JP2017187476A - Method of measuring modified antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of quickly and easily measuring the amount of antibody modified by stress and the like during manufacture.SOLUTION: A method comprises steps of (1) bringing an oligopeptide-bound carrier comprising a carrier and an oligopeptide that recognizes a modified antibody bound thereto, a sample containing an antibody under test, and a labelled antibody comprising a labeling substance directly or indirectly bound to an antibody that recognizes the antibody under test into contact with each other to produce a complex containing the oligopeptide-bound carrier, the modified antibody and the labelled antibody, and (2) detecting the labelled substance in the complex produced in the step (1) to measure the amount of the modified antibody in the sample.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for measuring the amount of denatured antibody. The present invention also relates to a method for evaluating the stability of an antibody using the method. Furthermore, the present invention relates to a kit for measuring the amount of denatured antibody.

  Antibodies used for pharmaceutical applications are usually produced by culturing cells capable of expressing the antibody, and then subjecting the obtained culture solution to purification operations such as centrifugation, filtration, and column chromatography. At this time, the obtained antibody may contain an aggregate of the antibody that is generated by being denatured due to stress during production. Since antibody aggregates may have immunogenicity (Non-patent Document 1), it is necessary to detect the aggregates with high precision, particularly when used for pharmaceutical applications.

  Known methods for detecting antibody aggregates include methods using size exclusion chromatography and field flow fractionation. However, these methods are methods for detecting antibody aggregates based on protein size, and are not methods based on structure and properties. In addition, X-ray crystal structure analysis and NMR-based methods are known as precise analysis methods of antibody higher-order structures, but these methods are very time-consuming methods, such as purity control during production. It is not suitable for applications that require quick and simple measurement.

  In recent years, molecules that specifically recognize antibodies denatured by stress or the like regardless of their molecular weight have been reported (Patent Document 1, Patent Document 2, Patent Document 3, and Non-Patent Document 2). However, the denatured antibody measurement method using the molecule described in these documents is a method using surface plasmon resonance, and an expensive dedicated device such as Biacore (manufactured by GE Healthcare) is required to perform the method. To do.

International Publication No. 2014/103203 International Publication No. 2014/115229 International Publication 2008/054030

S. K. Singh, J .; Pharm. Sci. , 100, 354-387, 2011 H. Watanabe et al. Biol. Chem. , 289, 3394-3404, 2014

  The present invention has been made in view of the above-described problems of the prior art, and an object of the present invention is to provide a method capable of quickly and easily measuring an antibody that has been denatured due to stress during production.

  As a result of intensive studies to achieve the above object, the present inventors have made an oligopeptide-binding carrier comprising an amino acid sequence represented by SEQ ID NO: 1 and an oligopeptide recognizing a denatured antibody bound to the carrier. And using a labeled antibody in which a labeling substance is bound to an antibody that recognizes the test antibody, the denatured antibody in the sample containing the test antibody can be quantitatively detected quickly and easily. . Further, in the method for measuring the amount of the denatured antibody, it has been found that a collagen peptide is useful in suppressing nonspecific adsorption to the carrier. Furthermore, it has also been clarified that highly hydrophobic plates or magnetic beads are preferably used as the carrier. In addition, according to such a method, the amount of denatured antibody can be measured regardless of the type of stress that causes degeneration, and in particular, heat treatment, freezing which could not be detected by existing protein property analyzers. The inventors have also found that antibodies denatured by melting treatment or stirring treatment can be detected quantitatively with high accuracy, and the present invention has been achieved.

That is, the first aspect of the present invention is a method for measuring a denatured antibody, comprising the steps shown in the following (1) and (2).
(1) An oligopeptide-binding carrier comprising the amino acid sequence set forth in SEQ ID NO: 1, wherein an oligopeptide that recognizes a denatured antibody is bound to a carrier, a sample containing the test antibody, and the test antibody A step of forming a complex containing an oligopeptide-binding carrier, a denatured antibody and a labeled antibody by contacting the antibody with a labeled antibody in which a labeled substance is bound directly or indirectly,
(2) A step of measuring the amount of the denatured antibody in the sample by detecting the labeling substance in the complex formed in the step (1).

The second aspect of the present invention is a method for evaluating the stability of an antibody, comprising the steps shown in the following (1) to (3).
(1) An oligopeptide-binding carrier comprising the amino acid sequence set forth in SEQ ID NO: 1, wherein an oligopeptide that recognizes a denatured antibody is bound to a carrier, a sample containing the test antibody, and the test antibody A step of forming a complex containing an oligopeptide-binding carrier, a denatured antibody and a labeled antibody by contacting the antibody with a labeled antibody in which a labeled substance is bound directly or indirectly,
(2) a step of measuring the amount of denatured antibody in the sample by detecting the labeling substance in the complex formed in step (1),
(3) A step of evaluating the stability of the test antibody in the sample based on the amount of the antibody measured in the step (2).

  Further, in the third aspect of the present invention, the step (1) is a step of first forming the complex by bringing the oligopeptide binding carrier and the sample into contact with each other and then contacting with the labeled antibody. The method according to the first or second aspect. The fourth aspect of the present invention is the method according to any one of the first to third aspects, wherein the oligopeptide-binding carrier is a carrier further blocked with a collagen peptide.

  The fifth aspect of the present invention is the method according to any one of the first to fourth aspects, wherein the carrier is a highly hydrophobic plate.

  The sixth aspect of the present invention is the method according to any one of the first to fourth aspects, wherein the carrier is a magnetic bead.

  Furthermore, the seventh aspect of the present invention is an oligopeptide binding carrier comprising the amino acid sequence set forth in SEQ ID NO: 1, wherein an oligopeptide recognizing a denatured antibody is bound to a carrier, and an antibody recognizing the antibody is labeled. It is a kit for measuring the amount of denatured antibody including a labeled antibody to which a substance is bound.

  The method for measuring the amount of denatured antibody of the present invention comprises (1) an oligopeptide-binding carrier comprising the amino acid sequence set forth in SEQ ID NO: 1, wherein an oligopeptide that recognizes the denatured antibody is bound to a carrier; A sample containing a test antibody is contacted with a labeled antibody in which a labeling substance is directly or indirectly bound to an antibody that recognizes the test antibody, thereby including an oligopeptide-binding carrier, a denatured antibody, and a labeled antibody. A step of forming a complex, and (2) a step of measuring the amount of denatured antibody in the sample by detecting a labeling substance in the complex formed in step (1). An antibody that has been denatured due to stress (eg, acid, alkali, heat, freeze-thaw, stirring) can be measured quickly and easily. Further, according to the method of the present invention, it is possible to measure the amount of denatured antibody relatively inexpensively without requiring an expensive dedicated device such as Biacore. Furthermore, antibody stability can also be evaluated based on the measurement results.

FIG. 6 is a diagram showing the results of Example 1. The white triangle is the result of the native human antibody solution, and the black circle is the result of the modified human antibody solution. In the result of Example 2, it is the figure which showed the result when 1% (w / v) collagen peptide was used as a blocking agent. In the graph, black indicates the result when the polypeptide (2A1) having the amino acid sequence described in SEQ ID NO: 1 is immobilized on the well, and white indicates the result when 2A1 is not immobilized on the well (graph). The same notation applies to FIGS. 2B to 2D below). In the result of Example 2, it is the figure which showed the result when 2% (w / v) skim milk was used as a blocking agent. In the result of Example 2, it is the figure which showed the result when 100 mM ethanolamine is used as a blocking agent. In the result of Example 2, it is the figure which showed the result when 0.5% (w / v) BSA is used as a blocking agent. It is the figure which showed the result of Example 3. The black rhombus shows the PolySorp plate, the white square shows the MediSorp plate, the white triangle shows the MaxiSorp plate, and the black circle shows the result using the MultiSorp plate. It is the figure which showed the result of Example 4. The black rhombus shows the PolySorp plate, the white square shows the MediSorp plate, the white triangle shows the MaxiSorp plate, and the black circle shows the result using the MultiSorp plate. It is the figure which showed the result of having evaluated the stability of the said antibody by the method of this invention for the human antibody which carried out the acid process or the alkali process among the results of Example 5. FIG. It is the figure which showed the result of having evaluated the stability of the said antibody with the commercially available apparatus for the human antibody which carried out the acid process or the alkali process among the results of Example 5. FIG. It is the figure which showed the result of having evaluated the stability of the said antibody by the method of this invention for the human antibody which carried out heat processing, the freeze-thaw process, or the stirring process among the results of Example 5. FIG. It is the figure which showed the result of having evaluated the stability of the said antibody with the commercially available apparatus for the human antibody which carried out heat processing, the freeze-thaw process, or the stirring process among the results of Example 5. FIG. It is the figure which showed the result of Example 6, and is the figure which showed the result when a human antibody is eluted with the buffer solution containing ammonium sulfate. It is the figure which showed the result of Example 6, and is the figure which showed the result when a human antibody is eluted with the buffer solution containing potassium chloride. It is the figure which showed the result of Example 7.

  Hereinafter, the present invention will be described in detail.

<Method for measuring the amount of denatured antibody>
The method for measuring the amount of the modified antibody of the present invention is a method comprising the steps shown in the following (1) and (2).
(1) An oligopeptide-binding carrier comprising the amino acid sequence set forth in SEQ ID NO: 1, wherein an oligopeptide that recognizes a denatured antibody is bound to a carrier, a sample containing the test antibody, and the test antibody A step of forming a complex containing an oligopeptide-binding carrier, a denatured antibody and a labeled antibody by contacting the antibody with a labeled antibody in which a labeled substance is bound directly or indirectly,
(2) A step of measuring the amount of the denatured antibody in the sample by detecting the labeling substance in the complex formed in the step (1).

  In the present invention, a denatured antibody refers to an antibody whose entire structure or partial structure has been changed by stress due to acid, alkali, heating, freeze-thawing, stirring, or the like. The modified antibody is not particularly limited as long as it has an Fc region, and includes, for example, immunoglobulin isotypes (IgG, IgA, IgD, IgE, IgM, etc.) and subclasses of those isotypes. In particular, there are nine classes of human immunoglobulins: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. The origin of the modified antibody is not particularly limited, and examples thereof include antibodies derived from humans, mice, rats, rabbits, goats, and the like, and antibodies derived from humans are particularly preferable. Furthermore, the denatured antibody is not limited to an antibody derived from such a single animal species, and may be an antibody formed by binding fragments of antibodies derived from different animal species. For example, a chimeric antibody (Fc region of a human antibody and a chimeric antibody containing a sequence (variable region, CDR, etc.) derived from an antibody derived from another animal (eg, mouse antibody, rat antibody, rabbit antibody, goat antibody)) ), May be a humanized antibody. The antibody according to the present invention may be a monoclonal antibody or a polyclonal antibody.

  The sample to be used in the method of the present invention is not particularly limited as long as it contains the above-mentioned test antibody regardless of whether it is denatured or undenatured, and is usually a solution containing an antibody, particularly containing a human antibody. Is preferred. Also, the solution is not particularly limited as long as it does not inhibit the binding between the modified antibody and the following oligopeptide.

  In the present invention, the oligopeptide that recognizes the denatured antibody only needs to contain at least the amino acid sequence set forth in SEQ ID NO: 1, and an oligopeptide consisting only of the amino acid sequence set forth in SEQ ID NO: 1 may be used. In addition, an oligopeptide added with an oligopeptide consisting of several amino acid residues to several tens of amino residues such as a linker peptide for binding to the carrier described later may be used on the N-terminal side or C-terminal side of the oligopeptide. The length of the oligopeptide according to the present invention is not particularly limited as long as it recognizes a denatured antibody, but is usually 50 to 25 amino acids, preferably 40 to 25 amino acids, more preferably 30 to 25 amino acids. And particularly preferably 25 amino acids (SEQ ID NO: 1 only amino acid sequence). In addition, the oligopeptide is usually composed of natural amino acids linked by peptide bonds. However, in the present invention, it may contain a non-natural amino acid and is modified. There may be.

  In the present invention, the carrier for immobilizing the oligopeptide comprising the amino acid sequence described in SEQ ID NO: 1 is not particularly limited, and includes, for example, a plate (a plate having a plurality of recesses (well plate, microplate, etc.). ), Beads, gels and the like. Examples of the carrier material include plastics (for example, styrene resins such as polystyrene, vinyl polymers such as polyvinyl chloride, acrylic resins such as polymethyl (meth) acrylate, polyolefin resins such as polyethylene, polyethylene terephthalate, etc. Polyester resins, polycarbonate resins), metals (gold, silver, aluminum, stainless steel, etc.), oxides (glass, silica, alumina, titania, zirconia, indium tin oxide (ITO), etc.), agar and the like.

  More specifically, the carrier in the present invention is a well plate used in an ELISA (Enzyme-Linked Immunosorbent Assay) method, glass beads, magnetic beads, metal particles, silica beads, zirconia beads, cellulose gel, agarose gel, TOYOPEARL. (Registered trademark, polymers obtained by introducing a hexyl group, a butyl group, a phenyl group, or an oligoethylene glycol group into a hydrophilic vinyl polymer, manufactured by Tosoh Corporation), and the like.

  When a plate such as a well plate is used as the carrier, it is preferable to use a highly hydrophobic plate because nonspecific adsorption can be further suppressed (the binding ability to the denatured antibody is low). Here, “highly hydrophobic” usually means that the water contact angle is 60 degrees or more, preferably 70 degrees or more, more preferably 80 degrees or more, still more preferably 85 degrees or more, and particularly preferably 90 degrees or more. There is (the water contact angle means a static contact angle with respect to a water droplet at room temperature in the atmosphere). Further, as a preferred example of the plate in the present invention, a polystyrene plate (PolySorp plate, manufactured by Thermo Scientific) treated with polysoap (registered trademark) can be cited (according to Japanese Patent Application Laid-Open No. 2014-224710) The water contact angle is 88.1 degrees).

  In order to carry out the method of the present invention more easily and rapidly, when using an automatic enzyme immunoassay device such as AIA-600II or AIA-CL2400 (both manufactured by Tosoh Corporation), magnetic beads are preferably used as a carrier. It is done.

  The carrier to which the oligopeptide used in the present invention is bound (oligopeptide binding carrier) can be obtained by chemically or physically immobilizing the oligopeptide on the above-mentioned carrier. The method for immobilizing the oligopeptide according to the present invention on the carrier is not particularly limited, and examples thereof include a method using physical adsorption, electrostatic interaction, hydrophobic interaction, a crosslinking agent and the like. What is necessary is just to adjust the density | concentration at the time of the fixation | immobilization of the oligopeptide concerning this invention suitably according to the material of a support | carrier, a shape, the immobilization method etc., for example, the density | concentration of 5-50 microgram / mL is mentioned, Preferably it is 10 ~ 40 μg / mL.

  Further, it is preferable to perform a blocking operation with a blocking agent on the carrier after oligopeptide immobilization, because nonspecific adsorption can be suppressed and the background during measurement can also be suppressed. In particular, as shown in Example 2 described later, it is preferable to use a collagen peptide as a blocking agent because the background during measurement can be further suppressed. In addition to collagen, a blocking agent containing a glycoprotein (such as Super Block (trademark) (TBS) Blocking Buffer (manufactured by Thermo Fisher)) can be used as a blocking agent.

  The collagen peptide is not particularly limited in its origin (for example, pig, cow, fish) as long as it is obtained by hydrolyzing collagen. The degree of hydrolysis is not particularly limited. For example, collagen may be heated, for example, at 50 ° C. for 180 minutes in the presence of a protein hydrolase such as papain, pepsin or trypsin. Moreover, there is no restriction | limiting in particular also about the weight average molecular weight, Usually, 2000-10000, Preferably it is 3000-5000. Furthermore, the amount of the collagen peptide used is preferably 0.5 to 10% by weight, more preferably 1 to 5% by weight in the solution state. Moreover, there is no restriction | limiting in particular also as a liquid for melt | dissolving a collagen peptide, For example, various buffer solutions (For example, pH 8-10 buffer solutions (a carbonate buffer solution, phosphate buffered saline (PBS) etc.)) are used, and blocking is carried out. It can be prepared as an agent.

  In the present invention, the antibody that recognizes the test antibody may be any antibody that can recognize the antibody, and may be any isotype such as IgG, IgA, IgD, IgE, IgM, IgA, and IgY. The form of the antibody is not particularly limited, and the antibody that recognizes the test antibody may be a functional fragment thereof. However, even if the antibody recognizing the test antibody is denatured, the denatured recognition antibody is difficult to bind to the oligopeptide-binding carrier, and only the denatured antibody in the test antibody is the oligopeptide-binding carrier. In view of facilitating binding to the antibody, the antibody recognizing the test antibody is preferably a functional fragment of an antibody not containing the Fc region. Examples of such a functional fragment of an antibody that does not contain the Fc region include Fab, Fab ', F (ab') 2, Fv, scFv, and diabody. The antibody recognizing the test antibody and the test antibody containing the denatured antibody are preferably derived from different species. For example, when the test antibody containing the denatured antibody is a human-derived antibody, Examples of the antibody recognizing the test antibody include antibodies derived from non-human animals (eg, mouse antibody, rat antibody, rabbit antibody, goat antibody).

  The labeling substance that binds to the antibody that recognizes the test antibody used in the present invention may be appropriately selected from substances usually used in the field of measurement using antigen-antibody reaction. As an example, a fluorescent substance such as fluorescein, , Enzymes such as alkaline phosphatase, and radioactive substances.

  In addition, the binding between the antibody recognizing the test antibody and the labeling substance is usually performed by a cross-linking reaction via a linker (for example, NHS ester, maleimide) or the like for the convenience of detection of the substance described later. It can also be performed using a secondary antibody to which a substance is bound. Here, the secondary antibody is an antibody that exhibits specific binding to an antibody that recognizes the test antibody. Then, by utilizing this specific binding property, the labeling substance can be indirectly bound to the antibody recognizing the test antibody via the secondary antibody. When a secondary antibody to which this labeling substance is bound is used, after forming a complex with the oligopeptide binding carrier and the denatured antibody, a labeling substance is added to the antibody that recognizes the antibody via the secondary antibody. It may be combined.

  In the present invention, the conditions for bringing the sample containing the oligopeptide-binding carrier and the test antibody into contact with the labeled antibody are conditions that allow the above-described oligopeptide and the denatured antibody in the sample to bind to each other. The conditions are sufficient as long as the test antibody and the labeled antibody can bind to each other, and are usually contact at a temperature of 0 to 45 ° C., preferably contact at a temperature of 0 to 40 ° C., more preferably 4 to 4 ° C. The contact is performed at a temperature of 37 ° C., more preferably 25 ° C. to 37 ° C. Further, the contacting time is not particularly limited, and may be appropriately adjusted depending on the type of oligopeptide binding carrier, the concentration of antibody and labeled antibody in the sample, the type of labeled substance, etc., but is usually 5 minutes to 6 hours. , Preferably 10 minutes to 2 hours, more preferably 20 minutes to 1 hour.

  Further, after this contact, a washing step may be provided in the method of the present invention in order to remove the antibody that could not bind to the oligopeptide, the labeled antibody that could not bind to the test antibody, and the like. The washing solution used for the washing is not limited to the binding between the oligopeptide and the denatured antibody and the binding between the test antibody and the labeled antibody, and can wash the antibody non-specifically adsorbed on the carrier. Well, for example, a buffer solution (pH 6-8), more specifically, a Tris buffer solution, a phosphate buffer solution, and a HEPES buffer solution may be mentioned. Moreover, these buffers may contain salts, surfactants, proteins, sugars, zwitterionic compounds and the like as appropriate. In addition, a cleaning liquid (for example, E test “TOSOH” II cleaning liquid, manufactured by Tosoh Corporation) included in a commercially available reagent kit for immune reaction is also preferably used.

  In addition, regarding the step (1) according to the present invention, as described above, the oligopeptide-binding carrier, the modified antibody, and the labeled antibody are obtained by bringing the sample containing the oligopeptide-binding carrier, the test antibody, and the labeled antibody together. Although the aspect which forms the composite_body | complex containing these was demonstrated, the process (1) concerning this invention is not limited to the said embodiment. For example, the oligopeptide binding carrier and the sample containing the test antibody are first contacted to form a complex containing the oligopeptide binding carrier and the denatured antibody, and then the labeled antibody is further contacted to obtain the oligopeptide. You may form the composite_body | complex containing a binding support | carrier, the modified | denatured antibody, and a labeled antibody. In the step (1) according to the present invention, a sample containing the test antibody and the labeled antibody are first contacted to form a complex containing the denatured antibody and the labeled antibody, and then the oligopeptide binding carrier May be further contacted to form a complex comprising the oligopeptide binding carrier, the denatured antibody and the labeled antibody. The contact conditions for the substances are as described above. In addition, the washing step described above may be appropriately provided in order to remove the antibody that could not be bound to the oligopeptide, the labeled antibody that could not be bound to the test antibody, and the like even after contact with each substance. Moreover, in such an embodiment, from the viewpoint that the detection sensitivity of the denatured antibody is more likely to be improved, the step (1) according to the present invention first involves contacting the oligopeptide-binding carrier with a sample containing the test antibody. Then, the step of contacting with a labeled antibody is preferable.

  Next, in the step (2) of the method of the present invention, the amount of the denatured antibody in the sample is measured by detecting the labeling substance in the complex formed through the contact in the above-mentioned step (1). To do.

  In the present invention, detection of a labeling substance means detection of a signal derived from the labeling substance, and the detection can be performed according to a known method in the art. For example, when a fluorescent substance is used as the labeling substance, the fluorescence emitted from the fluorescent substance can be detected and quantified using a plate reader or the like. In addition, when an enzyme is used as a labeling substance, it is possible to detect and quantify the coloration, luminescence, etc. using a plate reader or the like by adding a substrate that decomposes by the action of the enzyme to develop coloration, luminescence, etc. it can. When a radioactive substance is used as the labeling substance, the radiation dose emitted from the substance can be detected and quantified using a scintillation counter or the like.

  In the present invention, as shown in Examples described later, the signal intensity (absorbance, fluorescence intensity, etc.) derived from the labeling substance thus detected and the amount of denatured antibody in the sample are highly correlated. Showing gender. For this reason, the method of the present invention is carried out using a standard sample containing a denatured antibody with a known concentration, and correlation data with a measurement value obtained using a sample containing a test antibody is obtained. Based on this, the amount of denatured antibody present in the test sample can be measured (quantified). The correlation data is, for example, a calibration curve.

<Method for evaluating antibody stability>
As described above, in the modified antibody measurement method of the present invention, the signal (luminescence, fluorescence, etc.) derived from the labeling substance changes based on the amount of the modified antibody in the sample. Therefore, antibody stability can also be evaluated based on the signal.

That is, the method for evaluating the stability of the antibody of the present invention is a method including the following steps (1) to (3).
(1) An oligopeptide-binding carrier comprising the amino acid sequence set forth in SEQ ID NO: 1, wherein an oligopeptide that recognizes a denatured antibody is bound to a carrier, a sample containing the test antibody, and the test antibody A step of forming a complex in which an oligopeptide-binding carrier, a denatured antibody, and a labeled antibody react by contacting the antibody with a labeled antibody in which a labeling substance is bound directly or indirectly to the antibody;
(2) a step of measuring the amount of denatured antibody in the sample by detecting the labeling substance in the complex formed in step (1),
(3) A step of evaluating the stability of the test antibody in the sample based on the amount of the antibody measured in the step (2).

  Steps (1) to (2) are as described above. In step (3), the stability of the test antibody in the sample is not particularly limited. For example, if the antibody denatured in step (2) is not detected significantly, the antibody in the sample It can be evaluated that the stability of is extremely high. In addition, the measurement value obtained using the sample containing the test antibody is compared with the measurement value obtained by carrying out the method of the present invention using the sample containing only the denatured antibody at the same concentration, If it is 30% or less, preferably 20% or less, more preferably 10% or less, and even more preferably 5% or less (eg, 4%, 3%, 2%, 1%, 0%), the antibody in the sample is It can be evaluated that the stability is high. Also, as shown in the examples below, if the measured value after applying stress is not significantly different from that before applying stress (acid, alkali, heating, freezing and thawing, stirring, etc.) It can be evaluated that the stability of the antibody against each stress is extremely high.

<Kit for measuring the amount of denatured antibody>
A kit for measuring the amount of the modified antibody of the present invention comprises:
An oligopeptide binding carrier comprising the amino acid sequence set forth in SEQ ID NO: 1, wherein an oligopeptide recognizing a denatured antibody is bound to a carrier, and a labeled antibody in which a labeling substance is bound to an antibody recognizing the antibody The kit.

  The oligopeptide binding carrier and the labeled antibody are as described above. In addition to the above, the kit of the present invention has the above-described solution for dissolving or diluting the test antibody, blocking agent, washing solution, a substrate that decomposes by the action of an enzyme, develops color, emits light, etc., its color development, etc. It may contain a stop solution for stopping, a standard sample containing a modified antibody with a known concentration, and the like. Furthermore, the kit of the present invention can include instructions for using the oligopeptide-binding carrier and the like when the method of the present invention is carried out.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited by these Examples.

(Example 1) Denatured human antibody measurement using the method of the present invention (Part 1)
(1) In this example, an anti-CD20 antibody was used as the human antibody according to the present invention. A 10 mg / mL anti-CD20 antibody solution diluted to 1 mg / mL with a phosphate buffer (pH 7.4) is dialyzed overnight with a 100 mM glycine buffer (pH 2.0), thereby denatured human antibodies. (Hereinafter also referred to as “denatured human antibody solution”).
(2) Reactivity of the modified human antibody solution prepared in (1) and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (hereinafter referred to as 2A1) is measured using the ELISA method shown below. did. 2A1 was prepared by a method of organic synthetic chemistry.
(2-1) 4% (v / v) glutaraldehyde was applied to a 96-well microplate (PolySorp plate (Thermo Scientific)) at 300 μl / well, allowed to stand at 37 ° C. for 2 hours, and 2A1 was added at 2 μg / well. The polypeptide was immobilized on the well by allowing it to stand at 37 ° C. for 2 hours.
(2-2) 1% (w / v) collagen peptide (collagen or gelatin hydrolyzate derived from fish scale or fish skin, collagen peptide having a weight average molecular weight of 3000 to 5000, product name: Ikuos HDL, Nitta Gelatin Co., Ltd. A carbonate buffer solution (pH 9.6) containing 300 μl / well was added and allowed to stand at 37 ° C. for 2 hours to perform blocking treatment.
(2-3) Washing buffer A (0.05% (w / v) Tween 20, 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl) was prepared in (1). The denatured human antibody solution was added to each well and reacted at 25 ° C. for 1 hour.
(2-4) After completion of the reaction, the resultant was washed with washing buffer A and diluted to 10 ng / mL with PBS-T (Phosphate Buffered Saline-Tween 20). Anti-human IgG F (ab ′) 2 derived from alkaline phosphatase labeled goat Jackson ImmunoResearch, catalog number: 109-056-097) was added at 100 μL / well.
(2-5) The mixture was reacted at 25 ° C. for 1 hour, washed with washing buffer A, Phosphatase Substrate (manufactured by KPL) was added at 50 μL / well, and the mixture was reacted at 25 ° C. for 30 minutes.
(2-6) AStop Solution (manufactured by KPL) was added at 50 μL / well to stop the color reaction, and then the absorbance at 595 nm was measured with a microplate reader (manufactured by Tecan).
(3) As a comparison target, an unmodified human antibody solution not subjected to the operation of (1) was measured by an ELISA method similar to (2).

  FIG. 1 shows the results of absorbance (595 nm) measurement when various concentrations of denatured / native human antibody solutions were added. As the concentration of the denatured human antibody contained in the solution increases, the absorbance also increases. Therefore, it can be seen that the method of the present invention allows measurement based on the concentration of the denatured human antibody contained in the solution. Moreover, as a result of measuring the native human antibody solution, it showed an almost constant absorbance regardless of the concentration, indicating that the method of the present invention can specifically measure the modified human antibody.

(Example 2) Examination of blocking agent As a plate of Example 1 (2-1), a plate in which 2A1 is immobilized or a plate in which 2A1 is not immobilized is used in the blocking treatment of Example 1 (2-2). As an agent, 2% (w / v) skim milk (product name: Skim Milk, manufactured by Difco Laboratories), 100 mM ethanolamine (product name: 2-ethanolamine, manufactured by Wako Pure Chemical Industries, Ltd.) or 0.5% (w / V) Modified human antibody solutions at various concentrations were measured in the same manner as in Example 1 except that BSA (bovine serum albumin, product name: Bovine Serum Albumin, manufactured by Sigma-Aldrich) was used.

  The results are shown in FIGS. 2A to 2D. In these drawings, the black bar indicates the result when 2A1 is immobilized on the well, and the white bar indicates the result when 2A1 is not immobilized on the well. When collagen peptide was used as a blocking agent, the absorbance increased significantly due to the immobilization of 2A1 on the plate, and the absorbance varied depending on the concentration of the denatured human antibody (FIG. 2A). On the other hand, when skim milk, ethanolamine, or BSA was used as a blocking agent, a significant increase in absorbance due to immobilization of 2A1 could not be confirmed (FIGS. 2B to 2D). From this, it can be seen that a collagen peptide is preferable as a blocking agent to be used when preparing a carrier to which 2A1 is bound.

(Example 3) Examination of solid phase (part 1)
The same method as in Example 1 except that a PolySorp plate, MediSorp plate, MaxiSorp plate, and MultiSorp plate (all manufactured by Thermo Scientific) that do not immobilize 2A1 were used as the plate of Example 1 (2-1). Were used to measure various concentrations of denatured human antibody solutions and analyzed for non-specific adsorption of antibodies to the plate. The obtained result is shown in FIG. 3A. The hydrophobicity of each plate is higher in the order of PolySorp, MediSorp, MaxiSorp, and MultiSorp.

(Example 4) Examination of solid phase (part 2)
Except that the blocking treatment in Example 1 (2-2) was not performed, various concentrations of denatured human antibody solutions were measured in the same manner as in Example 4 and analyzed for nonspecific adsorption of antibodies to the plate. The obtained result is shown in FIG. 3B.

  As is clear from the results shown in FIG. 3B, it can be seen that the higher the hydrophobicity of the plate (in the order of PolySorp, MediSorp, MaxiSorp, MultiSorp), the more the increase in absorbance is suppressed when the denatured human antibody solution is added. . This shows that when a plate is used as a carrier for binding 2A1, a plate with low nonspecific adsorption and high hydrophobicity is preferable. Further, as is apparent from the results shown in FIG. 3A, the increase in absorbance when the denatured human antibody solution was added was further increased by blocking with a collagen peptide as compared to when blocking was not performed (FIG. 3B). You can see that it is suppressed. In particular, when a PolySorp plate, which is a highly hydrophobic plate, was used, it was also found that the increase in absorbance was sufficiently suppressed even when a solution having a high concentration of denatured human antibody (10 μg / mL) was added.

(Example 5) Denatured human antibody measurement using the method of the present invention (part 2)
(1) A modified human antibody solution was measured in the same manner as in Example 1 except that the preparation of the modified human antibody solution of Example 1 (1) was processed under any of the following conditions (a) to (e): did.
(A) Acid-treated / alkali-treated 10 mg / mL human monoclonal antibody solution prepared from glycine buffer (pH 2, pH 3), citrate buffer (pH 4, pH 5), phosphate buffer (pH 6, pH 7, pH 7.4), Treatment was carried out by allowing 100 μL of a 0.5 mg / mL human monoclonal antibody solution prepared by diluting with Tris-HCl buffer (pH 8) or carbonate buffer (pH 10) to stand at 37 ° C. overnight.
(B) Heat treatment 100 μL of a 0.5 mg / mL human monoclonal antibody solution was treated by standing overnight at 37 ° C. at 4 ° C., 25 ° C., 37 ° C. or 50 ° C.
(C) Freezing and thawing treatment 100 μL of a 0.5 mg / mL human monoclonal antibody solution was frozen (−30 ° C. or liquid nitrogen (−196 ° C.)) and thawed at 37 ° C. for 10 times.
(D) Stirring treatment, stirrer 600 μL of 0.5 mg / mL human monoclonal antibody solution was stirred at 25 ° C. overnight using a stirrer.
(E) Stirring treatment and shaker The treatment was performed by shaking 300 μL of a 0.5 mg / mL human monoclonal antibody solution at 25 ° C. overnight using a shaker.
(2) As a comparison object, the modified human antibody solution prepared in (1) was subjected to a commercially available protein physical property analyzer (Optim2, manufactured by Avacta), and the denaturation start temperature was measured.

  The results are shown in FIGS. In addition, the measurement result (FIG. 4A and FIG. 5A) by the method of this invention shows that stability of protein (antibody) is so high that a light absorbency is low (namely, the amount of denatured human antibodies is low). On the other hand, the measurement results (FIGS. 4B and 5B) using a commercially available apparatus (Optim2) indicate that the higher the denaturation start temperature, the higher the stability of the protein (antibody).

  When a human antibody is acid-treated or alkali-treated (FIGS. 4A and 4B), both the method of the present invention and the method using a commercially available apparatus reduce the stability of the human antibody. This shows that there is a correlation between the method of the present invention and the method using a commercially available apparatus. On the other hand, when the human antibody was heat-treated, freeze-thawed, or stirred, there was almost no change in the denaturation start temperature in the method using a commercially available apparatus (FIG. 5B), but in the method of the present invention, Heating, freeze-thawing, and stirring with a stirrer significantly increased the amount of denatured human antibody (ie, decreased human antibody stability) (FIG. 5A). From this, it has been clarified that the method of the present invention can measure the amount of denatured human antibody (that is, decreased stability of human antibody) with higher accuracy than the method of measuring denaturation start temperature using a commercially available apparatus. .

(Example 6) Denatured human antibody measurement using the method of the present invention (part 3)
The method of the present invention was applied to human antibody quality evaluation in a human antibody purification process.
(1) After subjecting human gamma globulin (trade name: gamma-globulin intramuscular injection, manufactured by Kakenken) to a protein A column (AF rProtein A-650F, manufactured by Tosoh Corporation), one of the following eluates A to E Was used to elute the human antibody.
Eluent A: acetate buffer (pH 3.3 to 4.0)
Eluent B: Acetate buffer solution (pH 3.3 to 3.9) containing 150 mM ammonium sulfate
Eluent C: acetate buffer (pH 3.3 to 3.9) containing 300 mM ammonium sulfate
Solution D: Acetate buffer solution (pH 3.1 to 3.9) containing 150 mM potassium chloride
Eluent E: Acetic acid buffer (pH 3.1 to 3.9) containing 300 mM potassium chloride
(2) Magnetic beads (particles made of ethylene-vinyl acetate copolymer (EVA) kneaded with ferrite) bound with 2A1 (polypeptide having the amino acid sequence described in SEQ ID NO: 1) are used as the human of (1) The antibody was added to the antibody eluate and stirred at 37 ° C. for 20 minutes. The method for immobilizing 2A1 on the magnetic beads is shown below.
(2-1) 4% (v / v) glutaraldehyde was added to the magnetic beads, stirred for 2 hours at 37 ° C, 2A1 was added, and the mixture was allowed to stand at 37 ° C for 2 hours to be immobilized on the magnetic beads. .
(2-2) A carbonate buffer solution (pH 9.6) containing 1% (w / v) collagen peptide (Ikuos HDL, Nitta Gelatin Co., Ltd.) is added at 300 μl / well and stirred at 37 ° C. for 2 hours. Thus, a blocking process was performed.
(3) After removing free components by washing with washing buffer B (trade name: E test “TOSOH” II washing solution, manufactured by Tosoh Corporation), alkaline phosphatase-labeled goat-derived anti-human IgG F (ab ′) 2 ( A solution containing Jackson ImmunoResearch, catalog number: 109-056-097) was added and stirred at 37 ° C. for 20 minutes.
(4) After removing excess labeled human antibody by washing with washing buffer B, 4-methylumbelliferyl phosphate as a substrate is added, and a fluorescent substance (4-methylumbelliferone) produced by an enzymatic reaction The amount of denatured human antibody bound to the magnetic beads was measured by measuring the production rate (rate value) of the protein with a fully automated enzyme immunoassay device AIA-600II (manufactured by Tosoh Corporation).

  The results are shown in FIGS. 6A and 6B. FIG. 6A shows the results when the eluent A, B, or C is eluted, and FIG. 6B shows the results when the eluent A, D, or E is eluted. The fluorescence intensity (that is, the concentration of the denatured human antibody) varies depending on the concentration of components (ammonium sulfate and potassium chloride) contained in the buffer solution and the pH of the buffer solution. Specifically, it can be seen that the component contained in the buffer is ammonium sulfate rather than potassium chloride, and the pH is acidic, and the stress on human antibodies is greater (conditions that are easily denatured).

(Example 7) Denatured human antibody measurement using the method of the present invention (Part 4)
(1) In this example, IgG1, IgG2, IgG3, and IgG4 (manufactured by Sigma) were used as human antibodies according to the present invention. Then, each 1 mg / mL antibody solution and 100 mM glycine buffer (pH 2.0) were mixed in equal amounts and reacted at 37 ° C. for 16 hours to prepare a denatured human antibody solution.
(2) The prepared modified human antibody solution was measured by the same method as in Example 1 (2).

  The results are shown in FIG. Since all the denatured human antibody solutions of IgG1, IgG2, IgG3, and IgG4 are detected in a concentration-dependent manner, it can be understood that the method of the present invention can be detected without depending on the IgG subclass.

  As described above, according to the present invention, the amount of an antibody that has been denatured due to stress (eg, acid, alkali, heating, freeze-thawing, stirring) or the like can be measured quickly and easily. Further, according to the method of the present invention, it is possible to measure the amount of denatured antibody relatively inexpensively without requiring an expensive dedicated device such as Biacore. Furthermore, antibody stability can also be evaluated based on the measurement results. Therefore, the present invention is useful for quality control of pharmaceuticals containing antibodies.

Claims (7)

(1) A method for measuring the amount of denatured antibody comprising the steps shown in (1) and (2) below: (1) An oligopeptide comprising the amino acid sequence of SEQ ID NO: 1 and recognizing the denatured antibody is used as a carrier. A bound oligopeptide binding carrier, a sample containing the test antibody,
A step of forming a complex comprising an oligopeptide-binding carrier, a denatured antibody, and a labeled antibody by contacting a labeled antibody having a labeled substance directly or indirectly bound to an antibody that recognizes the test antibody;
(2) A step of measuring the amount of the denatured antibody in the sample by detecting the labeling substance in the complex formed in the step (1). A method for evaluating antibody stability comprising the following steps (1) to (3): (1) An oligopeptide comprising the amino acid sequence of SEQ ID NO: 1 and recognizing a denatured antibody binds to a carrier An oligopeptide binding carrier, a sample containing the test antibody,
A step of forming a complex comprising an oligopeptide-binding carrier, a denatured antibody, and a labeled antibody by contacting a labeled antibody having a labeled substance directly or indirectly bound to an antibody that recognizes the test antibody;
(2) a step of measuring the amount of denatured antibody in the sample by detecting the labeling substance in the complex formed in step (1),
(3) A step of evaluating the stability of the test antibody in the sample based on the amount of the antibody measured in the step (2). The method according to claim 1 or 2, wherein step (1) is a step of forming the complex by first contacting the oligopeptide-binding carrier and the sample and then contacting the labeled antibody.   The method according to any one of claims 1 to 3, wherein the oligopeptide-binding carrier is a carrier further blocked with a collagen peptide.   The method according to any one of claims 1 to 4, wherein the carrier is a highly hydrophobic plate.   The method according to claim 1, wherein the carrier is a magnetic bead.   An oligopeptide-binding carrier comprising the amino acid sequence of SEQ ID NO: 1, wherein the oligopeptide recognizing a denatured antibody is bound to the carrier; and a labeled antibody in which a labeling substance is bound to the antibody recognizing the test antibody A kit for measuring the amount of denatured antibody.