WO2015079977A1 - Antibody separation method, antibody evaluation method, medicine evaluation method, and 2-d antibody electrophoresis kit - Google Patents

Abstract

The antibody separation method for separating antibodies while maintaining disulfide bonds includes: a first dimension electrophoresis step for separating antibody protein solubilized in a prepared buffer, which does not contain reducing agent, by a first dimension electrophoresis using a buffer that does not contain reducing agent; and a second dimension electrophoresis step for separating the antibody protein electrophoresed in said first dimension electrophoresis step by a second dimension electrophoresis using a buffer that does not contain reducing agent.

Description

Antibody separation method, antibody evaluation method, pharmaceutical evaluation method, and antibody two-dimensional electrophoresis kit

The present invention relates to an antibody separation method, an antibody evaluation method, a pharmaceutical evaluation method, and an antibody two-dimensional electrophoresis kit.

As a central role in the post-genomic era, proteome research is actively conducted. By “proteome” is intended the entire protein that is translationally produced in a particular cell, organ and organ. In proteome research, large-scale analysis of proteins, especially for structure and function, is performed.

Protein electrophoresis is one of the most frequently used methods for large-scale analysis of proteins. Since all proteins have unique charges and molecular weights, they can be separated into various proteins by separating the protein mixture present in the living body according to the charge or molecular weight.

Even for the same type of protein, there are protein species with different charges due to post-translational modification, and the molecular weights of these proteins are almost the same, so it is particularly important to separate the proteins according to the charge. In addition, by using a method that separates proteins according to electric charge and a method that separates proteins according to molecular weight in a two-dimensional manner (two-dimensional electrophoresis), more proteins can be separated with high resolution. Can be separated.

Two-dimensional electrophoresis is composed of two electrophoresis steps: isoelectric focusing that separates proteins according to charge, and slab gel electrophoresis that separates proteins according to molecular weight (especially SDS-PAGE). In two-dimensional electrophoresis, it is possible to separate a denatured protein in the presence of a denaturing agent, or to separate a protein in a non-denaturing state in the absence of a denaturing agent. It is an excellent technique because it can separate proteins at once.

Among the protein samples, since antibodies in particular have a high molecular weight, it is known to perform two-dimensional electrophoresis in the presence of a denaturing agent. Patent Document 1 describes that antibodies with different sialic acid modifications are detected using two-dimensional electrophoresis in the presence of a denaturing agent. It was impossible to detect such an antibody only by electrophoresis according to the molecular weight. Further, cited document 1 describes that two-dimensional electrophoresis is used to detect different antibodies such as nitration and phosphorylation.

Japanese Patent Publication “Japanese Unexamined Patent Application Publication No. 2009-244245 (published on October 22, 2009)”

As general electrophoresis conditions for two-dimensional electrophoresis, first, in the first-dimension isoelectric focusing, a denaturing agent such as Urea and Thiourea, a surfactant such as CHAPS, and a reducing agent such as DTT are added. Electrophoresis is performed under reduced conditions. In the second dimension, for example, SDS-PAGE, electrophoresis is performed under reducing conditions to which a reducing agent such as sodium dodecyl sulfate (SDS) and DTT is added.

The in vivo antibody is a tetramer in which two heavy chains (H chains) and two light chains (L chains) are covalently bonded by disulfide bonds. Therefore, the molecular weight of the antibody is about 160 kDa and a high molecular weight in the tetramer state. Of antibodies, IgG has a specific basic isoelectric point. As described above, since an antibody has a high molecular weight, has a specific isoelectric point, and its structure is easily changed by electrophoresis, it is difficult to suitably separate the antibody. Therefore, it would be beneficial to have a two-dimensional electrophoresis method that can favorably separate antibodies.

The present invention has been made in view of the above-described problems, and an object of the present invention is to provide an antibody separation method and an antibody separation method that can more suitably separate antibodies using two-dimensional electrophoresis. An object of the present invention is to provide an antibody evaluation method, a pharmaceutical evaluation method, and a two-dimensional electrophoresis kit for antibodies used in the antibody separation method.

In order to solve the above-described problem, an antibody separation method according to one embodiment of the present invention is the first-dimensional electrophoresis of an antibody protein dissolved in a preparation buffer not containing a reducing agent, using an electrophoresis buffer that does not contain a reducing agent. -Dimensional electrophoresis step for separating by the first-dimensional electrophoresis, and the second-dimensional electrophoresis for separating the antibody protein electrophoresed in the first-dimensional electrophoresis step by a second-dimensional electrophoresis using a migration buffer that does not contain a reducing agent. And a process.

According to one aspect of the present invention, the antibody protein is two-dimensionally electrophoresed under non-reducing conditions, so that the antibody can be separated more suitably.

It is a schematic diagram explaining the preparation instrument of the IPG gel used in the antibody separation method which concerns on one Embodiment of this invention. It is a figure which shows the two-dimensional electrophoresis pattern of the antibody sample isolate | separated on non-reducing conditions in the antibody separation method which concerns on one Embodiment of this invention. In the antibody separation method which concerns on one Embodiment of this invention, it is a figure which shows the two-dimensional electrophoresis pattern of the antibody sample isolate | separated on non-denaturing conditions. In the antibody separation method which concerns on one Embodiment of this invention, it is a figure which compares the two-dimensional electrophoresis pattern of the antibody sample isolate | separated on each of non-reducing conditions and non-denaturing conditions. It is a figure which shows the two-dimensional electrophoresis pattern of the other antibody sample isolate | separated on non-reducing conditions in the antibody separation method which concerns on one Embodiment of this invention. In the antibody separation method which concerns on one Embodiment of this invention, it is a figure which shows the two-dimensional electrophoresis pattern of the other antibody sample isolate | separated on non-denaturing conditions. It is a figure which compares the electrophoresis conditions in the antibody separation method which concerns on one Embodiment of this invention. It is a figure which compares the acrylamide density | concentration of an IPG gel. It is a figure which compares the separation pattern by the conventional two-dimensional electrophoresis.

[Antibody Separation Method]
Hereinafter, embodiments of the present invention will be described in detail. The antibody separation method according to the present invention is a method for separating antibody proteins by two-dimensional electrophoresis, and includes a first-dimensional electrophoresis step and a second-dimensional electrophoresis step. In one embodiment of the antibody separation method, a gel preparation step for preparing a gel for electrophoresis is performed before the first-dimensional electrophoresis step. In one embodiment of the antibody separation method, an equilibration step of equilibrating the antibody sample separated in the first dimension electrophoresis step is performed between the first dimension electrophoresis step and the second dimension electrophoresis step. . Furthermore, in one embodiment of the antibody separation method, a detection step of detecting the separated antibody protein is performed after the second-dimensional electrophoresis step. In this embodiment, when an immobilized pH gradient (IPG) gel is used, isoelectric focusing is performed in the first dimensional electrophoresis step, and SDS-PAGE is performed in the second dimensional electrophoresis step. Will be described as an example.

(Gel preparation process)
In the gel preparation process, an IPG gel that performs electrophoresis in a first-dimensional electrophoresis process described later and a slab gel that performs electrophoresis in a second-dimensional electrophoresis process are prepared.

The IPG gel is produced using, for example, the IPG gel production tool shown in FIG. FIG. 1 is a schematic diagram illustrating an IPG gel preparation tool used in the antibody separation method according to an embodiment of the present invention. As shown in FIG. 1, the IPG gel preparation device 100 includes a gel preparation jig 10, gradient mixers 20 and 30, a peristaltic pump 40, and a silicon tube 50.

First, an acidic acrylamide buffer mixed solution is added to the gradient mixer 20 as a low specific gravity solution, and a basic acrylamide buffer mixed solution is added to the gradient mixer 30 as a high specific gravity solution. And the gel solution which mixed the low specific gravity solution and the high specific gravity solution so that it may have a desired pH gradient is produced. The prepared gel solution is slowly filled into the gel preparation jig 10 via the silicon tube 50 by the peristaltic pump 40 so as not to disturb the gradient.

The low specific gravity solution and the high specific gravity solution may be prepared by a conventionally known composition, and an acidic acrylamide buffer mixed solution can be used as the low specific gravity solution, and a basic acrylamide buffer mixed solution can be used as the high specific gravity solution.

Examples of the acidic acrylamide buffer mixed solution include 941 μl of 0.2K immobiline pK3.6, 273 μl of pK6.2, 243 μl of pK7.0, 260 μl of pK8.5 and 282 μl of pK9.3, 30% acrylamide stock Mix 2.5 ml of aqueous solution (29.1% acrylamide, 0.9% bisacrylamide), 4.2 ml of 87% glycerol solution, 108 μl of 10% APS, and 7.6 μl of 100% TEMED with distilled water. What was measured up to 15 ml can be used.

As the basic acrylamide buffer mixture, 0.2 M immobiline pK3.6 100 μl, pK6.2 333 μl, pK7.0 361 μl, pK8.5 239 μl and pK9.3 326 μl, 30% acrylamide stock solution (2.5% of 29.1% acrylamide, 0.9% bisacrylamide), 108 μl of 10% APS, and 7.6 μl of 100% TEMED are mixed and made up to 15 ml with distilled water. it can.

A gel solution prepared by mixing a low specific gravity solution and a high specific gravity solution may contain a polymerization initiator, a polymerization accelerator, a specific gravity adjusting agent, a denaturing agent, a buffer buffer, an acrylamide derivative, and the like together with a gel material such as acrylamide. it can.

As the polymerization initiator, conventionally known polymerization initiators can be used, and examples thereof include ammonium persulfate (APS) and methylene blue.

As the polymerization accelerator, conventionally known ones can be used, and examples thereof include tetramethylethylenediamine (TEMED). Moreover, you may comprise so that superposition | polymerization may be accelerated | stimulated by UV light irradiation.

As the specific gravity adjuster, conventionally known ones can be used, and examples thereof include glycerol and sucrose.

As the modifier, conventionally known ones can be used, and examples thereof include urea.

As the buffer buffer, a conventionally known buffer can be used, and examples thereof include a Bis-Tris buffer, a Tris buffer, a MOPS buffer, and a MES buffer.

The roughness of the network structure of the gel to be prepared is determined by the concentration of acrylamide (% T) in the gel solution and the concentration of bisacrylamide (% C) in the acrylamide. Therefore,% T and% C may be optimized in order to separate antibody molecules having a high molecular structure and a high-order structure having an average molecular weight of 160 kD or more. Optimum% T and% C are not particularly limited. For example,% T is 3.0 or more and 4.0 or less, and% C is more preferably 2.0 or more and 3.0 or less. Is particularly preferably 3.2 or more and 3.7 or less, and% C is 2.5 or more and 3.0 or less.

Further, the concentration of the polymerization initiator, polymerization accelerator, specific gravity adjuster, modifier, buffer buffer, etc. in the gel solution is not particularly limited, and may be a conventionally known concentration. Furthermore, the range of the pH gradient provided in the IPG gel may be in a range including the isoelectric point (pI) of the antibody protein to be separated. For example, the pH gradient is from 6 to 10 and includes the isoelectric point of many antibody proteins. do it.

In the gel preparation jig 10, a gel plate on which a gel bond film is attached is installed, the gel solution is filled in the gel preparation container 10, and the gel solution is gelled on the gel plate. Then, the generated gel is washed to remove unreacted acrylamide and then dried. Although the temperature at the time of gelatinizing a gel solution is not specifically limited, For example, what is necessary is just 40 degreeC or more and 50 degrees C or less.

The dried IPG gel is cut into strips to produce an IPG dry strip gel, which is subjected to isoelectric focusing in the first dimensional electrophoresis step.

Next, production of a slab gel that performs SDS-PAGE in the second-dimensional electrophoresis step will be described. As the slab gel, a conventionally known slab gel can be used. For example, polyacrylamide gel, starch gel, agar gel and the like can be suitably used. Such a slab gel can be produced by a conventionally known method.

(First dimension electrophoresis process)
In the first dimension electrophoresis step, antibody proteins dissolved in a preparation buffer not containing a reducing agent are separated by first dimension electrophoresis using a buffer not containing a reducing agent.

<Preparation of antibody sample>
In the first dimension electrophoresis step, antibody proteins are separated by the first dimension electrophoresis. The antibody sample is obtained by dissolving an antibody protein to be separated in a preparation buffer. The antibody sample is preferably stored avoiding denaturation of the antibody protein by heat until separation by electrophoresis.

The antibody protein that can be separated in the antibody separation method according to the present embodiment is not particularly limited, and a conventionally known antibody can be suitably used. Examples of such antibodies include Infliximab, Trastuzumab, Cetuximab, Bevaczumab, Rituximab and the like.

The antibody is desalted using, for example, 2D clean-up kit (GE Healthcare) and then dissolved in the preparation buffer. Further, the antibody may be fluorescently labeled with a fluorescent dye such as IC5-OSu special packaging (Doujin Chemical Laboratory) for detection after separation. In the antibody separation method according to the present embodiment, any of a non-reducing preparation buffer that does not contain a reducing agent and a non-denaturing adjustment buffer that does not contain a reducing agent and a protein denaturing agent is used as the preparation buffer.

The preparation buffer in the conventional antibody electrophoresis method contains a reducing agent such as 2-mercaptoethanol, and the antibody is separated in a state where the disulfide bond is dissociated by the reducing action of the reducing agent. Therefore, in the conventional electrophoresis method, H chain and L chain are detected by electrophoresis as two separate bands or a ladder of two spots. That is, the antibody cannot be separated while maintaining the tetramer state.

Antibodies are produced from antibody-producing cells and used in biochemical experiments and the like, but heterogeneous antibodies also exist due to differences in protein post-translational modifications and the like. Therefore, it would be beneficial if there is a method for subjecting an antibody produced from antibody-producing cells to two-dimensional electrophoresis as it is and examining whether the produced antibody is uniform. However, until now, it has been considered impossible to perform two-dimensional electrophoresis of antibodies while maintaining disulfide bonds, and no attempt has been made.

In addition, since a plurality of sugar chains are bound to the antibody molecule and each may be subjected to different modifications, a very complex glycoform is formed. It is believed that the structure of these sugar chains determines the conformation and stability of the antibody molecule, thereby affecting the affinity with the target and the efficacy as a drug. Therefore, an antibody as a pharmaceutical is required to be a glycoform that is optimal for a certain disease. In conventional antibody electrophoresis methods, antibodies cannot be separated while maintaining such a sugar chain modification, and the glycoform cannot be analyzed appropriately.

On the other hand, the non-reducing preparation buffer and the non-denaturing preparation buffer used in the antibody separation method according to the present embodiment are buffers that do not contain a reducing agent that cleaves a disulfide bond of a protein. According to the antibody separation method according to the present embodiment, since the reducing buffer is not included in the preparation buffer, the antibody can be more preferably separated while maintaining the disulfide bond. In addition, since antibodies can be separated in a more nearly complete state, they are also suitable for analysis of glycoforms.

≪Non-reducing preparation buffer≫
The non-reducing preparation buffer contains a nonionic surfactant or an amphoteric surfactant. The antibody has a specific basic isoelectric point and has a high molecular weight of about 160 kDa in the tetramer state, so it is difficult to separate by electrophoresis while maintaining the disulfide bond. In addition, it is necessary to suppress structural changes caused by electrophoresis. Therefore, until now, it was considered impossible to separate antibodies while maintaining disulfide bonds, and no attempt was made. In the antibody separation method according to the present embodiment, the non-reducing preparation buffer contains a nonionic surfactant or an amphoteric surfactant, so that the antibody can be preferably separated while maintaining the disulfide bond. .

The nonionic surfactant contained in the non-reducing preparation buffer is not particularly limited, but 3-[(4-heptyl) phenyl-3-hydroxypropyl] dimethylammoniopropanesulfonate (C7BzO) or Triton X is used. It can be suitably used.

The amphoteric surfactant contained in the non-reducing preparation buffer is not particularly limited, but 3- (3-colamidopropyl) dimethylammonio-1-propanesulfonate (CHAPS), 3-[(3-colamidopropyl) Dimethylammonio] -2-hydroxy-1-propanesulfonate (CHAPSO) or 3- [N, N-dimethyl (3-myristoylaminopropyl) ammonio] propanesulfonate, amidosulfobetaine-14 (ASB-14) Can be suitably used.

The concentration of the nonionic surfactant or amphoteric surfactant in the non-reducing preparation buffer is not particularly limited, but is preferably 0.1 w / v% or more and 4 w / v% or less, more preferably 2 w. / V% or more and 4 w / v% or less are particularly preferable.

The non-reducing preparation buffer may further contain a denaturing agent such as urea or thiourea, an amphoteric carrier such as amphorite, glycerol (thickening agent), and the like. The concentration of these substances in the non-reducing preparation buffer is not particularly limited, and can be a conventionally known concentration. Specific examples of the non-reducing preparation buffer include those containing 8M urea, 2M thiourea, 4 w / v% CHAPS, and 0.5% ampholite (pH 6 to 11).

≪Non-denaturing preparation buffer≫
The non-denaturing preparation buffer does not contain a reducing agent and further does not contain a protein denaturing agent. A non-denaturing preparation buffer is a buffer that does not contain a protein denaturant that cleaves intermolecular bonds such as protein hydrogen bonds. A preparation buffer in a conventional antibody electrophoresis method contains a protein denaturing agent such as urea, and the antibody is separated in a state where intermolecular bonds such as hydrogen bonds are dissociated by the action of the protein denaturing agent. On the other hand, according to the antibody separation method according to the present embodiment, since the preparation buffer does not contain a reducing agent and a protein denaturant, intermolecular bonds such as disulfide bonds and hydrogen bonds are maintained, and higher-order structures are more maintained. The antibody can be separated in the state.

The non-denaturing preparation buffer contains an additive selected from the group consisting of non-surfactant sulfobetaines, polysorbates, polyoxyethylene alkyl ethers, and sugar alcohols. Since the antibody has a specific basic isoelectric point and has a high molecular weight of about 160 kDa in the tetramer state, electrophoresis is performed with a higher-order structure maintaining disulfide bonds and hydrogen bonds. In addition to being difficult to separate, it is also necessary to suppress structural changes due to electrophoresis. Therefore, until now, it was considered impossible to separate an antibody maintaining a higher-order structure, and no attempt was made. In the antibody separation method according to this embodiment, an additive selected from the group consisting of non-surfactant sulfobetaines, polysorbates, polyoxyethylene alkyl ethers, and sugar alcohols in the non-denaturing preparation buffer Therefore, the antibody can be preferably separated while maintaining the higher order structure.

Non-detergent sulfobetaines (NDSBs) contained in non-denaturing preparation buffers are non-surfactants and are added to protein preservation solutions and protein preparations as protein stabilizers. Techniques have been reported (Reference 1: JP 2006-189358 A and Reference 2: JP 2007-516281 A). In the non-denaturing preparation buffer, the non-surfactant sulfobetaines function as solubilizing and stabilizing agents, and the antibodies are dissolved and stabilized.

Examples of non-surfactant sulfobetaines include glycine betaine (Glycine).
and dimethylethylammonium propane sulfonate (NDSB-195), 3- (1-pyridino) -1-propane sulfonate (NDSB-201), dimethyl (2-hydroxyethyl) ammonium propane sulfonate (NDSB) manufactured by Calbiochem -211), 3- (1-methylpiperidinium) -1-propanesulfonate (NDSB-221), dimethylbenzylammonium propanesulfonate (NDSB-256), and 3- (4-tert-butyl-1-pyridino ) -1-propanesulfonate (NDSB-256-4T) can be suitably used. Among them, glycine betaine is more preferable because it is neutral in charge.

The polysorbate contained in the non-denaturing preparation buffer is a surfactant, and for example, Tween 80 (registered trademark) (Poly (Oxyethylene) sorbitan monomolecular 80) can be suitably used because it is electrically neutral.

Polyoxyethylene alkyl ethers contained in the non-modified preparation buffer are nonionic surfactants, and for example, polyethylene glycol (PEG) can be suitably used.

The sugar alcohols contained in the non-denatured preparation buffer are not particularly limited, but, for example, sorbitol can be suitably used.

The concentration of these additives in the non-denaturing preparation buffer is not particularly limited, but is preferably 0.1 w / v% or more and 10 w / v% or less, more preferably 1 w / v% or more and 6 w / v. % Or less is particularly preferable. When the concentration of an additive such as NDSB in the non-denaturing preparation buffer is 1 w / v% or more and 6 w / v% or less, the antibody can be separated in a more complete state.

The non-denaturing preparation buffer can further contain a specific gravity adjusting agent such as glycerol, an amphoteric carrier such as an ampholite, a surfactant having a low denaturing action, a dye, and the like. The concentration of these substances in the non-denaturing preparation buffer is not particularly limited, and can be a conventionally known concentration. Specific examples of the non-denaturing preparation buffer include those containing 4 w / v% NDSB, 15 v / v% glycerol, and 0.5% ampholite (pH 6 to 11).

When an antibody sample is prepared with a non-denaturing preparation buffer, the antibody is not reduced or denatured, so that the antibody that maintains the intact state can be separated. When an antibody sample is prepared with a non-reducing preparation buffer, the antibody is not reduced, but may be denatured. Therefore, even if the antibody is apparently intact, a part of the three-dimensional structure is maintained. It may not have been. Therefore, by comparing the results obtained by separating the antibody samples prepared with the non-reducing preparation buffer and the non-denaturing adjustment buffer, the impurities in the antibody sample, the antibody multimer, the antibody in a state other than the complete body, etc. The presence or absence can be confirmed.

That is, antibody samples prepared with a non-reducing preparation buffer and a non-denaturing adjustment buffer are separated by the antibody separation method according to the present invention, the patterns are compared, impurities in the antibody sample, antibody multimers, complete An antibody evaluation method including a step of determining the presence or absence of an antibody in a state other than the body is also included in the category of the present invention.

<Isoelectric focusing>
The isoelectric focusing in the first dimensional electrophoresis process and the SDS-PAGE in the second dimensional electrophoresis process to be described later may be automatically performed using, for example, Auto2D manufactured by Sharp Manufacturing System. In the present embodiment, a case where the first-dimensional electrophoresis step and the second-dimensional electrophoresis step are performed using the Auto2D will be described as an example.

When Auto2D is used, for example, an isoelectric focusing chip having a pH of 6 to 10 can be used, and a PAGE chip having an acrylamide concentration of 7.5% or 6.5% can be used. The processing time for isoelectric focusing alone may be several minutes or more and 16 minutes or less, for example, 45 minutes. The total processing time may be 30 minutes or more and 20 hours or less, for example, 130 minutes.

The antibody sample prepared as described above is introduced into the IPG dry strip gel prepared in the gel preparation step, and isoelectric focusing is performed. The introduction time of the antibody sample to the IPG dry strip gel may be 1 minute or more and 60 minutes or less, for example, 30 minutes.

After the antibody sample is introduced, the IPG dry strip gel is swollen using a swelling liquid. The swelling liquid is also used as an electrophoresis buffer for isoelectric focusing. As the swelling liquid to be used, a conventionally known swelling liquid can be suitably used, but it does not contain a reducing agent. Thus, antibody samples can be separated by isoelectric focusing under non-reducing conditions that do not contain a reducing agent. Moreover, it is preferable that a swelling liquid does not contain a protein denaturant. Specific examples of the swelling liquid include those containing 4% NDSB-195, 15% Glycerol, and 0.2% Amphorite. That is, a non-denatured preparation buffer can be used as the swelling liquid. The amount of the swelling liquid used is, for example, 100 μL, and the swelling time is, for example, 5 minutes or more and 10 minutes or less, but is not limited thereto.

The antibody is separated based on the difference in isoelectric point by applying a voltage to the swollen IPG gel. The voltage applied to the IPG gel is, for example, control 1 (Step 1: 200 V, constant for 5 minutes, Step 2: 1000 V, linear gradient for 5 minutes, Step 3: 1000 V, constant for 5 minutes, Step 4: 7000 V, linear gradient for 15 minutes, Step 5: 7000 V , 15 minutes constant), Control 2 (Step 1: 200 V, 5 minutes constant, Step 2: 1000 V, 5 minutes linear gradient, Step 3: 1000 V, 5 minutes constant, Step 4: 4000 V, 10 minutes linear gradient, Step 5: 4000 V, 10 minutes constant Step 6: 7000V, linear gradient for 10 minutes, Step 7: 7000V, constant for 20 minutes, or control 3 (Step 1: 200V, constant for 5 minutes, Step 2: 1000V, linear gradient for 10 minutes, tep3: 1000V, 10 min constant, Step4: 8000 V, 15 minutes linear gradient, Step5: 8000 V, may be controlled to 15 minutes a constant).

That is, in the first-dimensional electrophoresis, when the voltage is kept constant and the current is decreased to a predetermined current value larger than 0, the current is increased by increasing the current, and the current starts to decrease during the voltage increase. It is preferable to control the voltage so that the voltage is kept constant and the current value is larger than 0 and 100 μA or less. Here, the predetermined current value may be appropriately set to a predetermined value close to 0 according to the separation state of the sample by electrophoresis or the like. That is, while the voltage is controlled to be constant, the current shows a negative gradient, and when the gradient approaches 0, the voltage gradient is controlled while the voltage is boosted (linear gradient) control. When the voltage begins to decrease, the voltage is kept constant. Further, since it is preferable to boost the voltage so that the current value is as low as possible, the upper limit of the current value may be 100 μA.

In addition, other electrophoresis conditions such as temperature may be conventionally known electrophoresis conditions.

(Equilibration process)
In the equilibration step, the antibody sample separated in the first-dimensional electrophoresis step is equilibrated. The equilibration of the antibody sample can be performed by immersing the IPG gel after the first-dimensional electrophoresis step in an equilibration solution and shaking. Thereby, SDS processing can be performed on the antibody sample separated by the first-dimensional electrophoresis.

The equilibration time of the IPG gel is, for example, 10 minutes, but is not limited thereto. As the equilibration liquid used for equilibration of the gel, a conventionally known equilibration liquid can be used, and for example, Tris-HCl, SDS, EDTA, glycerol, BPB and the like can be included. Specific examples of the equilibration liquid include those containing 0.5 mM Tris-HCl (pH 8.8), 4.75% SDS, 0.5 mM EDTA, 20 v / v% glycerol, and 0.005% BPB. .

(Second dimension electrophoresis process)
In the second-dimensional electrophoresis step, the antibody protein separated in the first-dimensional electrophoresis step is separated by second-dimensional electrophoresis using a buffer that does not contain a reducing agent. In the second dimensional electrophoresis step, antibody samples separated by isoelectric focusing in the first dimensional electrophoresis step are separated based on the difference in molecular weight by SDS-PAGE.

The IPG gel that has been subjected to isoelectric focusing in the first-dimensional electrophoresis step is brought into contact with the slab gel for SDS-PAGE used in the second-dimensional electrophoresis step, and a current is passed through these gels. Thereby, the antibody sample separated in the IPG gel moves to the slab gel and is separated based on the difference in molecular weight. In the second-dimensional electrophoresis step, the current flowing through the gel may be controlled, for example, such that it flows for 10 minutes at a constant current of 10 mA and then flows for 30 minutes at a constant current of 20 mA.

A conventionally known electrophoresis buffer can be suitably used as the electrophoresis buffer used in SDS-PAGE in the second-dimensional electrophoresis step, but does not contain a reducing agent. Thus, antibody samples can be separated by SDS-PAGE under non-reducing conditions that do not contain a reducing agent. Specific examples of the electrophoresis buffer include those containing 25 mM Tris, 192 mM Glycin, and 0.5% SDS.

In addition, other electrophoresis conditions such as pH of the electrophoresis buffer may be conventionally known electrophoresis conditions.

(Detection process)
In the detection step, the antibody sample separated in the second-dimensional electrophoresis step is detected. If the antibody sample is fluorescently labeled, each spot of the separated antibody sample can be detected by tracking the fluorescence. These spots may be detected by staining each spot of the separated antibody sample with CBB staining, silver staining, or the like. Fluorescence detection of a fluorescently labeled antibody sample can be performed at a detection wavelength of 660 nm and a PMT of 400 V using, for example, an imager typhoon manufactured by GE Healthcare.

As described above, according to the antibody separation method of the present invention, the antibody protein is two-dimensionally electrophoresed under non-reducing conditions, so that the antibody can be separated while maintaining the disulfide bond. Particularly suitable for analysis.

[Antibody evaluation method]
The antibody evaluation method according to the present invention includes an evaluation step for evaluating whether or not the antibody separated by the above-described antibody separation method according to the present invention is the same as the antibody contained in the reference medicine.

In the evaluation step, for example, the pattern in which the antibody to be evaluated is separated by the antibody separation method according to the present invention is the same as the pattern in which the antibody contained in the reference medicine is separated by the antibody separation method according to the present invention. The antibody is evaluated by judging whether or not it exists. In addition, when the pattern in which the antibody to be evaluated is separated is also detected at a position different from the pattern in which the antibody contained in the reference medicine is separated, the antibody to be evaluated is included in the reference medicine. It can be determined that impurities that are not included, incomplete antibodies, and the like are included.

According to the antibody evaluation method according to the present invention, when the antibody to be evaluated is separated, the antibody is separated by the antibody separation method according to the present invention, so that a pattern in which the antibody is suitably separated while maintaining the disulfide bond can be obtained. . Therefore, the antibody can be detected as it is while maintaining the tetramer, and thus can be evaluated more appropriately.

[Pharmaceutical evaluation method]
The method for evaluating a medicament according to the present invention includes an evaluation step for evaluating whether or not the antibody contained in the medicament separated by the antibody separation method according to the present invention described above is uniform. The pharmaceutical evaluation method according to the present invention can be used, for example, for antibody drug evaluation, biopharmaceutical evaluation, and the like.

In the evaluation step, for example, whether or not a single spot (including a ladder-like spot detected in a single region) is detected when the drug to be evaluated is separated by the antibody separation method according to the present invention. By judging whether or not, the drug is evaluated. In the evaluation step, when the spot from which the medicine is separated is detected as a single spot, it is determined that the antibody contained in the medicine is uniform, and when a plurality of spots from which the medicine is separated is detected, the medicine is It can be determined that the antibody contained in the antibody is heterogeneous or contaminated with impurities.

According to the method for evaluating a drug according to the present invention, since the drug to be evaluated is separated by the antibody separation method according to the present invention, a pattern in which antibodies are suitably separated while maintaining disulfide bonds can be obtained. Therefore, the antibody can be detected as it is while maintaining the tetramer, and thus can be evaluated more appropriately.

[Antibody two-dimensional electrophoresis kit]
The antibody two-dimensional electrophoresis kit according to the present invention includes an additive selected from the group consisting of non-surfactant sulfobetaines, polysorbates, polyoxyethylene alkyl ethers, and sugar alcohols, and is reduced. A preparation buffer containing no agent, or a preparation buffer containing a nonionic surfactant or an amphoteric surfactant and no reducing agent is provided. That is, the antibody two-dimensional electrophoresis kit according to the present invention includes either a non-denaturing preparation buffer or a non-reducing preparation buffer.

The non-denaturing preparation buffer and the non-reducing preparation buffer included in the antibody two-dimensional electrophoresis kit according to the present invention are as described above in the section of “Antibody separation method” in the present specification. Description is omitted.

The kit for two-dimensional electrophoresis of antibodies according to the present invention can be used for preparing an antibody sample when separating antibodies by two-dimensional electrophoresis using a conventionally known electrophoresis apparatus.

The antibody two-dimensional electrophoresis kit according to the present invention comprises a non-denaturing preparation buffer and a non-reducing preparation buffer together with a first-dimensional electrophoresis gel material and a migration buffer, a two-dimensional electrophoresis gel material and a migration buffer, A fluorescent dye for detection, an instruction manual, and the like may be provided. The antibody two-dimensional electrophoresis kit according to the present invention may be provided together with an electrophoresis apparatus such as Auto2D manufactured by Sharp Manufacturing System.

[Summary]
In the antibody separation method according to aspect 1 of the present invention, the first-dimensional electrophoresis is performed by separating the antibody protein (antibody sample) dissolved in the preparation buffer not containing the reducing agent by the first-dimensional electrophoresis using the buffer not containing the reducing agent. An electrophoresis step and a second-dimensional electrophoresis step in which the antibody protein separated in the first-dimensional electrophoresis step is separated by second-dimensional electrophoresis using a buffer that does not contain a reducing agent.

According to the above configuration, a preparation buffer that does not contain a reducing agent is used as an antibody preparation buffer, and the first-dimensional electrophoresis step and the second-dimensional electrophoresis step are performed using a buffer that does not contain a reducing agent. That is, antibodies can be separated using a preparation buffer that does not contain a reducing agent that cleaves the disulfide bond of the protein, and a buffer that does not contain a reducing agent.

The preparation buffer in the conventional antibody electrophoresis method contains a reducing agent such as 2-mercaptoethanol, and the migration buffer also contains a reducing agent, so that disulfide is reduced by the reducing action of the reducing agent. The antibody is separated with the bond dissociated. That is, in the conventional method, the H chain and the L chain are detected by electrophoresis as two separate bands or a ladder of two spots.

On the other hand, according to the antibody separation method according to this aspect, electrophoresis is performed using a buffer that does not contain a reducing agent and does not contain a reducing agent in the preparation buffer, so that the antibody can be separated while maintaining a disulfide bond. .

The antibody separation method according to aspect 2 of the present invention is the antibody separation method according to aspect 1, wherein the preparation buffer is composed of a group consisting of non-surfactant sulfobetaines, polysorbates, polyoxyethylene alkyl ethers, and sugar alcohols. It may contain selected additives.

The antibody has a specific basic isoelectric point and has a high molecular weight of about 160 kDa in the tetramer state, so it is difficult to separate by electrophoresis while maintaining the disulfide bond. In addition, it is necessary to suppress structural changes caused by electrophoresis. Therefore, until now, it was considered impossible to separate antibodies while maintaining disulfide bonds, and no attempt was made.

On the other hand, in the antibody separation method according to this embodiment, an additive selected from the group consisting of non-surfactant sulfobetaines, polysorbates, polyoxyethylene alkyl ethers, and sugar alcohols is used as a preparation buffer. Since the non-denaturing preparation buffer is used, the antibody can be suitably separated while maintaining the disulfide bond.

The antibody separation method according to aspect 3 of the present invention is the antibody separation method according to aspect 2, wherein the non-surfactant sulfobetaines are glycine betaine, dimethylethylammonium propane sulfonate (NDSB-195), 3- (1-pyridino)- 1-propanesulfonate (NDSB-201), dimethyl (2-hydroxyethyl) ammonium propanesulfonate (NDSB-211), 3- (1-methylpiperidinium) -1-propanesulfonate (NDSB-221), dimethylbenzylammonium The polysorbate is selected from the group consisting of propanesulfonate (NDSB-256) and 3- (4-tert-butyl-1-pyridino) -1-propanesulfonate (NDSB-256-4T), and the polysorbate is Tween 80 ( Registered trademark) The polyoxyethylene alkyl ethers, polyethylene glycol, the sugar alcohols may be sorbitol.

According to the above configuration, the antibody can be separated in a more complete state.

In the antibody separation method according to aspect 4 of the present invention, in the above aspect 2 or 3, the concentration of the additive in the preparation buffer may be 1 w / v% or more and 6 w / v% or less.

According to the above configuration, the antibody can be separated in a more complete state.

In the antibody separation method according to aspect 5 of the present invention, in any of the above aspects 2 to 4, the preparation buffer may not contain a protein denaturant.

According to the above configuration, the preparation buffer includes an additive selected from the group consisting of non-surfactant sulfobetaines, polysorbates, polyoxyethylene alkyl ethers, and sugar alcohols, and a reducing agent and Since it is a non-denaturing preparation buffer that does not contain a protein denaturant, it is possible to suitably separate antibodies while maintaining a higher-order structure that maintains disulfide bonds, hydrogen bonds, and the like.

In the antibody separation method according to aspect 6 of the present invention, in the aspect 1, the preparation buffer may contain a nonionic surfactant or an amphoteric surfactant.

According to the above configuration, since the non-reducing preparation buffer containing a nonionic surfactant or an amphoteric surfactant is used as the preparation buffer, the antibody can be preferably separated while maintaining the disulfide bond.

The antibody separation method according to aspect 7 of the present invention is the antibody separation method according to aspect 6, wherein the nonionic surfactant is 3-[(4-heptyl) phenyl-3-hydroxypropyl] dimethylammoniopropanesulfonate, or Triton. X and the amphoteric surfactant is 3- (3-colamidopropyl) dimethylammonio-1-propanesulfonate, 3-[(3-colamidopropyl) dimethylammonio] -2-hydroxy-1- It may be propane sulfonate, or 3- [N, N-dimethyl (3-myristoylaminopropyl) ammonio] propane sulfonate, amide sulfobetaine-14.

According to the above configuration, the antibody can be more preferably separated while maintaining the disulfide bond.

The antibody separation method according to Aspect 8 of the present invention is the antibody separation method according to any one of Aspects 1 to 7, wherein the voltage is kept constant and the current is reduced to a predetermined current value greater than 0 in the first-dimensional electrophoresis. The voltage may be controlled such that the voltage is sometimes increased to increase the current, the voltage is maintained constant when the current starts to decrease during the voltage increase, and the current value is greater than 0 and equal to or less than 100 μA.

According to the above configuration, while the voltage is controlled to be constant, the current exhibits a negative gradient, and when the gradient approaches 0, the voltage is boosted (linear gradient) and the voltage is boosted. At the same time, the voltage is kept constant when the current gradient begins to decrease. Then, the voltage is controlled so that the current value changes as low as possible and the upper limit of the current value is 100 μA. Thus, suitable first-dimensional electrophoresis is possible by appropriately controlling the voltage.

The antibody evaluation method according to aspect 9 of the present invention is an evaluation step for evaluating whether or not the antibody separated by the antibody separation method according to any one of the above aspects 1 to 8 is the same as the antibody contained in the reference drug. Is included.

According to the above configuration, when the antibody to be evaluated is separated, the antibody is separated by the antibody separation method of any one of the above aspects 1 to 8, so that a pattern in which the antibody is suitably separated while maintaining the disulfide bond is obtained. It is done. Therefore, the antibody can be detected as it is while maintaining the tetramer, and thus can be evaluated more appropriately.

The pharmaceutical evaluation method according to the tenth aspect of the present invention includes an evaluation step for evaluating whether or not the antibody contained in the pharmaceutical separated by the antibody separation method according to any of the first to eighth aspects is uniform.

According to the above configuration, when separating the drug to be evaluated, the drug is separated by the antibody separation method according to any one of the above aspects 1 to 8, and thus a pattern in which the antibody is suitably separated while maintaining the disulfide bond is obtained. It is done. Therefore, the antibody can be detected as it is while maintaining the tetramer, and thus can be evaluated more appropriately.

The antibody two-dimensional electrophoresis kit according to aspect 11 of the present invention is an additive selected from the group consisting of non-surfactant sulfobetaines, polysorbates, polyoxyethylene alkyl ethers, and sugar alcohols And a preparation buffer that does not contain a reducing agent (non-denaturing preparation buffer).

According to the above configuration, a non-denaturing agent that does not contain a reducing agent and contains an additive selected from the group consisting of non-surfactant sulfobetaines, polysorbates, polyoxyethylene alkyl ethers, and sugar alcohols Since the preparation buffer is provided, it can be used to suitably separate antibodies while maintaining disulfide bonds.

The kit for two-dimensional electrophoresis of antibodies according to aspect 12 of the present invention includes a preparation buffer (non-reduction preparation buffer) containing a nonionic surfactant or an amphoteric surfactant and not containing a reducing agent.

According to the above-described configuration, since the non-reducing preparation buffer containing a nonionic surfactant or an amphoteric surfactant is provided without a reducing agent, the antibody is preferably separated while maintaining a disulfide bond. Can be used for

The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention. Furthermore, a new technical feature can be formed by combining the technical means disclosed in each embodiment.

[Example 1: Non-reducing two-dimensional electrophoresis of antibody (Infliximab)]
Two-dimensional electrophoresis (isoelectric focusing and SDS-PAGE) was performed under non-reducing conditions in order to detect glycoforms and aggregates of the whole antibody molecule.

Antibody dissolved in PBS (Infliximab, manufactured by Mitsubishi Tanabe) is labeled with a fluorescent IC5 labeling kit (IC5-OSu special packaging, manufactured by Dojindo Laboratories) without desalting, and adjusted to 2 μg / μL. This was subjected to two-dimensional electrophoresis. Two-dimensional electrophoresis was automatically performed using Auto2D manufactured by Sharp Manufacturing System. An IEF chip having a pH of 6 to 10 was used, and a PAGE chip having an acrylamide concentration of 7.5% was used. The isoelectric focusing time was 45 minutes and the total processing time was 130 minutes.

As an IPG dry strip gel, a gel prepared by mixing an acidic acrylamide buffer mixed solution and a basic acrylamide buffer mixed solution having the above-described composition and using the IPG gel preparation instrument 100 shown in FIG. 1 was used. The prepared antibody sample was introduced into an IPG dry strip gel. The sample introduction time was 30 minutes. After the IPG gel was swollen with 100 μL of the swelling solution for 5 minutes, voltage was applied to the IPG gel and isoelectric focusing was performed under non-reducing conditions. The non-reducing buffer was used as the swelling liquid.

The voltage application is controlled as follows: Step 1: 200 V, constant for 5 minutes, Step 2: 1000 V, linear gradient for 5 minutes, Step 3: 1000 V, constant for 5 minutes, Step 4: 7000 V, linear gradient for 15 minutes, Step 5: 7000 V, constant for 15 minutes did.

700 μL of the gel after isoelectric focusing (0.5 mM Tris-HCl (pH 8.8) (manufactured by Wako)), 4.75% SDS (manufactured by Bio-Rad), 0.5 mM
After equilibrating with EDTA (manufactured by Dojindo), 20 v / v% glycerol (manufactured by Wako), and 0.005% BPB (manufactured by Sigma) for 10 minutes, the IPG gel and acrylamide gel were contacted, SDS-PAGE was performed under non-reducing conditions. The current flowing through the gel was controlled as follows: a constant current of 10 mA for 10 minutes and a constant current of 20 mA for 30 minutes. As the electrophoresis buffer, a buffer containing Tris, Glysin and SDS was used.

In the acrylamide gel after SDS-PAGE, fluorescence was detected at a detection wavelength of 660 nm and a PMT of 400 V using an imager typhoon manufactured by GE Healthcare. The results are shown in FIG.

As shown in FIG. 2, a main antibody spot group was detected around 160 kD, and an isoform group considered to be a dimer was detected around 300 kD. In addition to the main antibody spot, a protein spot derived from an antibody (LC desorption, sugar chain desorption, etc.) having a slightly lower molecular weight than the main antibody spot was detected. It was shown that a large number of glycoforms and multimers contained in a pharmaceutical can be clearly separated by using a non-reducing preparation buffer containing Urea and CHAPS and no reducing agent.

[Example 2: Non-denaturing two-dimensional electrophoresis of antibody (Infliximab)]
A method of separating the antibodies by two-dimensional electrophoresis without completely denaturing the antibodies was examined.

Antibody (Infliximab, Mitsubishi Tanabe) 4w / v5 NDSB (Merck), 15v / v% Glycerol (Wako), and 0.5% Amphorite (pH 6-11) (Invitrogen) Two-dimensional electrophoresis was performed in Auto2D in the same manner as in Example 1 except that it was dissolved in a non-denaturing preparation buffer, and spots were detected. The results are shown in FIG.

As shown in FIG. 3, a spot group regularly arranged in the isoelectric point and molecular weight direction was detected. In the spot group in FIG. 3, the difference in the spot position in the vertical direction represents the difference in molecular weight due to the number of added sugar chains, and the difference in the spot position in the horizontal direction represents the difference in isoelectric point due to the number of sialic acid additions. ing. That is, it is considered that glycoforms having different numbers of sugar chains for modifying antibody molecules and different types of terminal modifications were detected. By using a non-denaturing preparation buffer that contains NDSB and Glycerol but does not contain reducing agents and protein denaturing agents, it prevents artifacts due to protein denaturation and allows antibody isoforms to remain intact without degradation or subunit elimination. It was shown that it can be clearly separated.

Here, we will analyze the difference in patterns by two-dimensional electrophoresis of antibody samples separated under non-reducing conditions and non-denaturing conditions. FIG. 4 shows the results of two-dimensional electrophoresis of antibody samples separated in each of non-reducing two-dimensional electrophoresis (a) of Example 1 and non-denaturing two-dimensional electrophoresis (b) of Example 2. FIG. 4C is a graph showing the fluorescence intensity between b and b ′ in FIG. 4B.

As shown in FIG. 4 (a), in non-reducing two-dimensional electrophoresis, spots presumed to be multimers of a plurality of antibodies are detected in the region A, and impurities (not derived from the target component) are detected in the region B. A spot presumed to be (protein) was detected. The complete antibody (target component) was detected in the X region near 160 kDa, but part of the three-dimensional structure was dissociated into the Y region near 110 to 140 kDa and the Z region near 60 to 80 kDa. A spot presumed to be derived from an antibody having a slightly small molecular weight was detected. On the other hand, in non-denaturing two-dimensional electrophoresis, as shown in FIG. 4 (b), spots were detected in the X region near 160 kDa. Further, although it is difficult to visually recognize in FIG. 4B, as shown in FIG. 4C, an increase in fluorescence intensity is seen in the A region separately from the X region. It can be seen that spots are also detected in the region.

That is, in FIG. 4 (b), as shown in FIG. 4 (a), in the B region, a spot that is supposed to be an impurity, a Y region near 110 to 140 kDa, and a Z region near 60 to 80 kDa. In addition, a spot having a slightly small molecular weight in which a part of the three-dimensional structure was dissociated was not detected. Therefore, it can be determined that the spots in the Y, Z, and B regions are proteins derived from the target component of the antibody. Thus, by confirming the pattern of (b) in FIG. 4, the presence or absence of the target component, multimer, protein derived from the target component, and impurities in the antibody sample can be determined.

Therefore, by comparing the two-dimensional electrophoresis patterns of antibody samples separated under non-reducing conditions and non-denaturing conditions, the presence or absence of impurities other than antibodies, the presence or absence of multimers, and antibodies other than intact The presence or absence of contamination can be evaluated.

[Example 3: Non-reducing two-dimensional electrophoresis of other antibodies]
As in Example 1, two-dimensional electricity was used except that Trastuzumab (manufactured by Chugai Pharmaceutical), Cetuximab (manufactured by BMS), Bevacizumab (manufactured by Roche), and Rituximab (manufactured by Chugai Pharmaceutical) were used as antibodies. Electrophoresis was performed in Auto2D and spots were detected. The results are shown in FIG. 5A shows the pH range 7 to 10, and FIGS. 5B to 5C show the pH range 6 to 9.

As shown in FIG. 5, for trastuzumab (a), cetuximab (b), bevacumab (c), and Rituximab (d), as well as the main antibody spot group shown in FIG. An isoform group was detected.

[Example 4: Non-denaturing two-dimensional electrophoresis of other antibodies]
Trastuzumab (manufactured by Chugai Pharmaceutical Co., Ltd.) and Cetuximab (manufactured by BMS) were dissolved in the non-denaturing preparation buffer (+ NDSB) of Example 2, and Trastuzumab (manufactured by Chugai Pharmaceutical Co., Ltd.) were mixed with 50 v / v% Glycerol (Wako). ), 0.1% Amphorite (pH 6-11) (manufactured by Invitrogen), and a solution dissolved in water-containing preparation buffer (-NDSB), two-dimensional electrophoresis was performed in the same manner as in Example 2. Performed in Auto2D to detect spots. The results are shown in FIG. 6A and 6C show the pH range 7 to 10, and FIG. 6B shows the pH range 6 to 9.

As shown in FIG. 6, Trastuzumab (a) and Cetuximab (b) dissolved in a non-denaturing preparation buffer containing NDSB are spots regularly arranged in the direction of isoelectric point and molecular weight as in Infliximab shown in FIG. A group was detected. For trastuzumab (c) dissolved in a preparation buffer that does not contain NDSB, spots presumed to be antibody multimers have been detected, and the detection of trastuzumab (c) dissolved in a preparation buffer that contains NDSB is more stable. Was shown to be possible.

[Example 5: Change in current value during first-dimensional electrophoresis due to difference in electrophoresis conditions]
The optimization of voltage control conditions during the first-dimension isoelectric focusing was investigated. Electrophoresis conditions 1 (Step 1: 200 V, constant for 5 minutes, Step 2: 1000 V, linear gradient for 5 minutes, Step 3: 1000 V, constant for 5 minutes, Step 4: 7000 V, linear gradient for 15 minutes, Step 5: 7000 V, constant for 15 minutes) and electrophoresis Condition 2 (Step 1: 200V, constant for 5 minutes, Step 2: 1000V, linear gradient for 5 minutes, Step 3: 1000V, constant for 5 minutes, Step 4: 4000V, linear gradient for 10 minutes, Step 5: 4000V, constant for 10 minutes, Step 6: 7000V, 10 The change in the current value when the voltage during isoelectric focusing is controlled by each of the linear gradient of the minute, Step 7: 7000 V, constant for 20 minutes) is shown in FIG. 7A is a graph showing changes in voltage value and current value when isoelectric focusing is performed under electrophoresis condition 1, and FIG. 7B is an isoelectric point under electrophoresis condition 2. It is a graph which shows the change of the voltage value and electric current value when performing electrophoresis. In FIG. 7, the numerical value on the right side of the vertical axis represents the current value, the numerical value on the left side of the vertical axis represents the voltage value, and the horizontal axis represents time (seconds).

As shown in FIG. 7, in the step of controlling the voltage to be constant, the current shows a negative gradient, and it is shown that it is ideal to perform step-up (linear gradient) control when the gradient approaches zero. It was. Further, it was shown that in the step of controlling the voltage to be boosted, it is ideal to keep the voltage constant after the current gradient starts to decrease (negative). Furthermore, it has been shown that the voltage is preferably boosted so that the current value is as low as possible, and the upper limit of the current value may be 100 μA.

[Reference Example 1: Examination of IPG dry strip gel]
In an IPG gel of isoelectric focusing in the first dimension, an acrylamide concentration (% T) is used in order to separate an antibody having an average molecular weight of 160 kD or more and a very high molecular complex in a higher order structure under non-reducing conditions. Optimization of the bisacrylamide concentration (% C) was examined. As the pH range to be prepared, pH 6 to 10 that covers the isoelectric point of many antibodies was selected.

Gels were prepared by changing each concentration downward from 4.0% T and 3.0% C, which are commonly used in two-dimensional electrophoresis, and the separation performance was evaluated from the current value and the separation pattern. . As a separated sample, mouse liver soluble protein was labeled with IC5 (IC5-OSu special packaging, manufactured by Dojindo Laboratories), and two-dimensional electrophoresis was performed using Auto2D (manufactured by Sharp Manufacturing System). As mouse liver soluble protein, mouse liver tissue was ground under a solubilization buffer (50 mM Tris-HCl (pH 7.6), 20% Glycerol, 0.3 M NaCl), and the supernatant was used.

The electrophoresis conditions were the same as the electrophoresis conditions 2 described above. FIG. 8 shows the results of using 4.0% T and 3.0% C gel (a) and 3.6% T and 2.7% C gel (b), respectively.

As shown in FIG. 8, it was shown that the separation of the polymer region and the sample introduction efficiency were improved by setting each concentration in the gel to 3.6% T and 2.7% C.

[Reference Example 2: Two-dimensional electrophoresis of antibody under conventional reducing conditions]
Using Cetuximab (manufactured by BMS), reducing condition 1 (performing first- and second-dimensional electrophoresis under electrophoresis conditions including a reducing agent (DTT)) and reducing condition 2 (reducing agent (DTT)) 2D electrophoresis using Auto2D (manufactured by Sharp Manufacturing System Co., Ltd.) for each of the first-dimensional electrophoresis under the electrophoresis conditions not included and the second-dimensional electrophoresis under the electrophoresis conditions including the reducing agent (DTT). Electrophoresis was performed. Cetuximab was labeled with IC5 (IC5-OSu special packaging, manufactured by Dojindo Laboratories). Other electrophoretic conditions were the same as the electrophoretic conditions 2 described above.

The results are shown in FIG. 9A shows the result of the reduction condition 1, and FIG. 9B shows the result of the reduction condition 2. When antibody molecules were separated under conventional reducing conditions 1, as shown in FIG. 9 (a), an H chain was detected at about 60 kD, and an L chain was detected at about 20 kD. Further, as shown in FIG. 9 (b), even when the first-dimensional electrophoresis is performed under non-reducing conditions and the second-dimensional electrophoresis is performed under reducing conditions, and antibody molecules are separated under reducing conditions 2, The H chain was detected at about 60 kD and the L chain was detected at about 20 kD, but showed a more complicated separation pattern than in the case of reducing condition 1.

The present invention can be used in the fields of medicine, agricultural chemicals, and the like.

DESCRIPTION OF SYMBOLS 10 Gel preparation jig | tool 20 Gradient mixer 30 Gradient mixer 40 Peristaltic pump 50 Silicon tube 100 IPG gel preparation instrument

Claims (12)

1. A first-dimensional electrophoresis step in which antibody protein dissolved in a preparation buffer not containing a reducing agent is separated by first-dimensional electrophoresis using an electrophoresis buffer that does not contain a reducing agent;
   A second-dimensional electrophoresis step of separating the antibody protein separated in the first-dimensional electrophoresis step by a second-dimensional electrophoresis using an electrophoresis buffer that does not contain a reducing agent. Separation method.
2. The said preparation buffer contains the additive selected from the group which consists of non-surfactant type sulfobetaines, polysorbates, polyoxyethylene alkyl ethers, and sugar alcohols, The claim 1 characterized by the above-mentioned. Antibody separation method.
3. Non-surfactant type sulfobetaines include glycine betaine, dimethylethylammonium propanesulfonate (NDSB-195), 3- (1-pyridino) -1-propanesulfonate (NDSB-201), dimethyl (2-hydroxyethyl) Ammonium propane sulfonate (NDSB-211), 3- (1-methylpiperidinium) -1-propane sulfonate (NDSB-221), dimethylbenzyl ammonium propane sulfonate (NDSB-256), and 3- (4-tert- Selected from the group consisting of butyl-1-pyridino) -1-propanesulfonate (NDSB-256-4T);
   The polysorbate is Tween 80 (registered trademark),
   The polyoxyethylene alkyl ethers are polyethylene glycols,
   The antibody separation method according to claim 2, wherein the sugar alcohol is sorbitol.
4. 4. The antibody separation method according to claim 2, wherein the concentration of the additive in the preparation buffer is 1 w / v% or more and 6 w / v% or less.
5. The antibody separation method according to any one of claims 2 to 4, wherein the preparation buffer does not contain a protein denaturant.
6. The antibody separation method according to claim 1, wherein the preparation buffer contains a nonionic surfactant or an amphoteric surfactant.
7. The nonionic surfactant is 3-[(4-heptyl) phenyl-3-hydroxypropyl] dimethylammoniopropane sulfonate or Triton X,
   The amphoteric surfactant is 3- (3-colamidopropyl) dimethylammonio-1-propanesulfonate, 3-[(3-colamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate, or The method for separating antibodies according to claim 6, which is 3- [N, N-dimethyl (3-myristoylaminopropyl) ammonio] propanesulfonate or amidosulfobetaine-14.
8. In the first dimension electrophoresis,
   Maintaining the voltage constant and increasing the current when the current decreases to a predetermined current value greater than 0, increasing the current, maintaining the voltage constant when the current begins to decrease during the boost; and The antibody separation method according to any one of claims 1 to 7, wherein the voltage is controlled so that the current value is greater than 0 and 100 μA or less.
9. An evaluation step of evaluating whether or not the antibody separated by the antibody separation method according to any one of claims 1 to 8 is the same as an antibody contained in a reference medicine is characterized. Antibody evaluation method.
10. A method for evaluating a medicine, comprising an evaluation step for evaluating whether or not the antibody contained in the medicine separated by the antibody separation method according to any one of claims 1 to 8 is uniform.
11. It comprises an additive selected from the group consisting of non-surfactant sulfobetaines, polysorbates, polyoxyethylene alkyl ethers, and sugar alcohols, and has a preparation buffer that does not contain a reducing agent. A kit for two-dimensional electrophoresis of antibodies.
12. An antibody two-dimensional electrophoresis kit comprising a preparation buffer containing a nonionic surfactant or an amphoteric surfactant and not containing a reducing agent.

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