WO2016194114A1 - モノクローナル抗体の定量方法 - Google Patents
モノクローナル抗体の定量方法 Download PDFInfo
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- WO2016194114A1 WO2016194114A1 PCT/JP2015/065806 JP2015065806W WO2016194114A1 WO 2016194114 A1 WO2016194114 A1 WO 2016194114A1 JP 2015065806 W JP2015065806 W JP 2015065806W WO 2016194114 A1 WO2016194114 A1 WO 2016194114A1
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- monoclonal antibody
- antibody
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- peptide
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- 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
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21001—Chymotrypsin (3.4.21.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21004—Trypsin (3.4.21.4)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
- G01N2333/7158—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
Definitions
- the present invention relates to a method for quantifying a monoclonal antibody using mass spectrometry, and more specifically, selectively digesting a peptide fragment containing a specific sequence of a monoclonal antibody, and detecting the obtained peptide fragment by mass spectrometry. And using a specific enzyme for selective digestion.
- ⁇ ⁇ Pharmacokinetics especially concentration monitoring (TDM)
- concentration monitoring has attracted attention for the development and administration of drugs that exhibit high efficacy with few side effects.
- Concentration monitoring makes it possible to confirm whether the administered drug is in an appropriate amount and whether it has reached the lesion site, evaluate the effectiveness of the drug, and adjust the dose appropriately.
- the effectiveness of molecularly targeted drugs which have become mainstream in areas such as cancer and autoimmune diseases, allows doctors themselves to quickly determine whether drugs accumulate in lesion sites and exert their effects. is important.
- Antibody drugs are attracting attention as molecular target drugs.
- Antibody drugs bind specifically to cancer and other focal antigens, target molecules, growth factors, etc., and are therefore attracting attention as drugs with low side effects and high efficacy, and clinical research has progressed with many antibodies. ing. While antibodies exhibit extremely high molecular specificity by nature, there are often cases where even the same type of cancer does not exert drug efficacy due to the difference in the location of the lesion, or does not work on metastatic lesions. . In addition, there is a problem of drug prices, and it has been argued that it is important to perform optimized medical care by proper use.
- ELISA Enzyme-Linked ImmunoSorbent Assay
- the group of the present inventors has found a method capable of obtaining an optimal peptide fragment for specific detection of a protein using mass spectrometry by selective protease digestion of a protein such as an antibody.
- This method realizes regioselective protease digestion of a monoclonal antibody by immobilizing both a substrate protein such as an antibody and a protease enzyme on a solid phase (Patent Document 1 and Non-Patent Document). 1).
- the target protein is cleaved in a position-selective manner to produce peptide fragments specific to the protein and reduce the production of other peptide fragments. Is efficient. Therefore, in the case of an antibody, it is preferable to regioselectively digest a Fab domain containing a specific sequence, particularly a variable region of the Fab domain, while suppressing digestion of the Fc domain.
- Patent Document 1 and Non-Patent Document 1 are epoch-making methods capable of performing selective protease digestion of monoclonal antibodies using a solid-solid reaction. More specifically, for example, the C-terminal side of an antibody is immobilized on a Protein® G or Protein® A resin having a pore diameter of about 100 nm, and the variable region of the antibody is always oriented to the solution side. Next, protease is immobilized on the surface of fine particles having a particle diameter of about 200 nm. By limiting the contact of the protease with the antibody, it is possible to form a reaction field for performing an antibody degradation reaction selective for the variable region.
- the inventors of the present invention are most effective for detecting a protease digestion fragment contained in the CDR2 region in the variable region of a monoclonal antibody for detection using restriction protease digestion of the antibody. I found.
- protease when the peptide fragment after protease digestion is subjected to mass spectrometry, it is considered to use a protease with low self-digestion and high selectivity of the cleavage sequence. Therefore, those skilled in the art use mass spectrometry grade or sequencing grade proteases when using commercially available proteases.
- native trypsin derived from living organisms is known to have low cleavage site specificity because it produces pseudotrypsin that exhibits chymotrypsin-like activity by autolysis. Therefore, as a mass spectrometry grade, a product obtained by reducing methylation of a lysine residue of trypsin to increase resistance to autolysis is commercially available.
- the present inventors consider that the above results suggest the possibility of interference due to the presence of endogenous antibodies to the obtained peptide fragment, and obtain a peptide fragment free from such interference. Further study was carried out for the purpose. As a result, surprisingly, by using an enzyme with a lower specificity than the conventionally used enzyme for structural analysis, the number of cleavage sites is increased, and peptide fragments of different sequences are generated, resulting in quantitative detection. It has been found that an optimal peptide can be obtained, and the present invention has been completed.
- the aspect of this invention includes the following invention.
- a selective protease of a monoclonal antibody by contacting a porous body in which the monoclonal antibody to be measured is immobilized in the pores and a fine particle having an average particle diameter larger than the average pore diameter of the porous body in which the protease is immobilized.
- a method for quantifying a monoclonal antibody comprising the step of digesting, and a step of detecting a peptide fragment containing the amino acid derived from the CDR2 region of the heavy chain or light chain of the monoclonal antibody obtained by the digestion, The method as described above, wherein a protease having trypsin activity and chymotrypsin activity is used.
- a kit for quantitative detection of monoclonal antibodies by liquid chromatography mass spectrometry (LC-MS), A porous body for immobilizing the monoclonal antibody to be measured; Microparticles immobilized with protease having trypsin activity and chymotrypsin activity; A reaction vessel for selectively digesting monoclonal antibodies by contacting the porous body and the fine particles; A buffer solution that is introduced into the reaction vessel together with the fine particles and the porous body, and causes a digestion reaction with the protease; A kit as described above.
- the above-mentioned recording medium containing the data of the voltage applied to the electrode of a parent ion, a fragment ion, and a mass spectrometer about the peptide obtained by protease digestion of an antibody.
- LC-MS liquid chromatography mass spectrometry
- the method of the present invention uses a limited protease reaction field, it is possible to selectively decompose and recover a limited region of an antibody, particularly the CDR2 region.
- the CDR2 region can be preferentially degraded because it is the site most exposed on the antibody surface. By utilizing this fact, it becomes possible to more efficiently detect a monoclonal antibody in mass spectrometry.
- the concentration of the antibody drug can be measured directly from the biological sample.
- the quantification of individual antibodies by mass spectrometry can be performed reliably.
- the doctor himself can be involved in the analysis of the specimen in the clinical setting, and the effectiveness of the administered antibody can be quickly evaluated.
- the amino acid sequences of the heavy and light chains of mogarizumab (SEQ ID NOs: 1 and 2), the sequence of the Trypsin Gold cleavage fragment used for quantification and the position in the amino acid sequence of the heavy or light chain are shown.
- MRM chromatograms of Trypsin Gold cleavage fragments of mogarizumab (SEQ ID NOs: 3-9) are shown.
- the upper figure shows the analysis result of mogarizumab bulk powder, and the lower figure shows the analysis result of the sample spiked with mogarizumab in plasma.
- the vertical axis represents peak intensity, and the horizontal axis represents retention time.
- the fragment derived from the same peptide is detected at the same retention time, and the difference in production efficiency of each fragment ion is indicated by the peak height.
- the calibration curve prepared using the Trypsin Gold-cleaved fragment of mogarizumab spiked in plasma (SEQ ID NOs: 3 to 9) is shown.
- the sequence of the Trypsin TPCK-treated cleavage fragment used for quantification of mogarizumab and the positions in the heavy and light chain amino acid sequences are shown.
- MRM chromatograms of Trypsin TPCK-treated cleavage fragments of mogarizumab SEQ ID NOs: 10-14 are shown.
- the upper figure shows the analysis result of mogarizumab bulk powder
- the lower figure shows the analysis result of the sample spiked with mogarizumab in plasma.
- the vertical axis represents peak intensity
- the horizontal axis represents retention time.
- the fragment derived from the same peptide is detected at the same retention time, and the difference in production efficiency of each fragment ion is indicated by the peak height.
- the standard curve produced using the Trypsin TPCK-treated cleavage fragment (SEQ ID NOs: 10 to 14) of mogarizumab spiked in plasma is shown.
- the structure identification result of mogarizumab target peptide SYYPDSVK (sequence number 10) in accurate mass spectrometry is shown.
- the present invention includes the following steps: A selective protease of a monoclonal antibody by contacting a porous body in which the monoclonal antibody to be measured is immobilized in the pores and a fine particle having an average particle diameter larger than the average pore diameter of the porous body in which the protease is immobilized.
- a method for quantifying a monoclonal antibody comprising the step of digesting, and a step of detecting a peptide fragment containing the amino acid derived from the CDR2 region of the heavy chain or light chain of the monoclonal antibody obtained by the digestion,
- the present invention relates to the above method, characterized in that a protease having trypsin activity and chymotrypsin activity is used.
- the method of the present invention is intended to reduce the analysis target population while maintaining the specificity of the measurement target.
- CDR2 region
- FR framework region
- Those skilled in the art can efficiently determine the specificity of the target antibody by confirming the multiple alignment of the amino acid sequence of the target antibody and the other antibody and the cross-reactivity between the target antibody and the endogenous antibody.
- An amino acid sequence containing amino acids derived from the CDR2 region for detection can be determined, and a peptide fragment to be detected can be predicted and selected.
- amino acid sequence including an amino acid derived from the CDR2 region means an amino acid sequence including one or more amino acids derived from the CDR2 region specific to the target antibody, in other words, It can also be referred to as an amino acid sequence including a part of the CDR2 region.
- amino acid sequence including amino acids derived from the CDR2 region does not intend any amino acid sequence including “amino acids derived from the CDR2 region”, but the amino acid sequence as a whole is an amino acid sequence of the target antibody. It is intended to constitute a continuous array of parts.
- Mass spectrometry In order to detect an antibody by mass spectrometry, it is necessary to first extract the antibody from a biological sample and dissolve it in an appropriate solvent. In addition, since an antibody has a large molecule for analysis as it is, it is decomposed into a peptide by protease and then separated by liquid chromatography, followed by mass spectrometry. The molecular weight of peptides suitable for analysis is about 1000 to 3000 Da.
- the measurement object in the method of the present invention is a monoclonal antibody.
- the heavy and light chains of the monoclonal antibody are each composed of a constant region and a variable region.
- the constant region has an amino acid sequence common to most antibodies from the same species.
- the variable region has three complementarity determining regions (CDRs) (CDR1, CDR2, CDR3). The three-dimensional structure defined by these regions is involved in specific binding to the antigen, thereby forming an antibody-antigen complex.
- the CDR region involved in the specific binding to the antigen is located almost outside the antibody molecule.
- the CDR2 region is located on the outermost side (FIG. 8), and is optimal as a target for the digestion of the limited protease of the present invention.
- the entire amino acid sequence and CDR sequence are disclosed.
- those skilled in the art can specify the CDR region based on the amino acid sequence information of the antibody.
- Monoclonal antibodies that can be measured in the method of the present invention include, but are not limited to, human antibodies such as mogamulizumab, panitumumab, ofatumumab, golimumab, ipilimumab; tocilizumab, omalizumab, mepolizumab, gemtuzumab, palivizumab, Examples include humanized antibodies such as ocrelizumab, mogamulizumab, and eculizumab; chimeric antibodies such as cetuximab, infliximab, and basiliximab.
- the molecular diameter of the monoclonal antibody is about 14.5 nm.
- the CDR2 region of a monoclonal antibody can be protease-selectively digested, and the resulting peptide fragment of the CDR2 region is detected by mass spectrometry, so that the antibody can be identified or quantitatively detected. It can be performed. Since the method can be applied regardless of the type of antibody, the method of the present invention is not limited to the above-exemplified antibodies, and can also be applied to newly developed monoclonal antibodies and the like.
- porous material The porous body used in the method of the present invention is not particularly limited as long as it has a large number of pores, and activated carbon, porous membranes, porous resin beads, metal particles, etc. can be used. . Among these, those capable of binding an antibody site-specifically are preferable.
- the shape of the pore is not particularly limited. Moreover, what formed the pore which penetrates a porous body like a porous film can also be used.
- the size of the pores in the porous body is not particularly limited, and considers the molecular diameter of the antibody so that when the antibody is immobilized, the site to be selectively digested is located near the surface of the pore. Is preferably determined.
- the average pore diameter D2 of the porous body is appropriately set within a range of, for example, about 10 nm to 200 nm and smaller than the average particle diameter D1 of the fine particles.
- the average pore diameter D2 of the porous body is preferably about 20 nm to 200 nm, more preferably in the range of 30 nm to 150 nm, 40 nm to 120 nm, and 50 nm to 100 nm, and even more preferably about 100 nm.
- those in which a linker molecule that interacts site-specifically with an antibody is immobilized in the pores of the porous body are preferably used.
- the linker molecule Protein A, Protein G, or the like that binds site-specifically to the Fc domain of an antibody is preferably used.
- the Fc domain of the antibody is immobilized in the pores, and the Fab domain is located near the surface layer of the pores. In this way, by controlling the orientation of the antibody in the pore, it is possible to perform site-selective digestion of the Fab domain, particularly CDR2, by a protease.
- the size of the linker molecule is selected so that the selective cleavage site of the antibody is located near the surface layer of the pore.
- the molecular size of the state in which the linker molecule is bound to the antibody is preferably about 0.5 to 1.5 times, more preferably about 0.6 to 1.2 times the pore diameter of the porous body. It is more preferably about 7 to 1.1 times, and particularly preferably about 0.8 to 1 times.
- the linker molecule is not fixed to the porous body and the antibody is directly bonded to the pore, it is preferable that the molecular diameter of the antibody and the pore diameter of the porous body satisfy the above relationship.
- the porous material that can be suitably used in the present invention is not particularly limited, and examples thereof include Protein® G® Ultralink resin (Pierce) and Toyopearl (TOSO).
- Protein® G® Ultralink resin it has been found that 95% of the Protein® G bound to the resin is in the pores.
- the method for immobilizing the antibody in the pores of the porous body is not particularly limited, and an appropriate method can be adopted depending on the characteristics of the antibody and the porous body or the linker molecule.
- an antibody is immobilized on a porous body in which protein A or protein G is immobilized in the pores, a mixture of the porous body suspension and the antibody-containing solution is mixed into the pores. It is possible to easily immobilize the antibody.
- the amount ratio of the porous body and the antibody can be appropriately set according to the purpose. For example, when performing quantitative analysis of an antibody, it is desired that almost the entire amount of the antibody in the sample is immobilized on the porous body. Therefore, it is preferable to set the quantity ratio so that the amount of the porous material is excessive with respect to the estimated content of the antibody in the sample.
- the microparticles are used for the purpose of controlling protease access to the antibody immobilized in the pores of the porous body by immobilizing protease on the surface thereof. Therefore, the average particle diameter D1 is larger than the average pore diameter D2 of the porous body so that the fine particles do not enter deeply into the pores of the porous body.
- the shape of the fine particles is not particularly limited, but spherical fine particles are preferred from the viewpoint of uniform protease access to the pores of the porous body.
- the fine particles preferably have a uniform average particle size.
- the average particle diameter D1 of the fine particles is preferably in the range of 50 nm to 500 nm, more preferably 1.2 times or more of the average pore diameter D2 of the porous body, further preferably 1.5 times or more, 1.8 times or more, for example About twice is particularly preferred.
- the average pore diameter of the porous body is about 30 to 150 nm
- the average particle diameter D1 of the fine particles is preferably 100 nm or more, and more preferably 150 nm or more.
- the average pore diameter of the porous body is about 50 nm to 100 nm
- the average particle diameter of the fine particles is preferably 120 nm or more, more preferably 150 nm or more, and particularly preferably 170 nm or more.
- the upper limit of the average particle diameter D1 of the fine particles is preferably 500 nm or less, and more preferably 300 nm or less, from the viewpoint of increasing digestion efficiency by protease.
- the fine particles are not particularly limited as long as the above-mentioned protease can be immobilized on the surface, and metals, resins, and the like are appropriately used. Moreover, what coated the metal surface with resin, what coated the resin surface with the metal, etc. can also be used.
- magnetic fine particles that can be dispersed or suspended in an aqueous medium and can be easily recovered from the dispersion or suspension by applying a magnetic field are preferable. Further, in view of the fact that aggregation does not easily occur, magnetic fine particles whose surfaces are coated with an organic polymer are more preferable.
- the base material of the magnetic fine particles include ferromagnetic alloys such as iron oxide (magnetite (Fe 3 O 4 ), maghemite ( ⁇ -Fe 2 O 3 )), and ferrite (Fe / M) 3 O 4 .
- M means a metal ion that can be used together with iron ions to form a magnetic metal oxide, typically Co 2+ , Ni 2+ , Mn 2+. Mg 2+ , Cu 2+ , Ni 2+ and the like are used.
- the organic polymer that covers the magnetic fine particles include polyglycidyl methacrylate (polyGMA), a copolymer of GMA and styrene, polymethyl methacrylate (PMMA), and polymethyl acrylate (PMA).
- polyGMA polyglycidyl methacrylate
- PMMA polymethyl methacrylate
- PMA polymethyl acrylate
- Specific examples of magnetic nanobeads coated with an organic polymer include FG beads, SG beads, Adembeads, and nanomag.
- FG beads manufactured by Tamagawa Seiki Co., Ltd. (polymer magnetic fine particles having a particle size of about 200 nm in which ferrite particles are coated with polyglycidyl methacrylate (polyGMA)) are preferably used.
- the spacer is preferably one that can bind to the protease and does not inactivate the protease. From the viewpoint of controlling the access range of the protease immobilized on the surface of the fine particles, the spacer preferably has a small molecular diameter.
- the spacer molecular diameter is preferably 5 nm or less, more preferably 3 nm or less, and even more preferably 2 nm or less. Further, the molecular weight of the spacer is preferably 2000 or less, more preferably 1500 or less, and further preferably 1000 or less.
- the spacer molecule capable of immobilizing protease with the above molecular diameter is preferably a non-protein, and has an amino group, carboxyl group, ester group, epoxy group, tosyl group, hydroxyl group, thiol group, aldehyde group, maleimide group, succinimide group at the terminal.
- Molecules having functional groups such as azide group, biotin, avidin, chelate and the like are preferable.
- spacer molecules having an activated ester group are preferred for immobilizing trypsin.
- spacer arm portions other than the above functional groups are polyethylene glycol and derivatives thereof, polypropylene glycol and derivatives thereof, polyacrylamide and derivatives thereof, polyethyleneimine and derivatives thereof, poly (ethylene oxide) and derivatives thereof, poly Hydrophilic molecules such as (ethylene terephthalic acid) and its derivatives are used.
- Such fine particles surface-modified with spacer molecules are also commercially available, and they can be used.
- fine particles modified with spacer molecules having ester groups (active ester groups) activated with N-hydroxysuccinimide are commercially available under the trade name “FG beads NHS” (Tamakawa Seiki Co., Ltd.).
- the particle diameter of FG beads NHS is about 200 nm ⁇ 20 nm, and it is very homogeneous as fine particles.
- protease having trypsin activity and chymotrypsin activity is used as the protease immobilized on the microparticles.
- Trypsin has high substrate specificity, and since Lys or Arg is present at the C-terminus of the peptide after cleavage, the charge amount and charge localization of the peptide can be made uniform, so that a sample for mass spectrometry can be prepared. It is suitable for. Trypsin has a small molecular diameter (about 3.8 nm) and an active site is present inside the molecule. Therefore, the region where the active site can access the antibody is limited, and the position selectivity of protease digestion can be enhanced.
- the present inventors have found that by using a protease having trypsin activity and chymotrypsin activity instead of the commonly used mass spectrometry grade trypsin, it is possible to obtain an optimal peptide fragment for quantitative detection of individual antibodies. .
- protease used in the method of the present invention for example, native trypsin derived from a living body known to exhibit chymotrypsin-like activity by autolysis can be used.
- a product obtained by reducing the lysine residue of trypsin to increase resistance to self-digestion is commercially available as a mass spectrometry grade trypsin.
- the trypsin used in the present invention is a reductive methylation treatment of such a lysine residue. It is preferable that the trypsin is not used.
- Trypsin® Gold (manufactured by Promega) is a recombinant trypsin that has been subjected to a reductive methylation reaction after TPCK treatment.
- Trypsin TPCK-treated (manufactured by Sigma) is a protease that has been reduced in chymotrypsin activity by treating TPCK with trypsin purified from cattle but has not undergone a reductive dimethylation reaction.
- the method of the present invention uses a protease having trypsin activity and chymotrypsin activity, so that a trypsin that does not have chymotrypsin activity, for example, a monoclonal antibody that cannot be quantitatively detected when using mass spectrometry grade trypsin, It enables quantitative detection using peptide fragments that cannot be obtained by digestion of analytical grade trypsin.
- protease activity As the “protease having trypsin activity and chymotrypsin activity”, use of trypsin exhibiting chymotrypsin-like activity as described above is assumed, but as another embodiment, a mixture of trypsin and chymotrypsin can also be used. In this case, for example, trypsin gold from Promega can be used as trypsin, and trypsin TPCK treatment from Sigma can be used as chymotrypsin. The ratio of trypsin to chymotrypsin is preferably 9: 1 to 1: 1 in terms of protease activity (U).
- proteases examples include TrypsinpsTPCK-treated (manufactured by Sigma).
- the method for immobilizing the protease on the surface of the microparticle is not particularly limited, and an appropriate method can be adopted depending on the characteristics of the protease and the microparticle (or the spacer molecule that modifies the microparticle surface).
- the protease is spacer-modified.
- the protease can be immobilized on the surface of the fine particles by mixing a suspension of the fine particles and a solution containing the protease.
- the amine coupling method of fine particles and protease via the functional group of the spacer molecule is preferable.
- the carboxyl group whose surface is modified on the fine particles can be esterified with N-hydroxysuccinimide (NHS) to form an activated ester group, and the amino group of the protease can be bound thereto.
- NHS N-hydroxysuccinimide
- EDAC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
- DCC N, N'-dicyclohexylcarbodiimide
- DIPC bis (2,6-diisopropylphenyl) carbodiimide
- amino group of the protease is modified by using a cross-linking agent such as glutaraldehyde, bifunctional succinimide, bis (sulfosuccinimidyl) suberate (BS3), sulfonyl chloride, maleimide, pyridyl disulfide, etc. May be combined.
- a cross-linking agent such as glutaraldehyde, bifunctional succinimide, bis (sulfosuccinimidyl) suberate (BS3), sulfonyl chloride, maleimide, pyridyl disulfide, etc. May be combined.
- the coupling method between the microparticles and the protease via the functional group of the spacer molecule can be performed by a simple operation of adding the protease solution to the microparticle suspension and mixing and stirring under certain conditions.
- the conditions for protease digestion in the present invention are not particularly limited, and conditions similar to those for general protease digestion can be appropriately employed. For example, it is preferable to incubate at a temperature of about 37 ° C. for about 4 to 20 hours in a buffer solution adjusted to near the optimum pH of the protease.
- the mixing ratio of the porous body on which the antibody is immobilized and the microparticles on which the protease is immobilized is not particularly limited, and may be set so that the amount of protease corresponds to the amount of antibody.
- the access between the antibody and the protease is physically restricted by the combination of the porous material and the fine particles, it is preferable to increase the amount of the protease as compared with general protease digestion.
- antibody: protease about 30: 1 to 3: 1 is preferable, about 15: 1 to 4: 1 is more preferable, and about 10: 1 to 5: 1 is more preferable.
- protease digestion is carried out with the antibody immobilized on the porous body. Since the peptide fragment produced by protease digestion exists in the liquid phase, the target peptide fragment can be obtained in a position-selective manner without performing antibody elution or denaturation operation. According to the method of the present invention, peptide fragments can be recovered in a position-selective manner with a simpler operation than the conventional method.
- the target peptide fragment obtained by protease digestion is detected.
- the porous body and fine particles can be easily removed by filtering using a polyvinylidene fluoride (PVDF) filtration membrane (Low-binding hydrophilic PVDF, pore size 0.2 ⁇ m, manufactured by Millipore).
- PVDF polyvinylidene fluoride
- LC-MS Liquid chromatograph mass spectrometry
- the sample before being subjected to mass spectrometry is separated and concentrated by liquid chromatography (LC).
- LC liquid chromatography
- the eluate from LC may be directly ionized and subjected to mass spectrometry.
- Analysis can also be performed by LC / MS / MS or LC / MSn, which combines LC and tandem mass spectrometry.
- the eluate from LC may be collected once and then subjected to mass spectrometry.
- the LC column is not particularly limited, and a hydrophobic column such as C30, C18, C8, or C4 generally used for peptide analysis, a carrier for hydrophilic affinity chromatography, or the like can be appropriately selected and used. .
- mass spectrometry can determine an amino acid sequence, it can be determined whether or not a peptide fragment is a peptide fragment derived from a specific protein such as an antibody. Further, the concentration of the peptide fragment in the sample can be determined based on the peak intensity. In the analysis, if necessary, the sample may be used for the analysis after treatment such as desalting, solubilization, extraction, concentration, and drying.
- the ionization method in mass spectrometry is not particularly limited. Electron ionization (EI) method, chemical ionization (CI) method, field desorption (FD) method, fast atom collision (FAB) method, matrix-assisted laser desorption ionization (MALDI) Method, electrospray ionization (ESI) method and the like can be employed.
- the analysis method of the ionized sample is not particularly limited. Magnetic field deflection type, quadrupole (Q) type, ion trap (IT) type, time of flight (TOF) type, Fourier transform ion cyclotron resonance (FT-ICR) type Etc. can be appropriately determined according to the ionization method.
- MS / MS analysis or multistage mass spectrometry of MS3 or higher can be performed using a triple quadrupole mass spectrometer or the like.
- Suitable mass spectrometers for carrying out the method of the present invention include, but are not limited to, liquid chromatographs such as LCMS-8030, LCMS-8040, and LCMS-8050 (all manufactured by Shimadzu Corporation).
- -Structural analysis mass spectrometers that analyze accurate mass such as triple quadrupole mass spectrometers, LCMS-IT-TOF, and LCMS-Q-TOF (all manufactured by Shimadzu Corporation).
- the target antibody can be identified and quantified.
- Existing databases can also be used to identify antibodies based on mass spectrometry results.
- An antibody can also be identified by specifying the amino acid sequence of a peptide fragment by multistage mass spectrometry or the like.
- the peptide to be detected preferably has about 5 to 30 amino acid residues, more preferably about 7 to 25.
- the amount of antibody can be calculated based on the peak area and peak intensity of the detected peptide fragment ions (in the case of multistage MS, fragment ions obtained by cleavage of the parent ion).
- a peptide fragment in a sample can be obtained by associating a calibration curve (calibration curve) obtained in advance with a peak area or by associating a peak area derived from an internal standard added to the sample with a peak area derived from the sample.
- the amount and concentration of antibody are calculated based on the peptide fragment concentration.
- high-sensitivity analysis of a specific peptide of an antibody drug can be performed by purifying with a cation exchange resin (WCX and MCX), for example, following protease digestion.
- a cation exchange resin WCX and MCX
- the peptide fragment to be detected in the method of the present invention has an amino acid sequence containing an amino acid derived from the CDR2 region of the heavy chain or light chain of a monoclonal antibody such as an antibody drug.
- the method of the present invention it is also possible to detect and quantify antibodies by simultaneously detecting a plurality of predicted peptide fragments in parallel. However, it is preferable to select an optimal peptide fragment for each antibody in order to make the measurement easier and to reduce the time and cost required for the measurement. If the optimal peptide fragment is known, mass spectrometry conditions suitable only for the detection of the peptide fragment can be determined, and such information can be provided.
- the method of the present invention includes, as one embodiment, selecting a peptide fragment containing an amino acid in the CDR2 region suitable for quantification of individual monoclonal antibodies.
- Another embodiment of the present invention also provides a method comprising detecting a peptide fragment comprising an amino acid of a CDR2 region selected as suitable for quantification of an individual monoclonal antibody.
- the peptide predicted to be suitable for mass spectrometry is SEQ ID NO: 3 Is a peptide represented by ⁇ 9.
- these peptides were unable to perform concentration-dependent quantitative detection in serum samples, possibly due to endogenous antibody interference.
- FSGVPDR (SEQ ID NO: 3) FTISR (SEQ ID NO: 4) FSGSGTDFTLK (SEQ ID NO: 5) NSLYLQMNSLR (SEQ ID NO: 6) HSDGNFAFGYWGQGTLVTVSSASTK (SEQ ID NO: 7) GLEWVATISSASTYSYYPDSVK (SEQ ID NO: 8) NIVHINGDTYLEWYLQPKGQSPQLLIYK (SEQ ID NO: 9)
- SYYPDSVK (SEQ ID NO: 10) GMSWVR (SEQ ID NO: 11) ISRVEAEDVGVY (SEQ ID NO: 12) YLQKPGQSPQLLIYK (SEQ ID NO: 13) NIVHINGDTYLEW (SEQ ID NO: 14)
- the monoclonal antibody to be measured is mogamulizumab
- detection can be performed by multiple reaction monitoring using a triple quadrupole mass spectrometer (for example, LCMS-8050, Shimadzu Corporation) using the conditions described in Table 1 as an index.
- the present invention is a kit for quantitative detection of a monoclonal antibody by high performance liquid chromatography mass spectrometry (LC-MS) as described above, A porous body for immobilizing the monoclonal antibody to be measured; Microparticles immobilized with protease having trypsin activity and chymotrypsin activity; A reaction vessel for selectively digesting monoclonal antibodies by contacting the porous body and the fine particles; A buffer solution that is introduced into the reaction vessel together with the fine particles and the porous body, and causes a digestion reaction with the protease; The kit.
- LC-MS liquid chromatography mass spectrometry
- Measures by mass spectrometry can be performed with very high accuracy, while appropriate sample preparation and setting of appropriate analysis conditions are very important.
- the present invention provides a kit that can be used to carry out the above-described method so that accurate test results can be obtained more easily in a clinical setting.
- the buffer solution contained in the kit of the present invention is introduced into the reaction container together with the fine particles and the porous body, and is used for digestion reaction with the protease, and has reaction conditions suitable for protease digestion. It is to provide.
- the reaction conditions can be appropriately determined depending on the selected protease and the like, and the composition of the buffer solution can also be appropriately determined.
- the kit of the present invention can also include a filtration membrane for removing the porous body and the fine particles after the protease digestion reaction and extracting the product of the digestion reaction together with the buffer solution. This is because it is necessary to remove the porous material and fine particles in order to use the target peptide fragment obtained by protease digestion for mass spectrometry.
- the filtration membrane functions as a “bottom of the reaction vessel” with almost no permeation of the buffer and the peptide produced by protease digestion under conditions where no pressure or centrifugal force is applied, and the buffer is used for operations such as centrifugation. It preferably functions as a “monkey” that can permeate liquid and peptides.
- the centrifugation conditions to be added because the filtration membrane can permeate the buffer solution and the peptide are not limited, but for example, a range of 3,000 to 10,000 g is preferable.
- the filtration membrane that can be suitably used in the kit of the present invention include a filtration membrane made of polyvinylidene fluoride (PVDF) (Low-binding hydrophilic PVDF, pore size 0.2 ⁇ m, manufactured by Millipore).
- PVDF polyvinylidene fluoride
- a filtration membrane is made of PVDF
- a housing material is polyacrylonitrile resin, for example, Barex (registered trademark) (made by Mitsui Chemicals Fine Co., Ltd.) can do.
- the kit of the present invention can also include instructions describing the method of using the kit and / or mass spectrometric conditions for detection of monoclonal antibodies.
- the kit of the present invention can also contain one or more internal standard peptides.
- the internal standard peptide provides more reliable analysis results by analyzing under the same conditions simultaneously with the sample or separately.
- the internal standard peptide includes a specific amino acid sequence of the monoclonal antibody to be measured, and is a peptide generated by digestion with a protease included in the kit of the present invention.
- the internal standard peptides may include those labeled with stable isotope amino acids. In that case, the mass spectrometric quantification conditions differ from those of peptides that do not contain isotopes. preferable.
- the internal standard peptide can include a peptide having the amino acid sequence of SEQ ID NO: 10.
- peptide fragment preparation for monoclonal antibody identification and quantification can be performed more easily, and automation with an apparatus can be easily achieved.
- the protease is provided as a component of the kit in a state where it is immobilized on the surface of the microparticles, the operation of preparing the peptide fragment can be further simplified.
- the present invention is also a computer-readable recording medium on which data for performing mass spectrometry is recorded for use in the method of the present invention, wherein the data is obtained by protease digestion of the monoclonal antibody.
- Monoclonal antibody by liquid chromatograph mass spectrometry (LC-MS) containing the recording medium, including the parent ion, fragment ion, and voltage applied to the electrode of the mass spectrometer for the peptide, and the recording medium Provides a method package for quantitative detection of.
- the “method package” refers to a package that includes liquid chromatograph mass spectrometry analysis conditions for a specific measurement target in a readable form and can be circulated independently. By importing the data included in the method package into LC-MS, it is possible to analyze under the optimal measurement conditions obtained after detailed examination.
- the method package may include instructions for using the recording medium.
- the present applicant has provided method packages for analysis by LC-MS in order to allow users to perform mass spectrometry of agricultural chemicals and veterinary drugs more easily. Therefore, the present invention also provides a method package for identifying and quantifying monoclonal antibodies in a sample by LC-MS.
- the present invention allows selective protease digestion of a monoclonal antibody by contacting a porous body in which the monoclonal antibody to be measured is immobilized in the pores with microparticles in which a protease having trypsin activity and chymotrypsin activity is immobilized. And data for performing the mass spectrometry was recorded for use in the method for detecting the monoclonal antibody by analyzing the obtained peptide fragments by liquid chromatography mass spectrometry (LC-MS).
- LC-MS liquid chromatography mass spectrometry
- a computer-readable recording medium wherein the data includes, but is not limited to, for example, at least a parent ion for a peptide having an amino acid sequence including an amino acid of CDR2 region obtained by protease digestion of the monoclonal antibody, Fragment ion, mass spectrometer power ,
- each of the triple quadrupole contains data of the voltage applied to provide the recording medium.
- the recording medium can also include additional data such as expected holding time.
- the expected holding time, voltage data, and the like described above are numerical values that vary depending on the equipment used, measurement conditions, etc., and are preferably provided according to the equipment. Further, as will be understood by those skilled in the art, it is preferable to provide the fluctuation range of the numerical value that varies depending on the conditions.
- the recording medium may be in any form and is not particularly limited.
- a disk or memory capable of recording information magnetically and optically can be mentioned.
- the above method package may include the following information and software functions, for example.
- optimized ⁇ been parent ion m / z values and optimized fragment ion m / z values, optimized Q1 pre bias voltage-Optimized Q2 collision energy voltage-Optimized Q3 pre Bias voltage value • Expected ion retention time and mass analysis time • Quantitative value conversion formula • Analysis result report output function *: Measures each condition item, the highest ion intensity, and the most reproducible m / z Adopt a value and make it the optimal value.
- the present invention also provides a method package for detection of monoclonal antibodies by liquid chromatography mass spectrometry (LC-MS), which includes the above recording medium.
- the method package may include instructions for using the recording medium.
- Examples of the above recording medium or method package include those containing only information limited to a specific monoclonal antibody. Therefore, when the monoclonal antibody is, for example, mogamulizumab, a recording medium or method package describing conditions suitable for the analysis can be provided.
- the data included in the recording medium can relate to analysis conditions for the peptide having the amino acid sequence of SEQ ID NO: 10, for example.
- the method package may describe data common to a plurality of mass spectrometers, or may describe various data suitable for analysis by a specific mass spectrometer.
- the recording medium or method package can be provided together with the kit of the present invention described above or separately from the kit.
- Enzyme bead washing Add 25 ml of 25 mM HEPES-NaOH, 50 mM NaCl, and pH 7.0 to 200 mg of FG bead precipitates, respectively, and check the suspension of FG beads using an ice-cooled ultrasonic washer. Then, vortex continuously at the minimum speed to keep the suspension. After 5 minutes of washing, centrifuge and remove the supernatant. 7. Storage Add 25 mM Tris-HCl, pH 8.0 to a final protease concentration of 0.5 ⁇ g / ⁇ l, and check the suspension of FG beads using an ice-cooled ultrasonic washer. Dispense into. Store at -80 ° C.
- Protease digestion of monoclonal antibodies in plasma samples was performed by the following steps. 1. Recovery of monoclonal antibody from plasma Take 20 ⁇ l of plasma and dilute with 180 ⁇ l PBS + 0.1% n-octyl- ⁇ -D-thioglycoside (Dojindo Laboratories). 40 ⁇ l of 50% suspension of Protein G Ultralink resin (Pierce) is added here. Vortex gently at room temperature for 1-2 hours or rotate with a rotary mixer to bind antibody molecules in plasma to the resin. The resin is precipitated by centrifugation and the supernatant is discarded.
- FIG. 1 shows the amino acid sequences of heavy and light chains of mogarizumab (SEQ ID NOs: 1 and 2), the sequence of the cleaved fragments by Trypsin Gold used for quantification (SEQ ID NOs: 3 to 9), and the amino acids of heavy or light chains. Indicates the position in the sequence.
- Fig. 2 shows the results of applying the Trypsin Gold cleaved fragment of mogarizumab (SEQ ID NOs: 3 to 9) to the MRM chromatogram under the above analysis conditions.
- SEQ ID NOs: 3 to 9 Trypsin Gold cleaved fragment of mogarizumab
- Fig. 3 shows the results of creating a calibration curve based on the peaks detected with the Trypsin Gold digested fragments (SEQ ID NOs: 3 to 9) spiked in plasma.
- SEQ ID NOs: 3 to 9 the Trypsin Gold digested fragments
- FIG. 3 shows the results of creating a calibration curve based on the peaks detected with the Trypsin Gold digested fragments (SEQ ID NOs: 3 to 9) spiked in plasma.
- FIG. 4 shows the sequences of Trypsin TPCK cleavage fragments (SEQ ID NOs: 10 to 14) used for quantification of mogarizumab and the positions in the heavy and light chain amino acid sequences.
- FIG. 5 shows the result of analyzing the trypsin TPCK cleavage fragment (SEQ ID NOs: 10 to 14) of mogarizumab under the same conditions as described above. From the results of FIG. 5, peaks derived from 5 types of peptide fragments were confirmed both when the mogalizumab bulk powder was analyzed as it was (upper figure) and when mogalizumab was spiked into the plasma (lower figure).
- a calibration curve was prepared using the Trypsin TPCK cleaved fragment spiked in plasma (SEQ ID NO: 10 to 14). As shown in FIG. 6, a suitable calibration for quantitative detection with the peptide SYYPDSVK (SEQ ID NO: 10). A line was obtained and found to be optimal for quantification of mogarizumab.
- FIG. 7 shows the results of selecting the peptide SYYPDSVK (SEQ ID NO: 10) as a mogarizumab target peptide in accurate mass analysis and performing structure identification by accurate mass analysis. From the result of FIG. 7, it was confirmed that the structure as predicted from the peptide sequence can be identified.
- the detected peptide SYYPDSVK (SEQ ID NO: 10) was derived from the Fv region and was confirmed to be located on the outermost side in the three-dimensional structure of the human immunoglobulin molecule obtained in ProteinProData Bank. .
- the antibody in the sample can be quantified by selectively digesting the antibody using a specific protease and mass-analyzing the obtained peptide fragment.
- the method of the present invention is easy to operate and can ensure reproducibility and quantitativeness.
- the present invention can provide an extremely versatile analytical method for such a wide variety of antibody drugs, and can contribute to the acceleration of the development of antibody drugs in the future.
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Abstract
Description
(1)以下のステップ:
測定対象のモノクローナル抗体を細孔内に固定化した多孔質体と、プロテアーゼを固定化した、平均粒径が多孔質体の平均細孔径よりも大きい微粒子とを接触させてモノクローナル抗体の選択的プロテアーゼ消化を行うステップ、及び
前記消化によって得られた、前記モノクローナル抗体の重鎖又は軽鎖のCDR2領域由来のアミノ酸を含むペプチド断片を検出するステップ
を含む、モノクローナル抗体の定量方法であって、前記プロテアーゼとして、トリプシン活性及びキモトリプシン活性を有するプロテアーゼを使用することを特徴とする、上記方法。
(2)トリプシン活性及びキモトリプシン活性を有するプロテアーゼが、還元メチル化処理を行っていないトリプシンである、(1)記載の方法。
(3)前記モノクローナル抗体がモガリズマブである、(1)又は(2)記載の方法。
(4)前記ペプチド断片が配列番号10に示すアミノ酸配列を有するペプチドである、(3)記載の方法。
(5)液体クロマトグラフ質量分析(LC-MS)によるモノクローナル抗体の定量的検出のためのキットであって、
測定対象のモノクローナル抗体を固定化するための多孔質体と、
トリプシン活性及びキモトリプシン活性を有するプロテアーゼを固定化した微粒子と、
前記多孔質体と前記微粒子とを接触させてモノクローナル抗体を選択的に消化するための反応容器と、
前記微粒子及び多孔質体と共に前記反応容器内に導入され、前記プロテアーゼによる消化反応をさせるための緩衝液と、
を含む、上記キット。
(6)トリプシン活性及びキモトリプシン活性を有するプロテアーゼが、還元メチル化処理を行っていないトリプシンである、(5)記載のキット。
(7)測定対象がモガリズマブであって、内部標準ペプチドが配列番号10に示すアミノ酸配列を有するペプチドである、(5)又は(6)記載のキット。
(8)(1)~(4)のいずれか記載の方法に使用するための、質量分析を実行させるためのデータが記録されたコンピュータ読み取り可能な記録媒体であって、前記データが、前記モノクローナル抗体のプロテアーゼ消化によって得られるペプチドについての、親イオン、フラグメントイオン、及び質量分析計の電極に印加する電圧のデータを含む、上記記録媒体。
(9)(8)記載の記録媒体と、前記記録媒体の使用説明書とを含む、液体クロマトグラフ質量分析(LC-MS)によるモノクローナル抗体の定量的検出のためのメソッドパッケージ。
測定対象のモノクローナル抗体を細孔内に固定化した多孔質体と、プロテアーゼを固定化した、平均粒径が多孔質体の平均細孔径よりも大きい微粒子とを接触させてモノクローナル抗体の選択的プロテアーゼ消化を行うステップ、及び
前記消化によって得られた、前記モノクローナル抗体の重鎖又は軽鎖のCDR2領域由来のアミノ酸を含むペプチド断片を検出するステップ
を含む、モノクローナル抗体の定量方法であって、前記プロテアーゼとして、トリプシン活性及びキモトリプシン活性を有するプロテアーゼを使用することを特徴とする、上記方法に関する。
[質量分析]
質量分析により抗体を検出するためには、まず生体試料より抗体を抽出し、適切な溶媒に溶解することが必要である。また、抗体はそのまま分析するには分子が大きいため、プロテアーゼによりペプチドに分解し、その後液体クロマトグラフで分離した後に質量分析を行う。分析に適したペプチドの分子量は約1000~3000 Da程度である。
本発明の方法における測定対象はモノクローナル抗体である。モノクローナル抗体の重鎖及び軽鎖はそれぞれ定常領域と可変領域から成る。定常領域は、同一種由来の抗体のほとんどで共通するアミノ酸配列を有している。一方、可変領域には、相補性決定領域(CDR)が各3つずつ(CDR1、CDR2、CDR3)存在する。これらの領域が規定する立体構造が抗原との特異的結合に関わっており、それによって抗体-抗原複合体が形成される。
本発明の方法に使用する多孔質体は、多数の細孔を有するものであれば、その材料は特に限定されず、活性炭、多孔質膜、多孔質樹脂ビーズ、金属粒子等を用いることができる。これらの中でも、抗体を部位特異的に結合可能なものが好ましい。
抗体を多孔質体の細孔内に固定化する方法は特に限定されず、抗体と多孔質体あるいはリンカー分子の特性等に応じて適宜の方法を採用できる。例えば、細孔内にprotein Aやprotein Gが固定化された多孔質体に抗体を固定化する場合は、多孔質体の懸濁液と抗体を含む溶液とを混合することにより、細孔内に抗体を容易に固定化できる。
本発明の方法において、微粒子は、その表面にプロテアーゼを固定化して、多孔質体の細孔内に固定化された抗体へのプロテアーゼのアクセスを制御する目的で用いられる。そのため、微粒子は、多孔質体の細孔の奥深くまで入り込まないように、その平均粒径D1が、多孔質体の平均細孔径D2よりも大きいものとする。
本発明の方法では、微粒子に固定化させるプロテアーゼとして、トリプシン活性及びキモトリプシン活性を有するプロテアーゼを用いる。
プロテアーゼを微粒子の表面に固定化する方法は特に限定されず、プロテアーゼと微粒子(あるいは微粒子表面を修飾するスペーサ分子)の特性等に応じて適宜の方法を採用でき、例えば、プロテアーゼをスペーサ修飾された微粒子表面に固定化する場合は、微粒子の懸濁液とプロテアーゼを含む溶液とを混合することにより、微粒子表面にプロテアーゼを固定化できる。上記のスペーサ分子の官能基を介した微粒子とプロテアーゼのアミンカップリング法が好ましい。例えば、微粒子に表面修飾したカルボキシル基をN-ヒドロキシスクシンイミド(NHS)でエステル化して活性化エステル基とし、これに、プロテアーゼのアミノ基を結合させることができる。このカップリング反応には、1-エチル-3-(3-ジメチルアミノプロピル)カルボジイミド(EDAC)、N,N'-ジシクロヘキシルカルボジイミド(DCC)、ビス(2,6-ジイソプロピルフェニル)カルボジイミド(DIPC)等のカルボジイミドを縮合剤の存在下に行うことができる。また、微粒子に表面修飾したアミノ基に、グルタルアルデヒド、2官能性スクシンイミド、ビス(スルホスクシンイミジル)スベレート(BS3)、スルホニルクロリド、マレイミド、ピリジルジスルフィド等の架橋剤を用いてプロテアーゼのアミノ基を結合させてもよい。
抗体が固定化された多孔質体と、プロテアーゼが表面に固定化された微粒子とを接触させることにより、抗体がプロテアーゼ消化され、ペプチド断片が産生される。接触は、液体中で行うことが好適である。ここで、「液体」とは、基質(固相)及び酵素(固相)が液相中で接触することを意味するものであり、またプロテアーゼ消化反応に適した水性媒体を意図する。
上記で得られたペプチド断片を含む試料を、LC-MSにより分析することで、抗体の同定や定量を行い得る。
本発明の方法における検出対象のペプチド断片は、抗体医薬等のモノクローナル抗体の重鎖又は軽鎖のCDR2領域由来のアミノ酸を含むアミノ酸配列を有するものである。
FTISR(配列番号4)
FSGSGSGTDFTLK(配列番号5)
NSLYLQMNSLR(配列番号6)
HSDGNFAFGYWGQGTLVTVSSASTK(配列番号7)
GLEWVATISSASTYSYYPDSVK(配列番号8)
NIVHINGDTYLEWYLQPKGQSPQLLIYK(配列番号9)
GMSWVR(配列番号11)
ISRVEAEDVGVY(配列番号12)
YLQKPGQSPQLLIYK(配列番号13)
NIVHINGDTYLEW(配列番号14)
本発明は、上記した高速液体クロマトグラフ質量分析(LC-MS)によるモノクローナル抗体の定量的検出のためのキットであって、
測定対象のモノクローナル抗体を固定化するための多孔質体と、
トリプシン活性及びキモトリプシン活性を有するプロテアーゼを固定化した微粒子と、
前記多孔質体と前記微粒子とを接触させてモノクローナル抗体を選択的に消化するための反応容器と、
前記微粒子及び多孔質体と共に前記反応容器内に導入され、前記プロテアーゼによる消化反応をさせるための緩衝液と、
を含む、上記キットに関する。
本発明はまた、本発明の方法に使用するための、質量分析を実行させるためのデータが記録されたコンピュータ読み取り可能な記録媒体であって、前記データが、前記モノクローナル抗体のプロテアーゼ消化によって得られるペプチドについての、親イオン、フラグメントイオン、及び質量分析計の電極に印加する電圧のデータを含む、上記記録媒体、並びに、上記記録媒体を含む、液体クロマトグラフ質量分析(LC-MS)によるモノクローナル抗体の定量的検出のためのメソッドパッケージを提供する。
・最適化されたフラグメントイオンm/z値
・最適化されたQ1 pre bias電圧値
・最適化されたQ2 collision energy電圧値
・最適化されたQ3 pre bias電圧値
・目的イオンの予想保持時間および質量分析時間
・定量値換算式
・解析結果レポート出力機能
※:各条件項目を実測し、最もイオン強度の高いもの、および最も再現性のあるm/z値を採択し、これを最適値とする。
以下のステップによって微粒子にプロテアーゼを固定化した。
1. FGビーズ洗浄
FGビーズNHS 200 mg(多摩川精機社製)を遠心分離(15,000 g X 5 分, 4 ℃)し、保存用上清イソプロパノールを除去する。上清は、沈殿しない浮遊物も含め、慎重に除去する。
2. 酵素準備
Trypsin Gold 1 mg(プロメガ社製)又はTrypsin TPCK 5 mg(シグマアルドリッチ社製)を開封し、4 ℃に冷却した25 mM HEPES-NaOH, pH7.0に溶解する。溶解後、50 ml遠沈管に取り、氷上に静置する。酵素は25 mM HEPES-NaOH, pH7.0を用いて1回共洗いし、できる限り回収する。最終緩衝液量は25 ml程度となる。
3. FGビーズ洗浄
それぞれ10 mlの氷冷したメタノールを加え、氷冷した超音波洗浄機の中で、FGビーズの懸濁を確認してから遠心分離(15,000 g X 5 分, 4 ℃)し、上清メタノールを除去する。
4. 酵素固定化反応
FGビーズの沈殿200 mgに、Trypsin Gold又はTrypsin TPCK溶液を加える。氷冷した超音波洗浄機を用いてFGビーズの懸濁を確認してから、懸濁を保てる最低限のスピードで30 分間連続してボルテックスを行う。
30分間反応させた後、遠心分離(15,000 g X 5 分, 4 ℃)を行い、上清を除去する。上清を回収し、BCAアッセイによりカップリング効率確認試験を行う。
5. 活性基ブロック
FGビーズの沈殿200 mgに、それぞれ25 mlの1M 2-アミノエタノール塩酸塩、pH8.0を加え、氷冷した超音波洗浄機を用いFGビーズの懸濁を確認してから、懸濁を保てる最低限のスピードで連続ボルテックスを行う。30分間反応させてブロッキングした後、遠心分離を行い、上清を除去する。
6. 酵素ビーズ洗浄
FGビーズの沈殿200 mgに、それぞれ25 mlの25 mM HEPES-NaOH、50 mM NaCl、pH7.0を加え、氷冷した超音波洗浄機を用いFGビーズの懸濁を確認してから、懸濁を保てる最低限のスピードで連続ボルテックスを行う。洗浄5分後、遠心分離を行い、上清を除去する。
7. 保存
プロテアーゼ終濃度0.5 μg/μlとなるように25 mM Tris-HCl、pH8.0を加え、氷冷した超音波洗浄機を用いてFGビーズの懸濁を確認してから、それぞれ500 μlに分注する。その後-80 ℃で保存する。
血漿試料中のモノクローナル抗体のプロテアーゼ消化は以下のステップによって実施した。
1. 血漿からのモノクローナル抗体の回収
血漿20μlをとり、180 μlのPBS + 0.1 % n-オクチル-β-D-チオグリコシド(同仁化学)で希釈する。
ここにProtein G Ultralink樹脂(Pierce社製)の50%懸濁液を40μl加える。室温で1~2時間緩やかにボルテックス、もしくはロータリーミキサーで回転させることで、血漿中の抗体分子を樹脂に結合させる。遠心分離により樹脂を沈殿させ、上清を捨てる。
200μlのPBS + 0.1 % n-オクチル-β-D-チオグリコシドを添加して3回洗浄することで、樹脂に非特異的に結合した血漿中タンパク質を洗浄する。次いで、200μlのPBSで1回洗浄し、界面活性剤を除去する。
2. プロテアーゼ反応
Protein G Ultralink樹脂が残っているチューブに、200μlの25 mM Tris-HCl、pH8.0を加える。次いで、これに上記で調製したトリプシン(Trypsin Gold又はTrypsin TPCK)を固定化したビーズを80μl加え、ローテータ-にセットし、37℃で6時間ゆっくり回転する。
反応後、0.2 μmの低結合性親水性PVDF膜(ミリポア社製)でろ過することで、Protein G樹脂とトリプシンを固定化したビーズを共に除去し、反応溶液を回収する。
市販の血漿(シグマ社製)に抗体医薬をスパイクし、プロテアーゼ消化によって得られるペプチドを検出した。モノクローナル抗体として、モガムリズマブ(医薬品名ポテリジオ、協和発酵キリン)を用いた。質量分析の条件は以下の通りである。
<HPLC条件(Nexera LC30A液体クロマトグラフシステム)>
溶媒A:0.1% ギ酸、溶媒B:0.1% ギ酸+アセトニトリル
流速:0.5 ml/分
グラジェント時間:1%B(1.5分)、1-40%Bグラジェント(5分)、95%B(5分)、1%B(5分)
カラム:L-column2 ODS, 2 x 50 mm(化学物質評価研究機構)
カラム温度:50℃
<MSインターフェイス条件(LCMS-8050(島津製作所))>
ネブライザーガス:3 L/分
ヒーティングガス:10 L/分
ドライイングガス:10 L/分
インターフェイス温度:300℃
DL温度:250℃
ヒートブロック温度:400℃
図1に、モガリズマブの重鎖及び軽鎖のアミノ酸配列(配列番号1及び2)、及び定量用に使用したTrypsin Goldによる切断断片の配列(配列番号3~9)及び重鎖又は軽鎖のアミノ酸配列中の位置を示す。
図4は、モガリズマブの定量用に使用したTrypsin TPCK切断断片の配列(配列番号10~14)、及び重鎖及び軽鎖のアミノ酸配列中の位置を示す。モガリズマブのTrypsin TPCK切断断片(配列番号10~14)を上記と同様の条件で分析した結果を図5に示す。図5の結果から、モガリズマブ原末をそのまま分析した場合(上図)、血漿中にモガリズマブをスパイクした場合(下図)のいずれにおいても、5種のペプチド断片に由来するピークが確認された。
Claims (9)
- 以下のステップ:
測定対象のモノクローナル抗体を細孔内に固定化した多孔質体と、プロテアーゼを固定化した、平均粒径が多孔質体の平均細孔径よりも大きい微粒子とを接触させてモノクローナル抗体の選択的プロテアーゼ消化を行うステップ、及び
前記消化によって得られた、前記モノクローナル抗体の重鎖又は軽鎖のCDR2領域由来のアミノ酸を含むペプチド断片を検出するステップ
を含む、モノクローナル抗体の定量方法であって、前記プロテアーゼとして、トリプシン活性及びキモトリプシン活性を有するプロテアーゼを使用することを特徴とする、上記方法。 - トリプシン活性及びキモトリプシン活性を有するプロテアーゼが、還元メチル化処理を行っていないトリプシンである、請求項1記載の方法。
- 前記モノクローナル抗体がモガリズマブである、請求項1又は2記載の方法。
- 前記ペプチド断片が配列番号10に示すアミノ酸配列を有するペプチドである、請求項3記載の方法。
- 液体クロマトグラフ質量分析(LC-MS)によるモノクローナル抗体の定量的検出のためのキットであって、
測定対象のモノクローナル抗体を固定化するための多孔質体と、
トリプシン活性及びキモトリプシン活性を有するプロテアーゼを固定化した微粒子と、
前記多孔質体と前記微粒子とを接触させてモノクローナル抗体を選択的に消化するための反応容器と、
前記微粒子及び多孔質体と共に前記反応容器内に導入され、前記プロテアーゼによる消化反応をさせるための緩衝液と、
を含む、上記キット。 - トリプシン活性及びキモトリプシン活性を有するプロテアーゼが、還元メチル化処理を行っていないトリプシンである、請求項5記載のキット。
- 測定対象がモガリズマブであって、内部標準ペプチドが配列番号10に示すアミノ酸配列を有するペプチドである、請求項5又は6記載のキット。
- 請求項1~4のいずれか1項記載の方法に使用するための、質量分析を実行させるためのデータが記録されたコンピュータ読み取り可能な記録媒体であって、前記データが、前記モノクローナル抗体のプロテアーゼ消化によって得られるペプチドについての、親イオン、フラグメントイオン、及び質量分析計の電極に印加する電圧のデータを含む、上記記録媒体。
- 請求項8記載の記録媒体を含む、液体クロマトグラフ質量分析(LC-MS)によるモノクローナル抗体の定量的検出のためのメソッドパッケージ。
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