WO2018038469A1 - Method for confirming selective production of desired bispecific antibody - Google Patents

Abstract

The present invention relates to a method for confirming a production of a bispecific antibody, which can confirm whether a desired bispecific antibody is selectively produced. Through this method, the production efficiency of the desired bispecific antibody from the bispecific antibody production method can be confirmed.

Description

 [Explanation of invention]

 [Name of invention]

 Selective Production Confirmation Method of Bispecific Antibodies [Technical Field]

 The present invention relates to a method for identifying bispecific antibody production capable of confirming whether a desired bispecific antibody is produced or whether a desired bispecific antibody is selectively made. Background Technology

 Bispecific antibodies (BsAb) are antibodies with two antigen binding sites, each of which recognizes a different target antigen, which collectively refers to an antibody or antigen binding fragment thereof that can simultaneously bind to the different target antigen. The dual targeting ability of such bispecific antibodies may provide new fields of application that are not applicable with monospecific antibodies (Monospeci ant ibodies). From a therapeutic point of view, (1) reliably introducing immune cells into the vicinity of the target cell, (2) synergistic effects by simultaneously inhibiting or activating two distant signal transduction pathways of the target cell, (3) Specific and controllable delivery of therapeutic, radioactive materials, medicines, toxins, etc. to target cells has emerged as an important concern.

 As can be seen in Figure 1, when preparing a bispecific antibody, there are 10 kinds of antibodies that can be made of a combination of two heavy and light chains, respectively, of which bispecific antibodies having two antigen binding sites of interest It is one species, circled in FIG. 1. At this time, the 10 combinations produced when the bispecific antibody is prepared with two heavy chains and two light chains have similar molecular weights and are not easily separated by electrophoresis, and thus a bispecific antibody of a desired type has been produced. Difficult to identify.

For the development of efficient bispecific antibody production techniques, the evaluation of the production of bispecific antibodies of the desired combination and / or the ratio of production of the bispecific antibodies of the desired combination, among 10 possible combinations as shown in FIG. Thus, the development of a means by which bispecific antibody preparation technology can evaluate the efficiency or selective preparation rate of a desired bispecific antibody is Required.

[Detailed Description of the Invention]

 Technical problem

 The inventors of the present invention prepared a antibody having one heavy chain and one light chain having no variable region in a wild-type antibody having two heavy chains and two light chains, and then confirming antigen binding, thereby determining the desired heavy chain-light chain and heavy chain-. The present invention has been completed with the discovery that it is possible to easily and accurately identify and / or evaluate whether bispecific enclosures with heavy chain combinations are selectively produced and / or selective production levels (volume or ratio of the desired bispecific antibody).

 One example provides a method for identifying and / or evaluating the selective production of a desired bispecific antibody.

 Another example provides a method of identifying and / or evaluating the selective production of a desired bispecific antibody in an antibody preparation method (bispecific antibody preparation method).

[Measures of problem]

When a bispecific antibody is prepared using the heavy chain A and the light chain a that bind one antigen, and the heavy chain B and the light chain b that binds the other antigen, an antibody including two heavy chains and two light chains is as shown in FIG. 1. 10 combinations can be made (in FIG. 1, the antibodies in which heavy chain A and light chain a and heavy chain B and light chain b are correctly paired are indicated by dotted circles). At this time, one heavy and light chains (for example, heavy chain B and light chain b) were prepared in such a manner that the variable portion was removed, thereby producing a combination of antibodies in the case of producing a bispecific antibody is shown in FIG. In FIG. 2, the desired bispecific antibody (ie, two pairs of heavy and two light chains that bind to different antigens are correctly matched to the antigen to which each binds (combination of Aa and Bb; indicated by dotted circles in FIG. 1). Antibody combinations (ie, heavy-light chain with variable region and heavy-light chain without variable region) are indicated by dotted circles. FIG. 3 schematically shows an antibody (indicated by a dotted circle in FIG. 2) in which two heavy and two light chains are correctly paired using a set of heavy and light chains lacking a variable portion. At light weight the antibody combinations illustrated in FIG. The heavy weights are grouped into groups 1 through 5, with the heaviest gains for a combination of two relatively heavy (long chain) heavy chains and two relatively heavy (long chain) light chains. In the case of a combination of two relatively light (short chain length) light chains and two relatively light (short chain length) heavy chains, the lightest weight is represented as one group (see FIG. 4). . In Figure 4, the bispecific antibodies of interest belong to group 3 because the combination of the relatively heavy and heavy light chains and the combination of the relatively light and heavy light chains form a dimer and have a medium weight (indicated by the dashed circle). ). Since the antibodies belonging to the three groups have almost the same molecular weight, they can be distinguished from other groups by conventional methods such as electrophoresis. However, even if it was confirmed that the antibodies belonging to the three groups were produced, since it contains mismatched dimers in addition to the desired bispecific antibody, there is a limit in accurately determining whether the desired bispecific antibody has been formed.

 In the present specification, in order to overcome the above limitation, the heavy chain (without the variable part) including the variable part in the antibody combination (Fig. 3) corresponding to the desired bispecific antibody (heavy chain of A chain in Fig. 3) and Affinity chromatography was performed using an antigen to which the light chain (light chain of A chain in FIG. 3) binds, and includes a combination of a heavy chain including a variable portion and a light chain including a variable portion among the antibodies belonging to the three groups shown in FIG. 4. By detecting only an antibody to be detected, a technique capable of selectively detecting only an antibody corresponding to a desired bispecific antibody (indicated by a dotted circle in FIG. 4) and / or confirming that an antibody corresponding to a desired bispecific antibody has been selectively generated. To provide.

 Methods for Confirming and / or Evaluating Selective Production and / or Evaluation of Bispecific Antibodies Provided herein, and / or Methods for Identifying and / or Evaluating Selective Production of Bispecific Antibodies of the Antibody Preparation Method (bispecific antibody preparation method) May be comprised of the following steps:

 (I) a first heavy chain and a crab 1 light chain that specifically bind to a first antigen, and

(ii) preparing an antibody using (expressed) a double heavy chain that does not include some or all of the variable parts and a second light chain that does not include some or all of the variable parts. step;

 (2) performing chromatography on the antibody prepared in step (1) using an adsorbent to which the first antigen is bound; And

 (3) eluting the antibody adsorbed on the adsorbent material after the step (2) to analyze the antibody contained in the eluate; And / or

 (4) analyzing the antibody contained in the f low through after performing step (2), and / or

 (5) analyzing the antibody contained in the result obtained in step (1).

 The preparation of the antibody in step (1) can be carried out by conventional antibody preparation methods, such as all methods commonly used for the preparation of bispecific antibodies (eg, recombinant methods, chemical methods, etc.). For example, the preparation of the antibody of step (1) may include a gene encoding the heavy chain, the crab-2 light chain, and the heavy chain which does not include some or all of the variable parts, and the light chains that do not include some or all of the variable parts. The recombinant vector obtained by cloning the vector can be carried out by a recombinant method comprising the step of expressing in a suitable host cell.

 The second heavy chain, which does not include some or all of the variable parts, and the second light chain, which does not include some or all of the variable parts, include a full-length heavy chain including the variable part by lacking the variable part of the second heavy chain and the crab light chain capable of binding to a second antigen, and It means having a molecular weight difference of a degree distinguishable from the full length light chain. The first and crab antigens may be different proteins or different parts of the same protein that are not overlapping (separated so as not to interfere with each other when combined with different antibodies), preferably different proteins. have.

 In the method, steps (3) and (4) are performed after the chromatography of step (2), and step (5) is performed after the antibody preparation step of step (1) and the chromatography step of step (2) It can be done before, after, or simultaneously. When performing two or more steps of the above steps (3) to (5), they may be performed simultaneously or sequentially.

 For example, the method is

(1) (i) a first heavy chain and a first light chain that bind to the crab antigen and (ii) Preparing an antibody using a second heavy chain not including some or all of the variable parts and a second light chain not including some or all of the variable parts;

 (2) performing chromatography on the antibody prepared in step (1) using an adsorbent to which the Cage-1 antigen is bound; And

 (3) eluting the antibody adsorbed to the adsorbent material after the step (2) to analyze the antibody contained in the eluate

 It may include.

 The eluate of step (3) is obtained by eluting the adsorbent after performing the chromatography of step (2) with a commonly used elution buffer, and includes an antibody adsorbed on the adsorbent.

 Analyzing the antibody contained in the eluate of step (3) may be performed by detecting an antibody including two heavy chains and two light chains, regardless of whether the eluate contains intact variable parts. The eluate contains an antibody capable of binding a first antigen, i.e., an intact C 1 antibody (half antibody; hal f-ant ibody) comprising a full l-length U heavy chain and a full length first light chain. Only included. At this time, only the antibody having a molecular weight corresponding to the desired bispecific antibody (that is, the antibody of the full length first heavy chain-full length crab 1 light chain and the variable part lacking second heavy chain-lacking variable crab 2 light chain combination) is detected. The higher the level of the antibody having the molecular weight detected (ie, the higher the concentration of the antibody having the molecular weight contained in the eluate; for example, as compared to the level (concentration) of the antibody having a molecular weight different from the molecular weight). Higher than this), the selective production rate of the desired bispecific antibody comprising not only intact Crab 1 antibodies but also well-matched 12 antibodies (including half variable chains lacking the variable region and two light chains lacking the variable region) This can be identified as high and / or evaluated.

In the step (3), if the eluate contains an antibody (whole antibody) having a molecular weight different from the molecular weight corresponding to the desired bispecific antibody, the selective production rate of the paired target bispecific antibody is confirmed to be high and / or In the evaluation, an antibody level having a molecular weight corresponding to a desired bispecific antibody in the eluate (ie, an antibody concentration having a molecular weight corresponding to a desired bispecific antibody included in the eluate) corresponds to the desired bispecific antibody. Antibody levels with a molecular weight different from the molecular weight The eluate is higher than the objective antibody concentration having a molecular weight different from the molecular weight of a bispecific antibody comprising a) may be a ^"premises.

 The molecular weight of the desired bispecific antibody (or the molecular weight corresponding to the desired bispecific antibody) includes a full length first heavy chain, a full length first light chain, a double heavy chain not including some or all of the variable parts, and some or all of the variable parts. A range obtained by summing the molecular weight of the second light chain which is not added (M) or the amount of molecular weight change (ΔΜ) due to all post-translat ion processing occurring during antibody production such as glycosylation to the numerical value (for example, M土 ΔΜ).

 Therefore, in the method, after step (3), only the antibody having a molecular weight corresponding to the desired bispecific antibody in the eluate is detected, or the molecular weight at which the level of the antibody having a molecular weight corresponding to the desired bispecific antibody is different. Higher than the level of the antibody having, the identification and / or evaluation that the desired bispecific antibody is selectively produced or that the method for producing the antibody used in step (1) selectively produces the desired bispecific antibody (step (3-1)) may be further included.

 In another example, the method

 (1) using (i) the first heavy chain and the first light chain to bind to the first antigen, and (ii) the second heavy chain not including some or all of the variable parts and the second light chain not including some or all of the variable parts, Preparing an antibody;

 (2) performing chromatography on the antibody prepared in step (1) using an adsorbent to which the Cage-1 antigen is bound; And

 (4) analyzing the antibody contained in the pass (f low, through) after performing step (2)

 It may include.

Analyzing the antibody contained in the flow-through of step (4) is carried out in the flow-through, whether or not including the intact variable region, the antibody comprising two heavy chains and two light chains ('intact ant ibody In the following, all antibodies not specifically referred to as 'half antibody' can be performed by detecting an antibody including two heavy chains and two light chains regardless of whether they contain variable regions. At this time, the antibody is not detected in the flow through, or the lower the detection level (that is, the lower the concentration of the antibody in the flow through), the impurities Of the desired bispecific antibody comprising a secret type first antibody (half antibody; including one heavy chain and one light chain) capable of binding to a first antigen with low production rate and a first heavy and crab light pair paired correctly High selective production rates can be identified and / or evaluated.

 Therefore, the method, after step (4), if the antibody is not detected in the flow-through solution or the level is low, the desired bispecific antibody is correctly paired with the first heavy chain and the first light chain constituting the Crab antibody Is optionally generated or the method for producing the antibody used in step (1) further comprises the step of confirming and / or evaluating the production of the desired bispecific antibody (step (4-1)). can do.

 In another example, the method

 (1) using (i) the first heavy chain and the crab 1 light chain that bind to the crab-1 antigen, and the (2) light chain that does not include some or all of the (Π) variable parts, using a second light chain that does not contain some or all of the variable parts Preparing an antibody;

 (2) performing chromatography on the antibody prepared in step (1) using an adsorbent to which the first antigen is bound;

 (3) eluting the antibody adsorbed on the adsorbent material after the step (2) to analyze the antibody contained in the eluate; And

 (4) analyzing the antibody contained in the f low through after performing step (2).

As described above, step (3) may be performed by detecting the antibody in the eluate, and step (4) may be performed by detecting the antibody in the flow through. By the above step (3), the desired duplex comprising not only the first paired antibody (half antibody) correctly paired, but also the second antibody (half antibody) paired correctly with the second heavy chain lacking the variable region and the second light chain lacking the variable region Selective generation and / or selective production rate of the specific antibody can be confirmed and / or evaluated, and by step (4), the half-antibody in its intact form capable of binding to the Cage-1 antigen with the Cage-1 and the first light chain paired correctly Selective production and / or selective production rate of the desired bispecific antibody comprising can be identified and / or evaluated more accurately (or supplementally). Thus, in the case of including (performing) both step (3) and step (4), a second antibody (half antibody) paired correctly with a first paired antibody (half antibody) It may be more advantageous to confirm and / or evaluate the selective production and / or evaluation of the desired bispecific antibody, and / or to identify and / or evaluate the selective production or selective production efficiency of the desired bispecific antibody in a method of preparing the bispecific antibody. In the method for confirming and / or evaluating the selective production of the desired bispecific antibody of the bispecific antibody production method, in order to confirm and / or evaluate the selective production of the desired bispecific antibody of the test method, the test subject The steps (1), (2), and (3) are performed for the production method and the comparison target production method, respectively, and the results obtained in step (3) are compared with each other.

 (a) an antibody level having a molecular weight corresponding to the desired bispecific antibody in the eluate detected in step (3) of the preparation method to be tested (ie, an antibody having a molecular weight corresponding to the desired bispecific antibody included in the eluate) Antibody levels having a molecular weight corresponding to the desired bispecific antibody in the eluate detected in step (3) of the preparation method to be compared (ie, an antibody having a molecular weight corresponding to the desired bispecific antibody included in the eluate). Concentration),

 (b) an antibody level having a molecular weight different from that corresponding to the desired bispecific antibody in the eluate detected in step (3) of the preparation method to be tested (ie, the molecular weight corresponding to the desired bispecific antibody included in the eluate). Antibody concentrations having a molecular weight different from the molecular weight corresponding to the desired bispecific antibody in the eluate detected in step (3) of the preparation method to be compared, i.e., the desired duplex contained in the eluate. Lower than the antibody concentration having a molecular weight different from that of the specific antibody), or

 (c) if it corresponds to both (a) and (b) above,

 The test subject manufacturing method can identify and / or evaluate that the selective production efficiency of the desired bispecific antibody or the production rate of the desired bispecific antibody is high as compared with the comparative subject manufacturing method.

 Therefore, the method of confirming and / or evaluating the selective production of the desired bispecific antibody of the bispecific antibody production method, after step (3),

(a) an antibody level (ie, having a molecular weight corresponding to the desired bispecific antibody in the eluate detected in step (3) of the preparation method to be tested) Antibody concentration having a molecular weight corresponding to the desired bispecific antibody contained in the eluate) (ie, the level of the antibody having a molecular weight corresponding to the desired bispecific antibody in the eluate obtained in step (3) using the preparation method of comparison) , The concentration of the antibody having a molecular weight corresponding to the desired bispecific antibody contained in the eluate)

 (b) an antibody level having a molecular weight different from that corresponding to the desired bispecific antibody in the eluate detected in step (3) of the preparation method to be tested (i.e., the molecular weight corresponding to the desired bispecific antibody in the eluate); Antibody levels having different molecular weights (i.e., the desired duplex contained in the eluate) have a molecular weight different from the molecular weight corresponding to the desired bispecific antibody in the eluate obtained in step (3) using the preparation method of comparison. Lower than the antibody concentration having a molecular weight different from that of the specific antibody), or

 (c) if it corresponds to both (a) and (b) above,

 The test subject manufacturing method may further comprise the step (3-2) of confirming and / or evaluating that the selective production efficiency of the desired bispecific antibody or the rate of production of the desired bispecific antibody is high as compared with the comparative subject manufacturing method. Can be. In addition, the method of confirming and / or evaluating the selective production of the desired bispecific antibody of the method of producing the bispecific antibody is tested after the step (3) and before the identification and / or evaluation (3-2). Comparing the antibody level having a predetermined molecular weight in the eluate obtained in step (3) using the subject preparation method with the antibody level having a predetermined molecular weight in the eluate obtained in step (3) using the subject preparation method It may further include. In one example, the method may include one or more of steps (3-1) and (3-2) in any order.

Further, in the method for confirming and / or evaluating the selective production of the desired bispecific antibody of the bispecific antibody production method, in order to identify and / or evaluate the selective production of the desired bispecific antibody of the production method under test, Perform the steps (1), (2), and (4) on the production method to be compared, and compare the result obtained in step (4) with the result obtained in step (4) using the test object production method, Antibody levels in the flow-through detected in step (4) of the preparation method to be tested (ie, the antibody contained in the flow-through) When the concentration is lower than the antibody level (ie, the antibody concentration contained in the flow through) detected in step (4) of the production method to be compared, the test target production method may It can be confirmed and / or evaluated that the selective production efficiency of the bispecific antibody or the production rate of the desired bispecific antibody is high.

 Therefore, the method of confirming and / or evaluating the selective production of the desired bispecific antibody of the bispecific antibody production method, after step (2), the analysis result in step (4) of the test target production method, the pass-through solution When no antibody is detected (ie, no antibody is present in the flow through), or the antibody level in the flow through is lower than the antibody level in the flow through obtained in step (4) using the preparation method to be compared. The production method to be tested may further include the step (4-2) of identifying and / or evaluating that the selective production efficiency of the desired bispecific antibody or the production rate of the desired bispecific antibody is high compared to the production method to be compared. have. In addition, the method for confirming and / or evaluating the selective production of the desired bispecific antibody of the method for producing a bispecific antibody is tested after the step (4) and before the step of identifying and / or evaluating (4-2). Comparing the antibody levels in the flow-through obtained in step (4) using the subject preparation method may further comprise comparing the antibody levels in the flow-through obtained in step (4) using the subject preparation method. In one example, the method may include one or more of steps (4-1) and (4-2) regardless of order.

 In addition, the method for confirming and / or evaluating the selective production of the desired bispecific antibody of the bispecific antibody production method is described in step (1) to

(4), one or more of steps (3-1) and (3-2), and one or more of steps (4-1) and (4-2) (eg, step (1), ( 2), (3), (3-1), (4) and (4- 1), or include steps (1), (2), (3), (3-2), (4) and ( 4-2)), optionally (when step (3-2) and / or (4-2) are included), and further comparing the results with the results of the corresponding comparison method respectively. It may include. Details are as described above.

 In another example, the method

(1) (i) the crab 1 heavy chain and the first light chain, and (ii) the second heavy chain and the second light that does not include some or all of the variable part. Preparing an antibody using a light chain; And

 (5) may comprise the step of analyzing the antibody contained in the result (manufacturing product) obtained in step (1).

 The product used in step (5) may be a product obtained in step (1), or may be subjected to a conventional antibody purification method (for example, protein A affinity chromatography, etc.) to increase the antibody concentration.

 It can be carried out by detecting the antibody according to the molecular weight in the result of the step (5). In the result, only the antibody having a molecular weight corresponding to the desired bispecific antibody as described above, or the higher the detection level of the antibody having the molecular weight (that is, the higher the concentration of the antibody having the molecular weight included in the result) For example, the higher the level (concentration) of an antibody having a molecular weight different from the above molecular weight, the higher the selective production rate of the desired bispecific antibody can be identified and / or evaluated.

 Thus, the method, after step (5), if only the antibody having a molecular weight corresponding to the desired bispecific antibody in the result is detected, or if the detection level is higher than the antibody level having a different molecular weight, the desired bispecific Confirming and / or evaluating that the antibody is selectively produced or that the method for producing the antibody used in step (1) selectively produces the desired bispecific antibody (step (5-1)) It can be included as.

 Since step (5) is performed on the result before the chromatography using the C antigen 1 of step (2), the antibody having a molecular weight corresponding to the desired bispecific antibody may be combined with other combinations in addition to the desired bispecific antibody. May be included (see group 3 in FIG. 4). However, an antibody having a molecular weight corresponding to the desired bispecific antibody is detected in step (5), and an antibody having a molecular weight corresponding to the desired bispecific antibody in the eluate is detected in step (3), and / or If no antibody is detected in the flow-through in step (4), the probability of selectively producing the desired bispecific antibody is increased. Therefore, step (5) may be further included in addition to steps (1) (2), and (3) and / or (4).

Another example is the coding gene of the crab 1 heavy chain, the coding gene of the first light chain, A recombinant vector comprising the coding gene of the second heavy chain lacking part or all of the variable portion, and the coding gene of the second light chain lacking part or all of the variable portion, and the coding gene of the first heavy chain, encoding of the first light chain Provided are a recombinant cell comprising a gene encoding a double heavy chain gene lacking part or all of a variable portion, and a coding gene of a double light chain lacking a portion or all of a variable portion. The recombinant vector may be a transformed host cell with the recombinant vector.

 Other examples include the selective generation and / or evaluation of a bispecific antibody comprising at least one selected from the group consisting of the recombinant vector and the recombinant cell, and / or the selection of the desired bispecific antibody in a method of preparing the bispecific antibody. Provide compositions for identifying and / or evaluating production.

As used herein, 'antibody' may be one or more selected from all kinds of immunoglobulins (immunoglobulin) derived from all kinds of mammals or birds. For example, antibodies used herein include IgG (eg, IgG type 1 (IgGl), IgG type 2 (IgG2), IgG type 3 (IgG3), and IgG type 4 (IgG4)), IgA (eg, IgA type 1 (IgAl) and I _g A type 2 (IgA2)), IgD, IgE, and IgM. The antibody may be an immunoglobulin derived from a mammal, such as a human, a primate including a mouse, a rodent including a mouse, a rat, and the like, for example, an immunoglobulin derived from a human. In one embodiment, the antibody comprises human IgGl (constant region; protein: GenBank Accession No. AAC82527.1, gene: GenBank Accession No. J00228.1), human IgG2 (constant region; protein: GenBank Accession No. AAB59393.1, Gene: GenBank Accession No. J00230.1), human IgG3 (constant region; protein: GenBank Accession No. P01860, gene: GenBank Accession No. X03604.1), human IgG4 (constant region; protein: GenBank Accession No. AAB59394. 1, gene: GenBank Accession No. K01316.1), human IgAl (constant region; protein: GenBank Accession No. AAT74070.1, gene: GenBank Accession No. AY647978.1), human IgA2 (constant region; protein: GenBank Accession No. AAB59396.1, Gene: GenBank Accession No. J00221.1), human IgD (constant region; protein: GenBank Accession No. AAA52771.1, AAA52770.1), human IgE (constant region; protein: GenBank Accession No. AAB59395.1, genes: GenBank Accession No. J00222. 1) and human IgM (constant region; protein: GenBank Accession No. CAB37838. 1, gene: GenBank Access ion No. X57086. 1). In one example, the antibody may be, for example, one or more selected from the group consisting of human, IgGl, IgG2, IgG3, and IgG4, but is not limited thereto. The amino acid sequences of the heavy and light chain constant regions of the antibody have a very high degree of subtype preservation.

 As used herein, 'an intact antibody' includes two heavy chains and two light chains, and includes one light chain and one heavy chain (Hal f-ant), regardless of whether they contain intact variable regions. i body) two means a disulfide-linked form of antibody in a heavy chain (eg, hinge region), and the term 'antibody' described herein without reference to two heavy chains and two light chains, regardless of whether they contain intact variable regions. And the same as the 'silver transfer antibody' comprising one light chain and one heavy chain. Used in the sense. In addition, the half antibody means a structure in which one heavy chain and one light chain are disulfide-linked regardless of whether the intact variable region is included, and the first antibody or the second antibody is included in the half antibody.

 As used herein, 'first antibody' and 'second antibody' are monovalent antibodies each comprising one heavy chain and one light chain, and recognize different antigens or different epitopes. Because of the nature of the methods provided herein, one of the 'first antibody' and 'second antibody' (assuming a second antibody herein) includes heavy and light chains that lack all or part of the variable portion. And substantially lost binding ability to the antigen (assumed herein as the second antigen). As described above, the first antibody or the second antibody is included in the half antibody, regardless of whether the secret variable portion is included. As used herein, 'bispecific antibody' refers to an antibody that recognizes and / or binds two different antigens or two distinct (non-duplicate) epitopes of one antigen.

As used herein, a 'bispecific antibody of interest' refers to a first antibody (half antibody) that recognizes a desired first antigen or first epitope and a second that recognizes a desired second antigen or second epitope. The antibody refers to an antibody to which an antibody (half antibody) is bound. The first heavy chain and the first light chain, which constitute the first antibody, and the second light. It means an antibody in which the second heavy chain and the second light chain, and the first heavy chain and the second heavy chain constituting the antibody are exactly matched. In the present specification, in order to confirm the production of the desired bispecific antibody, since the second heavy chain and the second light chain are each used in a state in which all or part of the variable part is missing, the ^first desired bispecific antibody 'is the full-length first heavy chain. And full length first light chain pairing, between a second heavy chain lacking a variable portion and a second light chain lacking a variable portion, and between a full length first heavy chain and a second heavy chain lacking a variable portion (between Fc domains) Means the antibody formed. ,

 The preparation of the bispecific antibody in step (1) may be by conventional recombinant methods. For example, the preparation of the bispecific antibody comprises a recombinant vector (expression vector) comprising a gene encoding the first heavy chain, the first light chain, the second heavy chain, and the second light chain, respectively, or two or more thereof, in a host cell. Expression (eg, co-expression in one cell). For example, the genes may be expressed by culturing a recombinant vector including the recombinant vector.

The "host cell ¹ " refers to a cell into which the recombinant vector is introduced, and the host cell into which the recombinant vector is introduced is called a recombinant cell. The host cell may be a viral cell, a bacterial cell, a eukaryotic cell, a nasal cell, or a plant. Cells, or animal cells, for example, E. coli G¾ \ coli, mouse cells (eg, COP, L, C127, S _P 2/0, NS-0, NS-1, At 20, NIH3T3, etc.), rat cells (PC12, PC12h, GH3, MtT, etc.), hamster cells (e.g., BHK, CHO, GS gene defect CHO, DHFR gene defect CH0, etc.), monkey cells (e.g., C0S1, C0S3, C0S7, CV1, etc.) , Vero et al.), Human cells (eg, Hela, HEK-293, HEK-293-producing cells, retinal-derived PER-C6, diploid fibroblasts, myeloma cells, HepG2, NS / 0 cells, and lymph Constituent cells, etc.), insect cells (eg Sf9 cells, Sf21 cells) From the group consisting of Tn-368 cells, such as BTI-TN-5B1-4 cells), hybridoma, it may more than one selected, without being limited thereto.

Such vectors include viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, retrovirus vectors and adeno-associated virus vectors. The recombinant vector comprises a first heavy chain, a first light chain, a second heavy chain lacking part or all of the variable portion, and As a vector operably introduced with a gene encoding a second light chain lacking part or all of the variable portion, plasmids used in the art (eg, pcDNA series, pSC10, _P GV1106, pACYC177, ColEl, pKT230, _P ME290, PBR322, _P UC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFRl, pHV14, pGEX series, ET series, pUC19, etc., phage (e.g., λ ^ 4 λ Β, λ -Charon, λ Δ ζΙ, M13, etc.) or viruses (for example, SV40, etc.).

 As used herein, 'heavy chain (or lacking a variable portion) heavy chain that does not include some or all of the variable portion' and 'light chain (or lacking a variable portion) that does not include some or all of the variable portion' refers to the full-length heavy chain and It means a state in which all or part of the variable part is removed from the light chain of the full length. In the present specification, the reason for the lack or elimination of the variable portion in the heavy and light chains is due to the difference in molecular weight, the full length first heavy chain and the full length first light chain, the second heavy chain lacking the variable portion and the second light chain lacking the variable portion, and The bispecific antibody in which the full length first heavy chain and the second heavy chain lacking the variable portion are correctly paired is intended to be distinguishable from other antibodies. Thus, the lack of 'variable portion' may be a variable portion fragment of a size that can cause a molecular weight difference that can be distinguished from full-length antibodies, including full-length heavy chain and full-length light chain, by conventional protein analysis techniques. The 'variable part' lacking in the first heavy chain or the first light chain may be fragments having the same or different sizes, but since the deletion of the variable part must occur in both the first heavy chain and the first light chain, All 11 light chains should be free of variable region fragments comprising at least one amino acid or variable region fragments having a molecular weight greater than 100 Da.

In one example, the portion of the variable region lacking in the first heavy chain and the first light chain means a contiguous or non-contiguous amino acid fragment in the first heavy chain variable region and / or the first light chain variable region, and includes a total size (molecular weight; The sum of the molecular weight of the heavy chain variable part and the molecular weight of the light chain variable part that is lacking) is at least IkDa, at least 2kDa, at least 4kDa, at least 6kDa, at least 8kDa, at least 10kDa, at least 12kDa, at least 14kDa, at least 16kDa, at least 18kDa, or at least 20kDa. (The upper limit may be the total molecular weight of the heavy chain variable part and the light chain variable part), and the molecular weight of the part of the heavy chain variable part or the part of the light chain variable part lacking in the first heavy chain and the first light chain is 0.5 kDa or more, IkDa or more, and 2 kDa, respectively. 3kDa or more ， 4kDa or more ， 5kDa or more ， 6kDa or more ， 7kDa or more Or more, 8 kDa or more, 9 kDa or more, or lOkDa or more (the upper limit is the molecular weight of the heavy chain variable part (for some heavy chain variable parts) or the molecular weight of the light chain variable part (for some light chain variable parts), and the sum of the molecular weights of each of the variable parts The total molecular weight range of some of the above-described variable parts). Given that the average amino acid molecular weight is about 110 Da, some of the variable parts lacking in the first heavy chain and the first light chain have a total number of amino acids of at least about 10, at least about 20, at least about 40, at least about 60, About 80 or more, or about 100 or more (the upper limit is the total number of amino acids in the heavy and light chain variable regions), and each of the variable regions lacking in the first heavy and first light chains has an amino acid number of about 5 At least about 10, at least about 20, at least about 30, at least about 40, or at least about 50 (the upper limit is the total number of amino acids in the heavy chain variable portion (for some of the heavy chain variable portions) or the total of the light chain variable portions The sum of the number of amino acids (for a part of the light chain variable part) and the number of amino acids of each of the part of the variable part may range from the total number of amino acids of the part of the variable part described above).

 The adsorbent used in step 2 may be selected from all the adsorbents used in the chromatography, for example, may be at least one selected from the group consisting of resin (eg, agarose resin, etc.), porous particles, and the like. In order to effectively layer an antigen on the adsorbent, the antigen may be a commonly used marker material (protein or peptide of 5 to 50 aa, 5 to 30 aa, 5 to 20 aa, 5 to 15 aa, or 5 to 10 aa). For example, a flag tag, etc.) may be tagged, and the adsorbent may be a conjugated substance (eg, a label-binding antibody) capable of binding to a label tagged with the antigen. The chromatography may be selected from all kinds of chromatography used to separate and / or purify the protein of interest, and may be, for example, affinity chromatography or the like, but is not limited thereto.

As used herein, the 'preparation method to be tested' can be selected from all bispecific antibody preparation methods to confirm the selective production of the desired bispecific antibody. 'Comparative subject manufacturing method' is not the same as the 'test subject manufacturing method', and may be selected from known methods for producing bispecific antibodies, for example, heavy chain constant region and / or light chain constant region of an antibody. Does not introduce mutations into (ie wild-type heavy chain Bispecific antibody production method using the constant region and / or light chain constant region, the existing dual antibody production method to compare the degree or degree of improvement of the production efficiency of the desired bispecific antibody in the development of the bispecific antibody production method, etc. It may be selected.

 Selective production (or production) of bispecific antibodies refers to the production (or production) or presence of an antibody comprising the exact combination of interest among the various combinations as shown in FIG. 1 in the preparation of the bispecific antibody, or the level (concentration) of the antibody. Or ratio).

 The methods used for antibody analysis in the above steps (3) to (5) may be independently selected from all protein detection methods and means, for example, may be selected from among methods and means for detecting proteins according to molecular weight differences. For example, the antibody analysis may be performed by a method selected from the group consisting of gel electrophoresis, si ze exclusion chromatography (SEC HPLC) and the like. At this time, whether or not the production of the antibody can be confirmed by the presence or absence of a band corresponding to the molecular weight of the antibody, the level of the antibody can be confirmed and / or evaluated and / or determined and / or determined by the concentration of the band (The thicker the band, the higher the level of the antibody can be identified and / or evaluated and / or determined and / or determined).

 In order to obtain more accurate results, the second step chromatography and the third to fifth antibody analysis performed in the preparation method to be tested and the second step chromatography and the third to five steps performed for the preparation method to be compared The antibody analysis of the steps can be performed under the same conditions (eg, the total amount of the antibody prepared in step (1), the chromatography conditions, the volumes of the eluate and the pass-through, the antibody analysis (eg, electrophoresis) conditions, etc.).

When explaining the above method in more detail, the type of antibody prepared by combining the variable portion of the heavy and light chain of one wild-type antibody and another wild-type antibody is shown in FIG. FIG. 2 schematically shows a mutation for removing all of the variable portions of heavy chain B and a corresponding mutation for removing all of the variable portions of light chain b, and shows a total of 10 combinations that may appear according to their combination. It was. The exact paired antibodies are shown as dotted circles. The exactly matched antibody indicated by the dotted circle is shown schematically in FIG. 3. These are grouped by weight (molecular weight) and are shown in FIG. Indicated. The dotted circles in FIG. 4 are the antibodies with the correct pairing. Antibodies with correct pairing are included in group 3. Group 3 contains three different combinations of antibodies other than those with exact pairing, but the remaining antibodies other than those with exact pairing do not have antigen-binding sites made from the combination of heavy chain A and light chain a. It cannot bind to its Daeung antigen. This can be used to determine how precisely the method for producing a bispecific antibody selectively produces the desired bispecific antibody. If the heavy and light chains are randomly bound when the bispecific antibody is prepared, a total of 10 antibodies may be generated as described above, and a total of 4 antibodies may be formed in Group 3 as well. However, on the contrary, if the formation of the bispecific antibody is specific binding of each heavy chain and the light chain only (that is, the binding is formed only between different heavy chains, so that only the bispecific antibody is formed solely, the bispecific antibody shown as circle in Fig. 3 or 4). Only the antibody having a weight corresponding to group 3 is produced, and only one species is included in the antibody having a weight of group 3. If so, the single antibody is not detected in the pass-through when the adsorption chromatography is filled with the resin to which the Daewoong antigen is bound, and only one band is formed in the eluate.

In other words, when bispecific antibodies were prepared using heavy and light chains in which one antigen-binding portion (variable portion) was removed according to the present invention, the adsorption chromatography was first performed when the bispecific antibodies were paired correctly. Antibodies are not detected in the pass-through solution of (the size of the detected antibody can be investigated by electrophoresis to predict what combinations are made between the two heavy chains and the two light chains. If you look at what combinations are present a lot), even if the antibody is eluted through the elution buffer to check the size of the eluted antibody can be confirmed whether the bispecific antibody is correctly paired. If the production method is efficient and correctly paired, only antibodies with only one elution peak appear in group 3, and if the production method is not efficient and not correctly paired, the flow-through fluid of group 3 4 and 5 Antibodies (see FIG. 4) are mixed, and in the eluate, groups 1, 2 and 3 sized antibodies (see FIG. 4) are mixed. 【Effects of the Invention】

According to the present invention it is possible to confirm the efficiency of the method of producing a bispecific antibody. In other words, according to the present invention, exactly paired bispecific antibodies are generated _. Whether it can be easily confirmed. This can be used to determine how good any method of producing a bispecific antibody is.

[Brief Description of Drawings]

 Figure 1 shows the type of combination between the heavy and light chains and heavy and heavy chains that may appear when producing a bispecific antibody, A and B represents a heavy chain, a is a light chain that binds to heavy chain A to create an antigen binding site, b is Each of the light chains that bind to heavy chain B to form another antigen-binding site ¾ (eg, A: giant U antibody heavy chain; B: second antibody heavy chain; a: first antibody light chain; b: crab 2 antibody light chain). Bispecific antibodies paired exactly in the desired combination are indicated by dotted lines. It is indicated by a circle.

 FIG. 2 is a diagram showing the case where the heavy and light chains of one half of Aa antibody (half antibody) and Bb antibody (half antibody) are not included in the variable region to determine whether bispecific antibodies are formed. The types of combinations between the heavy and light chains and between the heavy and heavy chains are shown, and the antibodies paired exactly in the desired combination are indicated by dotted circles. FIG. 3 woolly shows an antibody in which a half antibody comprising a full-length heavy chain and a full-length light chain (indicated by A chain) and a half antibody (indicated by a B chain) comprising a heavy and light chain having the variable portion removed.

 Figure 4 shows the molecular weight of the antibody that can be produced when the antibody is prepared by using the full-length heavy chain and full-length light chain, and heavy and light chains that do not include a variable portion in one embodiment, a total of 5 depending on the molecular weight It can be divided into groups.

Figure 5 shows the results when the antibody was prepared using the heavy and light chains, and full-length heavy and light chains that do not include the variable portion of the anti-TNF a antibody according to Example 1, L is condition media (condi t ion medium) was purified by Protein A, followed by electrophoresis, and FT was performed by electrophoresis of TNF a layered affinity chromatography and flow through. E is the result of electrophoresis of the eluate according to the affinity for TNF_alpha bound to ant i-Flag resin.

 FIG. 6 shows the results obtained when the antibody was prepared using the heavy and light chains from which the variable portion of the anti-TNF α antibody was removed and the full length heavy and light chains using the Chimps method illustrated in Example 2. L Is the electrophoresis of condition media, FT is the result of electrophoresis of flow through after affinity chromatography filled with TNF a, and E is the result of electrophoresis of eluate eluted with flag. .

 7 is a cleavage map of the pcDNA3 vector.

[Specific contents to carry out invention]

Hereinafter, the present invention will be described in more detail with reference to Examples. Example 1 Test of Antibody Production Method Without Variation of Constant Region Full-length heavy chain and full-length light chain of anti-TNF a antibody (adal imumab), heavy chain not including variable region (variable region lacking heavy chain) and variable region Antibodies were prepared by conventional methods (without introducing mutations into the constant regions) using a non-contained light chain (lacking variable region). In this embodiment, since various kinds of antibodies (see schematic diagram of FIG. 5) having different variable regions of heavy and light chains can be produced, the preparation of bispecific antibodies can be modeled. At this time, the antibody corresponding to the desired bispecific antibody is an antibody including a combination of a full length heavy chain and a full length light chain, and a combination of a variable region lacking heavy chain and a variable region lacking light chain. For the preparation of the antibody, a gene encoding the full-length heavy chain, full-length light chain, heavy chain not including the variable part (variable part lacking heavy chain) and light chain not including the variable part (variable part lacking light chain) of the anti-TNF a antibody, respectively After cloning into a pcDNA3 vector (Invi trogen; see FIG. 7), after transfection into Hek293 cel l (ATCC) and incubation in condition medium (condit ion medi a; CM; high-glucose DMEM (Welgene)) for 1 week, The medium was harvested and filtered and protein A chromatography was performed to produce antibodies. Full-length heavy chain, full-length light chain, heavy chain not including the variable part (variable part lacking heavy chain), and light chain not including the variable part (lacking variable part) of the anti-TNF ci antibody used above. Light chain) amino acid sequence is summarized in Table 1 below:

 [Table 1]

 (In Table 1, the variable part lacking an underlined part) A flag-tagged ΤΝΑα was added to a condition medium (CM; high-glucose DMEM; Welgene), and the anti-flag (ant i- Flag) resin (agarose bead resin conjugated with anti-Flag antibody; Sigma) was allowed to stratify the resin, TNFa antigen. Affinity chromatography was performed on the TNFci-filled resin for the condition medium containing the produced antibody (Binding buffer: 20 mM sodium phosphate, pH 7.0 buffer).

 After the condition medium containing the produced antibody, and the resin precipitated with the flow-through and the TNFa-bound antibody after performing the chromatography with a flag (elution solvent: 0.1 M glycine- SDS-PAGE (using 6% acrylamide gel, non-reducing conditions) was performed on HCl, pH 2.7 buffer) and the obtained eluate.

The obtained result is shown in FIG. In Figure 5, L lane Electrophoresis result of condition medium (Protein A purified medium), FT lane is the result of electrophoresis of flow through after affinity chromatography filled with TNF a, E lane is the flag (Fl ag) It is the result of electrophoresis of eluate eluted with).

 As shown in FIG. 5, when the antibody was produced using a heavy chain and a light chain in which a mutation did not occur in the constant region in a conventional manner, the condition medium of L lane was found to include all five groups of antibodies. The result is full length heavy chain, full length light chain, variable region. The lacking heavy and variable region lacking light chains show random binding. In the FT lane, the antibody (lOOkDa (group 5)) containing a combination of a variable region lacking heavy chain and a variable region lacking light chain does not include a combination of a full length heavy chain and a full length light chain, and does not include a full length heavy chain and a variable region missing light chain. Bands corresponding to antibodies (112 kDa (Group 4) or 125 kDa (Group 3)) including combinations and / or combinations of variable region lacking heavy and full length light chains were shown. These results show that antibodies that cannot bind to the antigen ΤΝΑ α are formed by passing the affinity chromatography because they do not include the combination of full-length heavy chain and full-length light chain. In addition, it can be seen from the results of the two bands in the FT lane that two or more kinds of antibodies are produced in the passage solution. On the other hand, in the E lane, three bands (150kDa (group l), 137kDa (group 2), and 125kDa (group 3)) appear in the eluent. It is shown that three or more different kinds of antibodies are produced, including combinations with full length light chains. In addition, in the 125 kD group, only antibodies of the desired pair that are exactly matched have a binding site capable of binding to TNF ci. Antibodies generated due to the presence of 125 kD in both E and FT lanes In addition to the desired antibody, it can be seen that contains a variety of antibodies.

Example 2 Test of Antibody Production Method Including Mutation of Constant Region Referring to Example 1, the full-length heavy chain (SEQ ID NO: 1) and full-length light chain (SEQ ID NO: 2) of the anti-TNF a antibody and the variable part were included. An antibody is prepared using a heavy chain (variable lacking heavy chain; SEQ ID NO: 3) and a light chain not including the variable region (variable lack light chain; SEQ ID NO: 4), and Chimps (Correl ated and Antibodies were prepared by introducing mutations into the constant region using a Harmonious Interfaci al Mutat ion between Protein Subuni ts) method (see Korean Patent Application No. 10-2017-0091758, which is incorporated herein by reference). The mutations of the constant regions introduced at this time are summarized in Table 2 below (values listed below are amino acid positions in the constant regions of antibodies expressed by the EU numbering system:

'ht tp: // www. i mgt. org / IMGTSc ient ifi cChart / Number i ng / Hu_IGHGnber .html ( heavy chain constant region), and ¹ 'http / www. imgt. org / IMGTSc i ent ifi cChar t / Number ing / Hu_IGKCnber .html (light chain kappa region) '

[Table 2]

이 때， 전장 중쇄 -전장 경쇄 및 가변부 결여 중쇄-가변부 결여 경쇄 조합을 갖는 항체가목적하는 이중특이성 항체에 해당한다고 할 수 있다. 실시예 1을 참조하여， 상기 제조된 항체에 대하여 SDS-PAGE를 수행한 결과를 도 6에 나타내었다. 도 6에서 L은 컨디션 배지의 전기영동 결과이고， FT는 TNF a가 충진된 친화크로마토그래피를 수행한 후 통과액 (Flow through)의 전기영동 결과이고, E는 condi t ioned medi a에 Flag tag이 달린 TNF a 을 넣어주고, ant i -Flag resin을 이용하여 TNF a 와 결합한 ant ibody를 침전시킨 후 플래그 (Flag)로 용출한 용출액의 전기영동 결과이다. 도 6의 L 레인에서 보여주듯이， 상기 표 1의 돌연변이를 도입시키는 Chimps 방법에 의하여 항체를 제조하는 경우， 정확하게 짝지어진 목적하는 조합의 항체가 속하는 3그룹에 해당하는 밴드만 나타나고, 다른 그룹에 해당하는 밴드는 나타나지 않았다. FT 레인에서는 어떠한 항체도 검출되지 않았으며， 이는 목적하는 조합의 항체와 무관한 4그룹 및 5그룹에 속하는 항체는 생성되지 않았음을 보여주며， 만들어진 모든 항체가 용출액에 포함되어 있음을 보여준다. 한편， E 레인에서는 L 레인에서와 같이 3그룹에 해당하는 밴드만 검출되었으며， 3그룹에 속하는 항체 중 TNF a가 층진된 레진에 결합할 수 있는 항체는 정확하게 짝지어진 목적하는 조합의 항체 (도 6의 점선 원)뿐이므로， E 레인과 L 레인의 밴드는 모두 정확하게 짝지어진 목적하는 조합의 항체를 나타내는 것이다. 따라서 위의 결과를 통하여 Chimps 방법은 특이적으로 정확히 짝지어진 이중특이성 항체를 매우 높은 효율 (약 100%에 가까움)로 생산할 수 있음을 보여준다.

At this time, it can be said that the antibody having a full-length heavy chain-full length light chain and a variable region lacking heavy chain-variable region lacking light chain combination corresponds to the intended bispecific antibody. Referring to Example 1, the results of performing SDS-PAGE on the prepared antibody are shown in FIG. 6. In Figure 6, L is the result of electrophoresis of the condition medium, FT is the result of electrophoresis of the flow (flow through) after performing affinity chromatography filled with TNF a, E is a flag tag on condi t ioned medi a TNF a was added and the ant ibody bound to TNF a was precipitated using ant i -Flag resin, followed by electrophoresis of the eluate eluted with flag. As shown in the lane L of Figure 6, when the antibody is prepared by the Chimps method of introducing the mutations of Table 1, only bands corresponding to the three groups belonging to the antibody of the desired combination matched exactly, and appear in other groups No band appeared. No antibodies were detected in the FT lanes, indicating that no antibodies belonging to groups 4 and 5 were irrelevant to the combination of antibodies of interest, indicating that all antibodies made were included in the eluate. Meanwhile, in lane E, only bands corresponding to group 3 were detected as in lane L. Of the antibodies, only TNF a-binding antibodies that can bind to the layered resin are exactly matched in the desired combination of antibodies (dotted circle in Fig. 6), so that the bands of the E and L lanes are all exactly matched. It represents. Therefore, the above results show that the Chimps method can produce highly precisely matched bispecific antibodies with very high efficiency (nearly 100%).

 Through the results of Example 1 and Example 2, the bispecific antibody production method according to the Chimps method of Example 2 compared with the conventional production method of the bispecific antibody (using the wild-type constant region of the antibody) of Example 1 It can be confirmed or evaluated that the selective production efficiency of the desired bispecific antibody is excellent.

Claims

[Range of request]  [Claim 1]  Methods for confirming the selective production of the desired bispecific antibody comprising the following steps:  (1) using (i) a first heavy chain and a crab 1 light chain that binds to the crab antigen, and (ii) a crab 2 heavy chain lacking some or all of the variable portion and a second light chain lacking some or all of the variable portion Preparing an antibody;  (2) performing chromatography on the antibody prepared in step (1) using an adsorbent to which the first antigen is bound; And  At least one step selected from the following steps (3) to (5):  (3) eluting the antibody adsorbed on the adsorbent material after the step (2) to analyze the antibody contained in the eluate;  (4) analyzing the antibody contained in the flow through after performing step (2); and  (5) analyzing the antibody contained in the result obtained in step (1).  [Claim 2]  The method of claim 1, wherein the method  (1) using (i) a crab 1 heavy chain and a crab 1 light chain capable of binding to the first antigen, and (ii) a crab 2 light chain lacking some or all of the variable portion, and a crab 2 light chain lacking some or all of the variable portion. Preparing an antibody;  (2) performing chromatography on the antibody prepared in step (1) using an adsorbent to which the first antigen is bound; And  (3) eluting the antibody adsorbed to the adsorbent material after the step (2) to analyze the antibody contained in the eluate  Which includes.  [Claim 3]  The method of claim 1, wherein the method  (1) using (i) a first heavy and a crab 1 light chain capable of binding to a first antigen, and (ii) a crab 2 light chain lacking some or all of the variable portion and a crab 2 light chain lacking some or all of the variable portion. Preparing an antibody; (2) the first antigen with respect to the antibody prepared in step (1) Performing chromatography using the bound adsorbate;  (3) eluting the antibody adsorbed on the adsorbent material after the step (2) to analyze the antibody contained in the eluate; And  (4) analyzing the antibody contained in the f low through after performing step (2);  Which includes.  [Claim 4]  The method according to claim 12 or 3, wherein  (5) further comprising the step of analyzing the antibody contained in the result obtained in step (1).  [Claim 5]  The method according to any one of claims 1 to 3, wherein the part of the variable part lacking in the second heavy chain and the second light chain is each a variable part fragment having a molecular weight of 5 kDa or more.  [Claim 6]  The method according to any one of claims U to C, wherein the method is for use in confirming the selective production of the desired bispecific antibody of the antibody production method used in step (1).  [Claim 7]  The coding gene of the first heavy chain, the coding gene of the crab 1 light chain, the crab which lacks part or all of the variable part, and the coding gene of the second heavy chain, and the coding gene of the second light chain lacking part or all of the variable part, And a portion of the variable portion lacking in the double heavy and crab 2 light chains is a variable region fragment each having a molecular weight of at least 5 kDa.  [Claim 8]  Crab 1 heavy chain coding gene, the first light chain coding gene, the second heavy chain coding gene lacking part or all of the variable part, and the crab 2 light chain coding gene lacking part or all of the variable part, And some of the variable regions lacking in the double heavy and crab 2 light chains are variable region fragments each having a molecular weight of at least 5 kDa.  [Claim 9] Comprising a recombinant vector of claim 17 or a recombinant cell of claim 8 Composition for confirming the selective production of bispecific antibodies.