[go: up one dir, main page]

WO2015079977A1 - Procédé de séparation d'anticorps, méthode d'évaluation d'anticorps, méthode d'évaluation médicale, et kit d'électrophorèse d'anticorps 2d - Google Patents

Procédé de séparation d'anticorps, méthode d'évaluation d'anticorps, méthode d'évaluation médicale, et kit d'électrophorèse d'anticorps 2d Download PDF

Info

Publication number
WO2015079977A1
WO2015079977A1 PCT/JP2014/080490 JP2014080490W WO2015079977A1 WO 2015079977 A1 WO2015079977 A1 WO 2015079977A1 JP 2014080490 W JP2014080490 W JP 2014080490W WO 2015079977 A1 WO2015079977 A1 WO 2015079977A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
electrophoresis
separation method
buffer
dimensional electrophoresis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2014/080490
Other languages
English (en)
Japanese (ja)
Inventor
公彦 矢部
英樹 木下
祥之 石田
圭介 福田
博史 山木
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sharp Corp
Original Assignee
Sharp Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sharp Corp filed Critical Sharp Corp
Priority to JP2015550664A priority Critical patent/JPWO2015079977A1/ja
Publication of WO2015079977A1 publication Critical patent/WO2015079977A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/24—Extraction; Separation; Purification by electrochemical means
    • C07K1/26—Electrophoresis
    • C07K1/28—Isoelectric focusing
    • C07K1/285—Isoelectric focusing multi dimensional electrophoresis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44747—Composition of gel or of carrier mixture
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44795—Isoelectric focusing

Definitions

  • the present invention relates to an antibody separation method, an antibody evaluation method, a pharmaceutical evaluation method, and an antibody two-dimensional electrophoresis kit.
  • proteome As a central role in the post-genomic era, proteome research is actively conducted.
  • proteome is intended the entire protein that is translationally produced in a particular cell, organ and organ.
  • 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.
  • 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).
  • isoelectric focusing that separates proteins according to charge
  • slab gel electrophoresis that separates proteins according to molecular weight (especially SDS-PAGE).
  • SDS-PAGE molecular weight
  • 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.
  • 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.
  • 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.
  • 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.
  • an antibody separation method 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
  • 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.
  • the antibody protein is two-dimensionally electrophoresed under non-reducing conditions, so that the antibody can be separated more suitably.
  • 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.
  • a gel preparation step for preparing a gel for electrophoresis is performed before the first-dimensional electrophoresis step.
  • 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.
  • a detection step of detecting the separated antibody protein is performed after the second-dimensional electrophoresis step.
  • IPG immobilized pH gradient
  • 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.
  • 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.
  • 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.
  • a 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 ⁇ l of 0.2K immobiline pK3.6, 273 ⁇ l of pK6.2, 243 ⁇ l of pK7.0, 260 ⁇ l of pK8.5 and 282 ⁇ l 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 ⁇ l of 10% APS, and 7.6 ⁇ l of 100% TEMED with distilled water. What was measured up to 15 ml can be used.
  • acrylamide buffer mixture As the basic acrylamide buffer mixture, 0.2 M immobiline pK3.6 100 ⁇ l, pK6.2 333 ⁇ l, pK7.0 361 ⁇ l, pK8.5 239 ⁇ l and pK9.3 326 ⁇ l, 30% acrylamide stock solution (2.5% of 29.1% acrylamide, 0.9% bisacrylamide), 108 ⁇ l of 10% APS, and 7.6 ⁇ l 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.
  • polymerization initiator conventionally known polymerization initiators can be used, and examples thereof include ammonium persulfate (APS) and methylene blue.
  • APS ammonium persulfate
  • methylene blue examples thereof include ammonium persulfate (APS) and methylene blue.
  • polymerization accelerator conventionally known ones can be used, and examples thereof include tetramethylethylenediamine (TEMED). Moreover, you may comprise so that superposition
  • TEMED tetramethylethylenediamine
  • specific gravity adjuster conventionally known ones can be used, and examples thereof include glycerol and sucrose.
  • 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.
  • 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.
  • 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.
  • the pH gradient is from 6 to 10 and includes the isoelectric point of many antibody proteins. do it.
  • 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.
  • 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.
  • slab gel that performs SDS-PAGE in the second-dimensional electrophoresis step
  • a conventionally known slab gel can be used.
  • 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.
  • 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.
  • 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.
  • a fluorescent dye such as IC5-OSu special packaging (Doujin Chemical Laboratory) for detection after separation.
  • 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.
  • a reducing agent such as 2-mercaptoethanol
  • 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.
  • an antibody as a pharmaceutical is required to be a glycoform that is optimal for a certain disease.
  • antibodies cannot be separated while maintaining such a sugar chain modification, and the glycoform cannot be analyzed appropriately.
  • 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.
  • 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.
  • antibodies can be separated in a more nearly complete state, they are also suitable for analysis of glycoforms.
  • 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.
  • 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.
  • CHPS 3- (3-colamidopropyl) dimethylammonio-1-propanesulfonate
  • CHPSO 3-[(3-colamidopropyl) Dimethylammonio] -2-hydroxy-1-propanesulfonate
  • ASB-14 amidosulfobetaine-14
  • 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.
  • 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 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.
  • 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.
  • an additive selected from the group consisting of non-surfactant sulfobetaines, polysorbates, polyoxyethylene alkyl ethers, and sugar alcohols.
  • 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).
  • the non-surfactant sulfobetaines function as solubilizing and stabilizing agents, and the antibodies are dissolved and stabilized.
  • non-surfactant sulfobetaines examples 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.
  • 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.
  • PEG polyethylene glycol
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • Auto2D manufactured by Sharp Manufacturing System.
  • 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.
  • 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.
  • the swelling liquid is also used as an electrophoresis buffer for isoelectric focusing.
  • a conventionally known swelling liquid can be suitably used, but it does not contain a reducing agent.
  • antibody samples can be separated by isoelectric focusing under non-reducing conditions that do not contain a reducing agent.
  • 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 ⁇ L, 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
  • 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 ⁇ A.
  • electrophoresis conditions such as temperature may be conventionally known electrophoresis conditions.
  • 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.
  • 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. .
  • 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.
  • 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.
  • the antibody sample separated in the IPG gel moves to the slab gel and is separated based on the difference in molecular weight.
  • 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.
  • 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.
  • electrophoresis conditions such as pH of the electrophoresis buffer may be conventionally known electrophoresis conditions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the antibody evaluation method 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 sorbi
  • the antibody can be separated in a more complete state.
  • the concentration of the additive in the preparation buffer may be 1 w / v% or more and 6 w / v% or less.
  • the antibody can be separated in a more complete state.
  • the preparation buffer may not contain a protein denaturant.
  • 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.
  • the preparation buffer may contain a nonionic surfactant or an amphoteric surfactant.
  • 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.
  • 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 ⁇ A.
  • the current 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 ⁇ A.
  • 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.
  • the antibody 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.
  • the drug 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).
  • 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.
  • the antibody is preferably separated while maintaining a disulfide bond.
  • 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 ⁇ g / ⁇ L. 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a buffer containing Tris, Glysin and SDS was used as the electrophoresis buffer.
  • a main antibody spot group was detected around 160 kD, and an isoform group considered to be a dimer was detected around 300 kD.
  • 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.
  • a spot group regularly arranged in the isoelectric point and molecular weight direction was detected.
  • the difference in the spot position in the vertical direction represents the difference in molecular weight due to the number of added sugar chains
  • 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.
  • 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.
  • 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.
  • 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.
  • non-denaturing two-dimensional electrophoresis as shown in FIG.
  • 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.
  • Example 3 Non-reducing two-dimensional electrophoresis of other antibodies
  • 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.
  • trastuzumab 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.
  • Cetuximab manufactured by BMS
  • Trastuzumab manufactured by Chugai Pharmaceutical Co., Ltd.
  • 0.1% Amphorite pH 6-11
  • 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.
  • 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.
  • 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)
  • 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.
  • FIG. 7A is a graph showing changes in voltage value and current value when isoelectric focusing is performed under electrophoresis condition 1
  • 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.
  • 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
  • the horizontal axis represents time (seconds).
  • 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 ⁇ A.
  • mice 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).
  • 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.
  • 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.
  • FIG. 9A shows the result of the reduction condition 1
  • FIG. 9B shows the result of the reduction condition 2.
  • FIG. 9 (a) an H chain was detected at about 60 kD
  • an L chain was detected at about 20 kD.
  • 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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Dispersion Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

Cette invention concerne un procédé de séparation d'anticorps permettant de séparer les anticorps tout en conservant les liaisons disulfure, ledit procédé comprenant : une étape d'électrophorèse de première dimension pour séparer la protéine d'anticorps solubilisée dans un tampon préparé qui ne contient pas d'agent de réduction, par une électrophorèse de première dimension utilisant un tampon qui ne contient pas d'agent de réduction ; et une étape d'électrophorèse de seconde dimension pour séparer la protéine d'anticorps soumise à électrophorèse dans ladite étape d'électrophorèse de première dimension par une électrophorèse de seconde dimension utilisant un tampon qui ne contient pas d'agent de réduction.
PCT/JP2014/080490 2013-11-26 2014-11-18 Procédé de séparation d'anticorps, méthode d'évaluation d'anticorps, méthode d'évaluation médicale, et kit d'électrophorèse d'anticorps 2d Ceased WO2015079977A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2015550664A JPWO2015079977A1 (ja) 2013-11-26 2014-11-18 抗体分離方法、抗体評価方法、医薬の評価方法、及び、抗体の２次元電気泳動用キット

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2013-244150 2013-11-26
JP2013244150 2013-11-26

Publications (1)

Publication Number Publication Date
WO2015079977A1 true WO2015079977A1 (fr) 2015-06-04

Family

ID=53198922

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2014/080490 Ceased WO2015079977A1 (fr) 2013-11-26 2014-11-18 Procédé de séparation d'anticorps, méthode d'évaluation d'anticorps, méthode d'évaluation médicale, et kit d'électrophorèse d'anticorps 2d

Country Status (2)

Country Link
JP (1) JPWO2015079977A1 (fr)
WO (1) WO2015079977A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107515303A (zh) * 2017-09-30 2017-12-26 广州万孚生物技术股份有限公司 检测尿液中hiv抗体的试纸条、检测杯及其制备方法

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0222297A (ja) * 1988-07-11 1990-01-25 Hidechika Okada 新規な糖蛋白質
JPH04505709A (ja) * 1989-11-07 1992-10-08 ブリストル―マイアーズ　スクイブ　カンパニー オリゴマー免疫グロブリン
JPH11511973A (ja) * 1996-02-02 1999-10-19 ルードヴィッヒ・インスティテュート・フォア・キャンサー・リサーチ 大腸細胞および大腸癌細胞関連核酸分子、タンパク質およびペプチド
JP2005027671A (ja) * 1998-05-20 2005-02-03 Kyowa Hakko Kogyo Co Ltd 遺伝子組換え抗体
JP2006189358A (ja) * 2005-01-07 2006-07-20 Nippon Meat Packers Inc 蛋白質含有液の保存方法及びそれに使用される希釈液
JP2007516281A (ja) * 2003-12-23 2007-06-21 レツク・フアーマシユーテイカルズ・デー・デー 有効成分とスルホベタインとを含む医薬組成物
JP2007535296A (ja) * 2003-06-27 2007-12-06 バイオジェン・アイデック・エムエイ・インコーポレイテッド 異種抗体溶液の生成のための疎水性相互作用クロマトグラフィーまたはヒンジ領域改変の使用
WO2013066866A1 (fr) * 2011-10-31 2013-05-10 Genentech, Inc. Formulations d'anticorps
WO2013094735A1 (fr) * 2011-12-22 2013-06-27 シャープ株式会社 Procédé de commande, dispositif de commande, système de commande, et programme de commande

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0222297A (ja) * 1988-07-11 1990-01-25 Hidechika Okada 新規な糖蛋白質
JPH04505709A (ja) * 1989-11-07 1992-10-08 ブリストル―マイアーズ　スクイブ　カンパニー オリゴマー免疫グロブリン
JPH11511973A (ja) * 1996-02-02 1999-10-19 ルードヴィッヒ・インスティテュート・フォア・キャンサー・リサーチ 大腸細胞および大腸癌細胞関連核酸分子、タンパク質およびペプチド
JP2005027671A (ja) * 1998-05-20 2005-02-03 Kyowa Hakko Kogyo Co Ltd 遺伝子組換え抗体
JP2007535296A (ja) * 2003-06-27 2007-12-06 バイオジェン・アイデック・エムエイ・インコーポレイテッド 異種抗体溶液の生成のための疎水性相互作用クロマトグラフィーまたはヒンジ領域改変の使用
JP2007516281A (ja) * 2003-12-23 2007-06-21 レツク・フアーマシユーテイカルズ・デー・デー 有効成分とスルホベタインとを含む医薬組成物
JP2006189358A (ja) * 2005-01-07 2006-07-20 Nippon Meat Packers Inc 蛋白質含有液の保存方法及びそれに使用される希釈液
WO2013066866A1 (fr) * 2011-10-31 2013-05-10 Genentech, Inc. Formulations d'anticorps
WO2013094735A1 (fr) * 2011-12-22 2013-06-27 シャープ株式会社 Procédé de commande, dispositif de commande, système de commande, et programme de commande

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HONG JI ET AL.: "Electrophoretic analysis of the novel antigen for the gastrointestinal-specific monoclonal antibody, A33", ELECTROPHORESIS, vol. 18, 1997, pages 614 - 621 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107515303A (zh) * 2017-09-30 2017-12-26 广州万孚生物技术股份有限公司 检测尿液中hiv抗体的试纸条、检测杯及其制备方法
CN107515303B (zh) * 2017-09-30 2024-06-07 广州万孚生物技术股份有限公司 检测尿液中hiv抗体的试纸条、检测杯及其制备方法

Also Published As

Publication number Publication date
JPWO2015079977A1 (ja) 2017-03-16

Similar Documents

Publication Publication Date Title
El Rassi Electrophoretic and electrochromatographic separation of proteins in capillaries: an update covering 2007–2009
US12379345B2 (en) Method of purity determination by capillary electrophoresis
Chevalier Highlights on the capacities of" Gel-based" proteomics
JP4503433B2 (ja) 電気泳動法
US20150162177A1 (en) Method For Analyzing Sample Components
Drabik et al. Gel electrophoresis
US20160025676A1 (en) Compositions and methods for improving resolution of biomolecules separated on polyacrylamide gels
JP4660653B2 (ja) キャピラリー電気泳動法によるヘモグロビンの分析方法およびそれに用いる添加剤
CN1630816A (zh) 通过毛细管电泳分离蛋白质的方法和毛细管电泳用的缓冲组合物
Zhang et al. Separation of monoclonal antibody charge state variants by open tubular capillary electrochromatography with immobilised protein as stationary phase
Yagi et al. Application of microchip electrophoresis sodium dodecyl sulfate for the evaluation of change of degradation species of therapeutic antibodies in stability testing
EP2183583B1 (fr) Système tampon pour gel d'électrophorèse précoulé longue durée
WO2015079977A1 (fr) Procédé de séparation d'anticorps, méthode d'évaluation d'anticorps, méthode d'évaluation médicale, et kit d'électrophorèse d'anticorps 2d
JP2008292492A (ja) 電気泳動分離方法
Song et al. Application of capillary electrophoresis-sodium dodecyl sulfate in assessing the purity of monoclonal antibody biopharmaceuticals
Miller et al. Other than IPG‐DALT: 2‐DE variants
Andrási et al. Analysis of rituximab, a therapeutic monoclonal antibody by capillary zone electrophoresis
Kaya et al. Recent biopharmaceutical applications of capillary electrophoresis methods on recombinant DNA technology‐based products
CN106061992A (zh) 通过用烷基阳离子处理来降低蛋白质制剂中的染色质含量的方法
Williams et al. Analysis of Bence Jones proteinuria by high resolution two‐dimensional electrophoresis
CN105651848A (zh) 一种含有保护剂的毛细管凝胶电泳检测试剂盒
KR20160117628A (ko) 아릴 음이온들의 처리에 의한 단백질 제제들 내의 응집체 함량을 감소시키기 위한 방법들
Vidanapathirana et al. Cationic ionic liquid surfactant-polyacrylamide gel electrophoresis for enhanced separation of acidic and basic proteins with single-step ribonuclease b glycoforms separation
Ramnani Method Development and Validation of Reverse-Phase HPLC for Purity Analysis and Size-Exclusion HPLC for Aggregate Analysis of Monoclonal Antibody, SUNmAb
JP2021510811A (ja) 電気泳動分離用分解剤としてのトリ−およびテトラ−ヒドロキシル第四級アンモニウム化合物の使用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14865843

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2015550664

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14865843

Country of ref document: EP

Kind code of ref document: A1