JP2018128408A - Antibody aggregate formation prediction method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for predicting formation of an antibody aggregate.SOLUTION: To solve the problem by a method including: (1) a process of bringing an oligopeptide bound carrier including an amino acid sequence of seq id no: 1 and recognizing a modified antibody, a specimen including the antibody to be detected, and an index antibody in which an index substance is directly or indirectly bonded to the antibody, into contacted with one another to form a composite in which the oligopeptide bound carrier, the modified antibody, and the index antibody are reacted; (2) a process of measuring an amount of the modified antibody in the specimen by detecting the index substance in the composite formed in the process (1); and (3) a process of predicting the formation of the aggregate of the antibody to be detected in the specimen, based on the amount of the modified antibody measured in the process (2).SELECTED DRAWING: Figure 12

Description

  The present invention relates to a method for predicting antibody aggregate formation.

  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. In that case, 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. In addition, aggregates may be formed in the storage state after preparation, and there is a demand for prediction of aggregate formation. As one of the formation mechanisms of antibody aggregates, it is first reported that a monomer having a partially broken three-dimensional structure is formed, and this serves as a nucleus to form an antibody aggregate (Non-patent Documents 2 and 3). . Therefore, it is considered that the formation of an antibody aggregate can be predicted by detecting an antibody having a partially broken three-dimensional structure that causes the formation of an antibody aggregate.

  As a method for detecting an antibody having a partially broken three-dimensional structure, a precise analysis method of the higher order structure of the antibody, that is, a method using X-ray crystal structure analysis or NMR is known. This method is very time-consuming and is not suitable for applications that require quick and simple measurement, such as purity control during production. In recent years, molecules that specifically recognize antibodies denatured by stress or the like regardless of their molecular weight have been reported (Patent Documents 1 to 3, and Non-Patent Documents 4 and 5). 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 C. J. et al. Roberts, Trends Biotechnol. , 32, 372-380, 2014 C. J. et al. Roberts, Curr. Opin. Biotechnol. , 30, 211-217, 2014 H. Watanabe et al. Biol. Chem. , 289, 3394-3404, 2014 H. Watanabe et al., Anal. Chem. , 88, 10095-10101, 2016

  The present invention has been made in view of the above-described problems of the prior art, and provides a method capable of quickly and easily predicting the formation of aggregates by measuring an antibody that has been denatured due to stress during production. For the purpose.

As a result of intensive studies to achieve the above object, the inventors of the present invention formed an aggregate of the antibody when stressed to cause denaturation was continuously applied to the antibody. Modified antibody using an oligopeptide-binding carrier in which an oligopeptide that recognizes a denatured antibody is bound to a carrier, and a labeled antibody in which a labeling substance is bound directly or indirectly to an antibody that recognizes a test antibody It was found that the aggregate formation of the test antibody can be predicted quickly and easily by measuring the amount of the antibody. Further, it has been found that a collagen peptide is useful in suppressing nonspecific adsorption to the carrier in a method for predicting the formation of an antibody aggregate. 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, regardless of the type of stress that causes the denaturation, antibody aggregate formation can be predicted, and in particular, by heat treatment, freeze-thaw treatment, or stirring treatment, which cannot be predicted with existing protein property analyzers. It was found that the formation of aggregates of the resulting antibody can be performed with high accuracy.

That is, the first aspect of the present invention is a method for predicting the formation of antibody aggregates, which comprises the following steps (1) to (3).
(1) an oligopeptide-binding carrier in which an oligopeptide that recognizes a denatured antibody comprising the amino acid sequence of SEQ ID NO: 1 is bound to a carrier;
A sample containing 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 a labeled antibody in which a labeled substance is bound directly or indirectly to an antibody that recognizes a 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 predicting the formation of an aggregate of the test antibody in the sample based on the amount of the denatured antibody measured in the step (2). The method according to the first aspect, in which a sample containing a test antibody is contacted and then contacted with a labeled antibody.

  The third aspect of the present invention is the method according to the first or second aspect, wherein the oligopeptide-binding carrier is a carrier further blocked with a collagen peptide.

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

  A fifth aspect of the present invention is the method according to any one of the first to third aspects, wherein the carrier is a magnetic bead.

According to a sixth aspect of the present invention, there is provided an oligopeptide-binding carrier in which an oligopeptide that recognizes a denatured antibody containing the amino acid sequence of SEQ ID NO: 1 is bound to a carrier;
A kit for predicting the formation of antibody aggregates, comprising a labeled antibody in which a labeling substance is bound directly or indirectly to an antibody recognizing a test antibody.

  The method for predicting antibody aggregate formation according to the present invention is a method for quickly and easily predicting aggregate formation of an antibody that has been denatured due to stress (eg, acid, alkali, heating, freeze-thawing, stirring) during production. can do. Further, according to the method of the present invention, aggregate formation of denatured antibodies can be predicted relatively inexpensively without requiring an expensive dedicated device such as Biacore (manufactured by GE Healthcare).

FIG. 6 is a diagram showing the results of Example 1. 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 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. It is the figure which showed the result of Example 4. It is the figure which showed the result of having evaluated the stability of the said antibody by the modified | denatured human antibody measuring method used by this invention for the human antibody which carried out acid treatment or alkali treatment 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 in the modified | denatured human antibody measuring method used by 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. . 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 was the figure which showed the result measured with the method of this invention for the heat-treated human antibody. It is the figure which showed the result of Example 6, and was the figure which showed the result of having measured the amount of the aggregates of the said antibody with the commercially available cell counting device aiming at the heated human antibody. It is the figure which showed the result of Example 6, and was the figure which showed the result of having measured the amount of the aggregates of the said antibody with the commercially available biopharmaceutical aggregation evaluation system for the heated human antibody.

  Hereinafter, the present invention will be described in detail.

The method for predicting antibody aggregate formation in the present invention measures the amount of denatured antibody by a method comprising the steps shown in the following (1) and (2), and based on the amount, antibody aggregate formation is determined. It is a method of prediction.
(1) an oligopeptide-binding carrier in which an oligopeptide that recognizes a denatured antibody comprising the amino acid sequence of SEQ ID NO: 1 is bound to a carrier;
A sample containing 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 a labeled antibody in which a labeled substance is bound directly or indirectly to an antibody that recognizes a 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).

  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 origin of the antibody is not particularly limited, and examples thereof include antibodies derived from humans, mice, rats, rabbits, etc., and antibodies derived from humans are particularly preferable. The type of the antibody is not particularly limited, and examples thereof include IgG1, IgG2, IgG3, and IgG4. Moreover, as long as the antibody has the Fc region of the antibody in addition to the complete antibody, a sequence (CDR) derived from an antibody derived from another animal (for example, human antibody, mouse antibody, rat antibody, rabbit antibody). Etc.) and a humanized antibody. Further, the antibody may be a monoclonal antibody or a polyclonal antibody. The sample used for the method of the present invention is not particularly limited as long as it contains the test antibody, regardless of whether it is denatured or undenatured, and is usually a solution containing an antibody, particularly a human antibody. The inclusion is preferred. 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 recognizing the denatured antibody only needs to contain at least the amino acid sequence described in SEQ ID NO: 1, and may consist of only the amino acid sequence described in SEQ ID NO: 1, An oligopeptide in which several amino acid residues to several tens of amino acid residues are added to the N-terminal or C-terminal of the amino acid sequence of No. 1 such as a linker peptide for binding to the carrier described later may be used. The length of the oligopeptide that recognizes the denatured antibody used in the present invention is not particularly limited as long as it recognizes the denatured antibody, but is usually 50 to 25 amino acids, preferably 40 to 25 amino acids, more Preferably it is 30 to 25 amino acids, and particularly preferably 25 amino acids (consisting only of the amino acid sequence described in SEQ ID NO: 1). 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, there is no particular limitation on the carrier on which the oligopeptide comprising the amino acid sequence shown in SEQ ID NO: 1 is immobilized. For example, a plate (including a plate having a plurality of recesses (well plate, microplate, etc.)) , Beads and gels. 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 resin, polycarbonate resin), metal (gold, silver, aluminum, stainless steel, etc.), oxide (glass, silica, alumina, titania, zirconia, indium tin oxide (ITO), etc.), and agar.

  As the carrier in the present invention, more specifically, well plates used in ELISA (Enzyme-Linked Immuno Sorbent Assay) method, glass beads, magnetic beads, metal particles, silica beads, zirconia beads, cellulose gel, agarose gel, Examples include polymer gels such as TOYOPEARL (registered trademark, a polymer in which a hexyl group, a butyl group, a phenyl group, or an oligoethylene glycol group is introduced into a hydrophilic vinyl polymer, manufactured by Tosoh Corporation).

  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 quickly, 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 the carrier.

  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. The concentration at the time of immobilization of the oligopeptide according to the present invention may be appropriately adjusted according to the material, shape, immobilization method and the like of the carrier. For example, the concentration is 5 to 50 μg / mL, preferably 10 To 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. Besides collagen, a blocking agent containing glycoprotein, Super Block ^™ (TBS) Blocking Buffer (manufactured by Thermo Fisher), etc. can be used as the 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. The weight average molecular weight is not particularly limited, but is usually 2000 to 10,000, preferably 3000 to 5000. Further, the amount of collagen peptide used is preferably 0.5 to 10% by weight, more preferably 1 to 5% by weight in the solution state. In addition, there is no particular limitation on the solution for dissolving the collagen peptide, and various buffer solutions (for example, pH 8 to 10 buffer solutions (carbonate buffer solution, phosphate buffered saline (PBS), etc.)) are used for blocking. It can be prepared as an agent.

  In the present invention, an oligopeptide-binding carrier in which an oligopeptide that recognizes a denatured antibody containing the amino acid sequence shown in SEQ ID NO: 1 is bound to a carrier, a sample containing the test antibody, and an antibody that recognizes the test antibody The order of contact with the labeled antibody in which the labeling substance is bound directly or indirectly is not particularly limited. All may be contacted simultaneously, or the oligopeptide-binding carrier and the sample containing the test antibody may be contacted first. Further, the sample containing the test antibody and the labeled antibody may be contacted first. Preferably, the oligopeptide binding carrier and the sample containing the test antibody are first contacted, and then the labeled antibody is contacted. Hereinafter, it demonstrates along the method.

  The conditions for contacting the oligopeptide-binding carrier with the sample containing the test antibody may be any conditions that allow the above-described oligopeptide and the denatured antibody in the sample to bind to each other, and usually a temperature of 0 to 45 ° C. Contact at a lower temperature, preferably at a temperature of 4 to 37 ° C., more preferably at a temperature of 25 ° C. to 37 ° C. Further, the contacting time is not particularly limited and may be appropriately adjusted depending on the kind of the oligopeptide binding carrier, the concentration of the antibody in the sample, etc., but is usually 5 minutes to 6 hours, preferably 10 minutes to 2 hours, Preferably it is 20 minutes to 1 hour.

  In addition, a washing step may be provided in the method of the present invention in order to remove the antibody that could not be bound to the oligopeptide after this contact. The washing solution used for the washing may be any washing solution that does not inhibit the binding between the oligopeptide and the denatured antibody and can wash the antibody non-specifically adsorbed on the carrier, for example, a buffer solution (pH 6 to 8). More specifically, a Tris buffer solution, a phosphate buffer solution, and a HEPES buffer solution may be mentioned. In addition, these buffers may appropriately contain salts, surfactants, zwitterionic compounds such as proteins, sugars, amino acids, and the like. 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.

  Next, by contacting the complex formed through the above contact with a labeled antibody in which a labeling substance is bound to an antibody that recognizes the test antibody, the oligopeptide binding carrier, the denatured antibody, and the labeled antibody A complex consisting of is formed.

  The antibody that recognizes the test antibody used in this step may be any antibody that can recognize the antibody, and particularly about isotype (IgG, IgM, IgE, etc.) and form (Fab, Fv, scFv, diabody). There is no limit.

  The labeling substance that binds directly or indirectly to the antibody recognizing 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. For example, fluorescein and the like Fluorescent substances, 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, a complex is formed with the oligopeptide binding carrier and the denatured antibody, and then the antibody that recognizes the test antibody is labeled via the secondary antibody. Substances may be bound.

  In this method, the condition for contacting the complex consisting of the oligopeptide-binding carrier and the denatured antibody with the labeled antibody described above may be any condition that allows them to bind, and usually 0 to 40 ° C. The contact is preferably performed at a temperature of 4 to 37 ° C, more preferably a contact at a temperature of 25 to 37 ° C. Further, the contacting time is not particularly limited and may be appropriately adjusted depending on the concentration of the complex and the labeled antibody, but is usually 5 minutes to 6 hours, preferably 10 minutes to 2 hours, more preferably 20 minutes to 1 It's time. Further, even after this contact, a washing step may be provided in order to remove the labeled antibody that could not be bound to the complex, and the above-mentioned washing solution is also preferably used in that step.

  Next, in the method of the present invention, the amount of denatured antibody in the sample is measured by detecting the labeling substance in the complex formed through the contact.

  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.

  As described above, in the denatured antibody measurement method used in the present invention, the signal (luminescence, fluorescence, etc.) derived from the labeling substance changes based on the amount of denatured antibody in the sample. Therefore, antibody aggregate formation can be predicted based on the signal. There is no particular limitation on the prediction method based on the amount of the denatured antibody measured by the method described above. For example, if the denatured antibody is not detected significantly in the step (2), the stability of the antibody in the sample is extremely high. It can be estimated that no aggregate is formed. In addition, the measurement value obtained using the sample containing the test antibody is usually 30% or less compared to the measurement value obtained by measuring in the same manner using a sample containing only the denatured antibody at the same concentration. The antibody in the sample is stable if it is preferably 20% or less, more preferably 10% or less, and even more preferably 5% or less (eg, 4% or less, 3% or less, 2% or less, 1% or less). Can be evaluated as high, and it can be predicted that aggregates will not be formed. In addition, as shown in the examples described later, if the measured value after applying stress is not significantly different from that before applying stress (heating), the stability of the antibody in the sample against stress is extremely high. It can be predicted that no aggregate will form.

  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 (Part 1)
(1) In this example, an anti-CD20 antibody was used as a human antibody. 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) The reactivity of the modified human antibody solution prepared in (1) with a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (hereinafter referred to as 2A1) was measured using the ELISA method shown below. . 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 (fish scale or fish skin-derived collagen or gelatin hydrolyzate, weight average molecular weight: 3000 to 5000 collagen peptide, product name: Ikuos HDL, manufactured by Nitta Gelatin ) Containing carbonate buffer (pH 9.6) was added at 300 μL / well and allowed to stand at 37 ° C. for 2 hours for 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, 100 μL of alkaline phosphatase-labeled anti-human IgG antibody (manufactured by Jackson ImmunoResearch) washed with washing buffer A and diluted to 10 ng / mL with PBS-T (Phosphate Buffered Saline-Tween 20) / 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. In the figure, 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. 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 this example 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 this Example 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. 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. 2). On the other hand, when skim milk, ethanolamine, or BSA was used as a blocking agent, no significant increase in absorbance due to immobilization of 2A1 could be confirmed (FIGS. 3 to 5). 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. In the figure, the black rhombus indicates the PolySorp plate, the white square indicates the MediSorp plate, the white triangle indicates the MaxiSorp plate, and the black circle indicates the result when the MultiSorp plate is used. The hydrophobicity of each plate is in the order of PolySorp, MediSorp, MaxiSorp, and MultiSorp from the highest to the lowest.

(Example 4) Examination of solid phase (part 2)
Except that the blocking treatment of Example 1 (2-2) was not performed, various concentrations of denatured human antibody solutions were measured in the same manner as in Example 3 and analyzed for nonspecific adsorption of antibodies to the plate. The obtained results are shown in FIG. In the figure, the black rhombus indicates the PolySorp plate, the white square indicates the MediSorp plate, the white triangle indicates the MaxiSorp plate, and the black circle indicates the result when the MultiSorp plate is used.

  As is clear from the results shown in FIG. 7, the higher the hydrophobicity of the plate (from higher to lower, in order of PolySorp, MediSorp, MaxiSorp, MultiSorp), when the modified human antibody solution was added It can be seen that the increase in absorbance is suppressed. 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 clear from the results shown in FIG. 6, the increase in absorbance when the denatured human antibody solution was added was further increased by blocking with the collagen peptide as compared with the case where the blocking treatment was not performed (FIG. 7). 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 (2)
(1) A modified human antibody solution prepared in the same manner as in Example 1, except that the modified human antibody solution of Example 1 (1) was prepared under the conditions (a) to (e) below. The solution was measured.
(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 performed 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 0.5 mg / mL human monoclonal antibody solution was treated by leaving it overnight 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 (freezing temperature: −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 treated by stirring overnight at 25 ° C. 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. 8 and FIG. 10) by the modified | denatured human antibody measuring method used by this invention shows that the 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. 9 and 11) 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. 8 and 9), both the modified human antibody measurement method used in the present invention and the method using a commercially available apparatus both reduce the stability of the human antibody. . This shows that there is a correlation between the modified human antibody measurement method used in the present invention and a 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. 11), but the denatured human antibody measurement method used in the present invention. Then, the amount of denatured human antibody significantly increased (ie, the stability of the human antibody decreased) by heating at 50 ° C., freeze-thawing (−30 ° C.), freeze-thawing (liquid nitrogen), and stirring with a stirrer (FIG. 10). ). From this, the modified human antibody measurement method used in 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 device. Became clear.

(Example 6) Prediction of antibody aggregate formation using the method of the present invention (1) Preparation of the modified human antibody solution of Example 1 (1) was conducted at 4 ° C using 2000 µL of a 0.5 mg / mL human monoclonal antibody solution. Alternatively, the modified human antibody solution was measured in the same manner as in Example 1 except that it was prepared by allowing it to stand at 50 ° C. overnight.
(2) As a comparison object, the modified human antibody solution prepared in the same manner as (1) except that the treatment time was 1 to 25 days was subjected to a commercially available cell counter (Coulter Counter Z series, manufactured by Beckman Coulter), The number of particles from 3 μm to 10 μm corresponding to the antibody aggregate was measured over time from the treatment time of 1 day to 25 days.
(3) Further, as a comparison target, the modified human antibody solution prepared in the same manner as (1) except that the treatment time was 1 to 21 days was subjected to a commercially available biopharmaceutical aggregation system (Aggregates Sizer, manufactured by Shimadzu Corporation). The particle concentration of 0.04 μm to 20 μm corresponding to the antibody aggregate was measured over time from the treatment time of 1 to 21 days.

  The results are shown in FIGS. FIG. 12 shows the results of measuring the treated antibody using the method of the present invention. FIG. 13 shows the results of measuring the treated antibody using a commercially available cell counter. FIG. 14 shows the results of measuring the treated antibody using a commercially available biopharmaceutical aggregation evaluation system. In the method of the present invention, a difference is observed in the measured values at 4 ° C. and 50 ° C. at the stage where the treatment is performed overnight, so that denatured human antibodies can be confirmed, and as a result, the formation of aggregates can be predicted. (FIG. 12). On the other hand, the difference between the measured values at 4 ° C. and 50 ° C. in the commercially available cell measurement device and biopharmaceutical aggregation evaluation system was observed after 10 days of treatment (FIGS. 13 and 14). From this, it became clear that the method of the present invention can predict the formation of aggregates more quickly than the method using the conventional apparatus.

  As described above, according to the present invention, aggregate formation by an antibody that has been denatured due to stress (for example, acid, alkali, heating, freeze-thawing, stirring) or the like can be predicted quickly and easily. Further, according to the method of the present invention, it is possible to predict the formation of an aggregate of an antibody that has been denatured relatively inexpensively without requiring an expensive dedicated device such as Biacore (manufactured by GE Healthcare). Therefore, the present invention is useful in quality control of pharmaceuticals including human antibodies.

Claims (6)

A method for predicting the formation of an antibody aggregate, comprising the following steps (1) to (3).
(1) an oligopeptide-binding carrier in which an oligopeptide that recognizes a denatured antibody comprising the amino acid sequence of SEQ ID NO: 1 is bound to a carrier;
A sample containing 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 a labeled antibody in which a labeled substance is bound directly or indirectly to an antibody that recognizes a 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 predicting the formation of an aggregate of the test antibody in the sample based on the amount of the denatured antibody measured in the step (2). The method according to claim 1, wherein the oligopeptide-binding carrier is first contacted with a sample containing the test antibody, and then contacted with a labeled antibody. The method according to claim 1 or 2, wherein the oligopeptide-binding carrier is a carrier further blocked with a collagen peptide. The method according to any one of claims 1 to 3, wherein the carrier is a highly hydrophobic plate. The method according to any one of claims 1 to 3, wherein the carrier is a magnetic bead. An oligopeptide-binding carrier in which an oligopeptide that recognizes a denatured antibody containing the amino acid sequence of SEQ ID NO: 1 is bound to a carrier;
A kit for predicting antibody aggregate formation, comprising a labeled antibody in which a labeling substance is bound directly or indirectly to an antibody recognizing a test antibody.

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