main components of eimeria-lizumab were separated on a chromatograph in a state in which the disulfide bond between the heavy chain and the light chain was reduced, while the protected disulfide isoform was detected with a unique peak representing a state in which the disulfide bond between the heavy chains remained unreduced (fig. 3B and fig. 3D).

[ example 4]]Isolation of protected disulfide isoforms (IdeS digestion, denaturation, reverse phase high performance liquid chromatography)

The samples were diluted with buffer, digested with IdeS protease and PNGase-F, and the proteins were denatured using denaturing buffer. Then, the sample diluted with TFA solution was poured into a reverse phase high performance liquid chromatography column and separated. As a result, it was found that the F (ab') 2 portion of the protected disulfide isoform was isolated after a longer retention time compared to the main component of eimeria-lizumab (fig. 4C and 4D).

[ example 5]Isolation of protected disulfide isoforms (IdeS digestion, reverse phase high performance liquid chromatography)

The samples were diluted with buffer and digested with IdeS protease and PNGase-F. The sample diluted with TFA solution was separated by reverse phase high performance liquid chromatography. As a result, it was found that the F (ab') 2 portion of the protected disulfide isoform was isolated after a longer retention time compared to the main component of eimeria-lizumab (fig. 4A and 4B).

[ example 6]Q-CDR-spliced variantsAnd evaluation of the biological Activity of protected disulfide isoforms

Color development assay

The amount of activated coagulation factor x (fxa) produced by the reaction of eimeria-vizumab in the presence of FIXa and FX in a reaction field provided with phospholipids is quantitatively determined using a specific chromogenic substrate, the amount of activated coagulation factor x (fxa) produced by the reaction of eimeria-vizumab is specifically determined by adding a solution containing tris-hydroxymethyl aminomethane, sodium chloride and BSA (TBSB) to the sample, preparing solutions of eimeria-vizumab diluted at various concentrations, adding each diluted solution to a respective well of a 96-well microplate, adding a coagulation factor solution containing FIXa, FX, calcium chloride, magnesium chloride, phospholipid and TBSB thereto, after shaking, leaving the plate for 30 minutes, adding an ethylenediaminetetraacetic acid solution to each well, shaking the plate, and adding the chromogenic substrate solution to each well (N-benzoyl-L-isoleucyl-L-glutamyl-glycyl-L-arginine-p-nitroanilide and its methyl ester), after shaking the plate is left for 35 minutes, after shaking the plate is added to each well, the absorbance of the solution is determined using a biological activity scale showing by a biological activity profile obtained from a standard curve obtained using an abx (1 nm), showing absorbance versus a biological activity curve obtained using an abx-18 nm.

Blood coagulation assay

In this assay, in a system for reconstructing an endogenous coagulation activation mechanism using human plasma deficient in factor VIII, the time until coagulation due to fibrin formation is measured based on the change in turbidity as an index. Specifically, a solution containing tris-hydroxymethylaminomethane, sodium chloride and BSA was added to a sample to prepare solutions of eimeria-lizumab diluted at various concentrations. Using an automated coagulation measurement device, factor VIII deficient plasma was added to the diluted solution and incubated, then APTT reagent was added and incubated, and finally calcium chloride solution was added and measured to determine the clotting time. The specific activity of the sample relative to the standard was calculated by parallel line measurement. As a result, the Q-CDR-cleaved variant showed a biological activity of 18 +/-1% and the protected disulfide isoform showed a biological activity of 16 +/-1% relative to the standard solution of eimeria-lizumab.

[ example 7]Evaluation of the Effect of culture parameters on Q-CDR-scissioned variant ratios

[ initial Medium ]

Plant-derived hydrolysates, amino acids, and the like are added and dissolved in commercially available basal media. The mixture was then sterilized by filtration.

[ feed Medium ]

Glucose, amino acids, and the like were added and dissolved in a commercially available basal medium. The mixture was then sterilized by filtration.

[ cell ], [

The use of eimeria-mab to generate CHO cells (DXB-11 strain) comprising incorporated therein a gene encoding eimeria-mab.

[ culture method ]

Production medium (to +/-10% of standard concentration) was poured into a 1L-scale cell culture apparatus and the CHO cell line described above was inoculated therein to give 2 to 6 × 10⁵Individual cells/m L the cell culture was started at a temperature of 36-38 ℃, a dissolved oxygen concentration of 40%, an initial pH of 7.20, the feed medium was added at a constant flow rate from day 1 to day 3 of the culture (to +/-10% of the standard concentration), and on day 3 of the culture the pH was changed to 6.70-7.10. the culture was continued for 13-15 days.

The culture was performed under a total of 56 conditions according to an experimental plan designed based on a central complex design including 12 central points (6 factors: concentration of production medium, concentration of feed medium, initial cell density, temperature, time to start feeding of feed medium, pH after shift). For all conditions, media were sampled on days 13, 14 and 15 of culture (168 samples in total) and centrifuged (3000rpm for 5 minutes). The supernatant was subjected to protein a purification and then used to measure the ratio of Q-CDR-cleaved variants.

[ analysis method ]

Viable cell number and viable cell ratio were measured by trypan blue staining. The Q-CDR cleaved variant was detected as a peak by cation exchange high performance liquid chromatography using a cation column (ProPac WCX-10).

[ results ]

The results are shown in fig. 5 and 6. The ratio of Q-CDR-spliced variants is influenced by temperature and pH after the transition. Within the range tested (temperature 36-38 ℃, pH after transition 6.70-7.10), the ratio of Q-CDR-cleaved variants decreased more in cultures at 36 ℃ and at transition pH of 7.10. There is an interaction between temperature and pH after the transition (transition pH). It has been found that the rate of Q-CDR-cleaved variants can be reduced when the shift pH is as low as 6.70, even under culture conditions of 38 ℃.

The Q-CDR-spliced variant can be successfully controlled at 4% or less by controlling the culture temperature at 36 to 38 ℃ and the pH at or after 3 days of culture at 6.70 to 7.10.

Example 8 removal of Q-CDR cleavage variants by cation exchange chromatography

Methods for isolating the Q-CDR cleavage variants and the antibody eimeria-vizumab were established using differences in their electrostatic properties. Examples are described below.

After loading, the column was washed with phosphate buffer containing sodium chloride, and then, only the variants on the acidic side including the Q-CDR-cleavage variants were specifically eluted so as to separate and remove them from the antibody eimeria-globin, to show how the removal effect was, CE-HP L C analysis was performed on the wash fraction, the elution fraction, and the load fraction before separation (fig. 7).

The loading, washing and elution conditions are as follows.

Loading Tris-HCl buffer containing the antibody eimeria-zumab adjusted to pH 5.0 was loaded under the condition of 33g of the antibody eimeria-zumab per 1L resin.

Washing phosphate buffer containing 25 mmol/L sodium chloride adjusted to pH7.2 was passed through a 3.5CV column at room temperature.

Elution phosphate buffer containing 100 mmol/L sodium chloride adjusted to pH6.5 was passed through a 6.5CV column at room temperature.

EXAMPLE 9 molecular Structure analysis of protected disulfide isoforms

The present invention relates to a method for correcting the scattering effect of anti-igg antibodies, which comprises preparing an antibody preparation of eimerichsizumab and protected disulfide isoforms (antibody concentration of 7.54mg/m L, 150 mmol/L arginine, 20 mmol/L histidine-aspartic acid, ph6.0), SAXS measuring using a line-collimated X-ray beam (Cu K α, λ 0.1542nm) generated by SAXSess mc2 system (Anton Paar, Graz, Austria) measuring temperature set at 25 ℃, detecting using a two-dimensional imaging plate set for 30 minutes, converting two-dimensional scattering intensity on SAXSQuant software (antpaar) into one-dimensional scattering intensity i (Rg), where q is a scattering vector (scattering vector), defined as q (4 pi/λ) sin (θ/2) (θ is an angle), for a light beam transmitted through a beam stop, the scattering curve is normalized for the scattering intensity at q 0, then normalized for sin (θ/2) (θ is an angle), obtaining a calibration curve using a white light scattering system (r) and a white light scattering system (r) as calibration curve, obtaining a calibration curve obtained by applying the correlation between the scattering curves obtained by applying the scattering curve obtained by applying the calibration curve of the scattering curves of the scattering system (i) to a scattering system (r) and the calibration curve obtained by applying the calibration curve obtained by the method when the calibration curve obtained by applying the calibration curve of the calibration curve obtained by the method when the calibration curve of the calibration curve obtained by applying (i) to the calibration curve obtained by applying (i) of the calibration curve obtained by applying (r) under the calibration curve obtained by the method under the conditions of the experimental peak of the calibration curve of the experimental particle size of the experimental peak of the anti-igg under the calibration curve, the calibration curve obtained by the experimental probe system (r) and the calibration curve, the calibration curve obtained by the method of the calibration curve obtained by the method of the method under the method of the calibration.

As a result, the protected disulfide isotype has an average Rg value of 4.8nm or less (a value of 0.3nm or more less than that of eimeria-lizumab) and an average Dmax value of 16.5nm or less (a value of 1.4nm or more less than that of eimeria-lizumab), and exhibits an average Rg value of 6% or more less and an average Dmax value of 7.5% or more less than that of Emcizumab it is reported that the Dmax value of an IgG4 antibody molecule, which is the same antibody subclass as eimeria-lizumab, corresponds to the distance between the tips of two Fab domains (NP L10), and thus, it is considered that the distance between the tips of two Fab domains of the protected disulfide isotype, which represents the distance from the centroid of the molecule (the center of the Rg) is shortened as compared to eimeria-bead, it is confirmed based on the above that the protected disulfide isotype has such a molecular structure that it has a distance between the ends of the two Fab domains of the J/Q chains, and thus, it is an important role for the two isotype antibody isotypes of the three-dimensional antibody classes to control of the interaction, and thus, the antibody type of the antibody, and the antibody, and.

[ example 10]Molecular Structure analysis by HDX-MS (Hydrogen deuterium exchange Mass Spectrometry)

Antibody preparations of eimeutralizumab and each protected disulfide isoform were prepared (antibody concentration 1mg/m L, 150 mmol/L arginine, 20 mmol/L histidine-aspartic acid, ph6.0) and HDX-MS measurements (measured at deuterium exchange times 30s, 60s, 120s, 240s, 480s, 960s, 1920s and 3840s) were performed using a HDX-MS apparatus (Orbitrap Fusion L umos (thermo Fisher scientific) with HDX-PA L (L EAP Technologies), UltiMate30000RS L cnano (thermo Fisher scientific)).

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

Industrial applicability

The antibody variants and isotypes of the invention have extremely reduced FVIII mimicking activity compared to eimeria-vizumab; thus, the pharmaceutical compositions of the invention comprising eimeria-zumab and having reduced content ratios of such antibody variants and isotypes can be used as a means of treating hemophilia. The method for analyzing antibody variants and isotypes of the present invention is useful for evaluating the quality of an eimeria-zumab preparation, and is also useful for developing an eimeria-zumab preparation with a reduced content ratio of antibody variants and isotypes or developing a method for inhibiting formation of antibody variants and isotypes.

Claims (21)

1. Antibody variants comprising a variable region comprising amino acid sequence SISPSGQSTYYRREVKG (SEQ ID NO: 2), wherein

(a) An amino acid residue R at position 12 from the N-terminal side of the sequence; or

(b) An amino acid residue YYR at positions 10 to 12 from the N-terminal side of the sequence,

is deleted and the variable region is cleaved at the site of deletion.

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 comprised in a heavy chain.

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

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

7. A method of detecting an antibody variant of any of claims 1-6, comprising the step of separating a sample comprising an antibody comprising a variable region comprising amino acid sequence SISPSGQSTYYRREVKG (SEQ ID NO: 2) by affinity chromatography, ion exchange chromatography, normal phase chromatography, reverse phase chromatography, hydrophilic interaction chromatography (HI L IC), Hydrophobic Interaction Chromatography (HIC), charge-based separation, Size Exclusion Chromatography (SEC), Gel Permeation Chromatography (GPC), or a combination thereof.

8. The assay of claim 7 which uses the antibody variant of any one of claims 1 to 6 as a reference standard.

9. A pharmaceutical composition comprising the antibody variant of any one of claims 1-6, wherein in said pharmaceutical composition the percentage of said antibody variant in total antibody molecules is 5% or less.

10. The pharmaceutical composition of claim 9, wherein the antibody is eimeria-lizumab.

11. 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 according to any one of claims 1-6, comprising the step of culturing antibody-producing cells at a pH of 7.1 or higher and/or at a culture temperature of 36 ℃ or lower.

13. Isotype of 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; and

(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 therein

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; and

(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.

14. The bispecific antibody isotype of claim 13, wherein disulfide bonds are formed in (1a) and (1 b).

15. An isotype of a bispecific antibody comprising a first heavy chain and a second heavy chain characterized in that it elutes in a region closer to the basic side than said bispecific antibody when separated using cation exchange chromatography.

16. The bispecific antibody isotype of any one of claims 13-15, which is an isotype of eimeislizumab.

17. A method of detecting the antibody isotype of any of claims 13-16, 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 (HI L IC), 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, which uses the antibody isotype according to any one of claims 13 to 16 as a reference standard.

19. A pharmaceutical composition comprising a bispecific antibody isotype of any of claims 13-16, wherein in the pharmaceutical composition the percentage of the antibody isotype in the total antibody molecules is 2% or less.

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

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