WO2018180205A1 - 免疫グロブリン精製用アフィニティー分離マトリックス - Google Patents
免疫グロブリン精製用アフィニティー分離マトリックス Download PDFInfo
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- WO2018180205A1 WO2018180205A1 PCT/JP2018/008051 JP2018008051W WO2018180205A1 WO 2018180205 A1 WO2018180205 A1 WO 2018180205A1 JP 2018008051 W JP2018008051 W JP 2018008051W WO 2018180205 A1 WO2018180205 A1 WO 2018180205A1
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
- B01D15/3828—Ligand exchange chromatography, e.g. complexation, chelation or metal interaction chromatography
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
- B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
- B01J20/3204—Inorganic carriers, supports or substrates
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
- B01J20/3206—Organic carriers, supports or substrates
- B01J20/3208—Polymeric carriers, supports or substrates
- B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
- B01J20/3206—Organic carriers, supports or substrates
- B01J20/3208—Polymeric carriers, supports or substrates
- B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3268—Macromolecular compounds
- B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
- B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/34—Regenerating or reactivating
- B01J20/3433—Regenerating or reactivating of sorbents or filter aids other than those covered by B01J20/3408 - B01J20/3425
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/34—Regenerating or reactivating
- B01J20/345—Regenerating or reactivating using a particular desorbing compound or mixture
- B01J20/3475—Regenerating or reactivating using a particular desorbing compound or mixture in the liquid phase
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- 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
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
Definitions
- the present invention relates to an affinity separation matrix having an immunoglobulin-binding peptide with improved chemical stability against an alkaline solution as a ligand, and a method for producing an antibody or antibody fragment using the affinity separation matrix.
- a protein A affinity separation matrix (hereinafter referred to as “SpA”) is used for purifying (capturing) antibody drugs from animal cell cultures at a high purity at a time. (May be abbreviated as ")" (Non-Patent Documents 1 and 2).
- SpA protein A affinity separation matrix
- Monoclonal antibodies are basically developed as antibody drugs, and are produced in large quantities using recombinant cultured cell technology.
- “Monoclonal antibody” refers to an antibody obtained from a clone derived from a single antibody-producing cell.
- Most antibody drugs currently on the market are immunoglobulin G (IgG) subclass in terms of molecular structure.
- SpG protein G
- group G streptococci Streptococcus sp.
- SpG affinity separation matrix product immobilized as a ligand (manufactured by GE Healthcare, product name “Protein-G Sepharose 4 Fast Flow”, Patent Document 1).
- SpG is characterized in that it binds strongly to the Fc region of IgG and binds strongly to IgG of a wider range of animal species than SpA.
- Non-Patent Documents 3 and 4 it has been found that it binds to the Fab region even though it is weak (Non-Patent Documents 3 and 4), and efforts have been made to improve the binding force to the Fab region (Patent Documents 2 to 4, Non-Patent Document 5). .
- the SpA affinity separation matrix is more widely used for purification of antibody drugs, and one of the reasons is that SpA has higher stability to alkaline solution than SpG (non-patented). Reference 1). If the stability against alkali is high, the affinity separation matrix can be regenerated and reused by washing with an aqueous sodium hydroxide solution that has a high cleaning and sterilizing effect and is inexpensive.
- protein engineering techniques such as introducing amino acid mutations. Specifically, amino acid substitution mutations to asparagine residues known to be susceptible to deamidation reactions under alkaline conditions and glycine residues after asparagine residues are effective in improving chemical stability.
- Non-Patent Document 1 For all asparagine residues in SpA, such a mutation does not show an improvement effect (Non-patent Documents 1 and 6).
- Non-patent Documents 7 and 8 studies have been made to introduce mutations into asparagine residues (Patent Document 5, Non-Patent Documents 7 and 8), but not all mutations are effective as in SpA. In addition, there is still room for improvement because the alkali stability comparable to that of SpA has not been achieved.
- the inventor of the present invention has a high amino acid sequence identity with the immunoglobulin binding domain of SpG, but the protein amino acid sequence (Jonsson H, Lindmark H, Guss B., Infect Immun., 1995, vol. 8, 2968-75), by evaluating the physical properties / functions of those amino acid sequences using protein engineering methods and genetic engineering methods, The present invention has been completed. Hereinafter, the present invention will be described.
- An immunoglobulin-binding peptide comprising an amino acid sequence defined by SEQ ID NO: 1 or an amino acid sequence having 94% or more sequence identity with the amino acid sequence defined by SEQ ID NO: 1 and a water-insoluble carrier, An affinity separation matrix, wherein an immunoglobulin-binding peptide is immobilized as a ligand on a water-insoluble carrier.
- the immunoglobulin-binding peptide is a deletion, substitution and / or addition of one or several amino acids located at the N-terminal and / or C-terminal.
- the affinity separation matrix according to the above [1] comprising an amino acid sequence obtained.
- [5] A method for producing a protein comprising an Fc region and / or Fab region of an immunoglobulin, Contacting the affinity separation matrix according to any one of [1] to [4] above with a liquid sample containing the protein, and separating the protein bound to the affinity separation matrix from the affinity separation matrix.
- the chromatographic support for affinity purification on which the peptide obtained in the present invention is immobilized has a small decrease in immunoglobulin binding activity due to alkali treatment damage. Therefore, in repeated use, cleaning with a sodium hydroxide aqueous solution at a high concentration or for a long time is possible.
- An affinity separation matrix comprises an amino acid sequence defined by SEQ ID NO: 1, or an immunoglobulin-binding peptide comprising an amino acid sequence having 94% or more sequence identity with the amino acid sequence defined by SEQ ID NO: 1
- a water-insoluble carrier is included, and the immunoglobulin-binding peptide is immobilized on the water-insoluble carrier as a ligand.
- the sequence of SEQ ID NO: 1 is shown below.
- Immunoglobulin is a glycoprotein produced by B cells of lymphocytes and has a function of recognizing and binding molecules such as specific proteins.
- An immunoglobulin has a function of specifically binding to a specific molecule called an antigen, and a function of detoxifying and removing a factor having an antigen in cooperation with other biomolecules and cells.
- Immunoglobulin is generally called “antibody”, which is a name that focuses on such a function. All immunoglobulins basically have the same molecular structure, and each has a basic structure of a four-chain “Y” -shaped structure composed of two light chain and heavy chain polypeptides.
- Immunoglobulin G is a monomeric immunoglobulin and is composed of two heavy chains ( ⁇ chains) and two light chains, and has two antigen-binding sites.
- the place corresponding to the vertical bar of the lower half of the “Y” of immunoglobulin is called the Fc region, and the “V” of the upper half is called the Fab region.
- the Fc region has an effector function that induces a reaction after the antibody binds to the antigen, and the Fab region has a function of binding to the antigen.
- the heavy chain Fab region and the Fc region are connected by a hinge part, and the proteolytic enzyme papain contained in papaya decomposes this hinge part and cleaves it into two Fab regions and one Fc region.
- the domain near the tip of the “Y” is called a variable region (V region) because various changes in the amino acid sequence are seen so that it can bind to various antigens.
- variable region of the light chain is called the VL region
- variable region of the heavy chain is called the VH region
- the Fab region and the Fc region other than the V region are regions with relatively little change, and are called constant regions (C regions).
- the constant region of the light chain is referred to as the CL region
- the constant region of the heavy chain is referred to as the CH region.
- the CH region is further divided into three, CH1 to CH3.
- the heavy chain Fab region consists of a VH region and CH1, and the heavy chain Fc region consists of CH2 and CH3.
- the hinge part is located between CH1 and CH2.
- peptide includes all molecules having a polypeptide structure, and includes not only so-called proteins, but also fragments and those in which other peptides are linked by peptide bonds. Shall be.
- a “domain” is a unit of protein conformation, consisting of a sequence of tens to hundreds of amino acid residues, sufficient to express some physicochemical or biochemical function.
- the phrase “having a (specific) amino acid sequence” means that the peptide only needs to contain the specified amino acid sequence, and the function of the peptide is maintained. To do. Examples of sequences other than the amino acid sequence specified in the peptide include a histidine tag, a linker sequence for immobilization, and a cross-linked structure such as an —SS— bond.
- the position can be specified using the alignment function of GENETYX (https://www.genetyx.co.jp/), which is genetic information processing software.
- the sequence identity required for the identification of the position is preferably 94% or more, more preferably 95% or more or 96% or more, still more preferably 98% or more or 99% or more, 99.5% or more or 99 More preferably, it is 8% or more.
- Ligand refers to a substance or functional group that selectively binds and captures a target molecule from a set of molecules based on the affinity between specific molecules represented by the binding between an antigen and an antibody.
- the term is used in the present invention to refer to a peptide that specifically binds to an immunoglobulin.
- affinity ligand is also synonymous with “ligand”.
- water-insoluble carrier used in the present invention examples include inorganic carriers such as glass beads and silica gel; organic carriers; and organic-organic and organic-inorganic composite carriers obtained by a combination thereof.
- organic carrier examples include synthetic polymer carriers such as crosslinked polyvinyl alcohol, crosslinked polyacrylate, crosslinked polyacrylamide, and crosslinked polystyrene, and polysaccharide carriers such as crystalline cellulose, crosslinked cellulose, crosslinked agarose, and crosslinked dextran.
- porous cellulose gel GCL2000 Sephacryl S-1000 covalently cross-linked allyldextran and methylenebisacrylamide
- Toyopearl acrylate carrier Sepharose CL4B agarose cross carrier
- Examples thereof include Cellufine, which is a cellulosic crosslinking carrier.
- the water-insoluble carrier in the present invention is not limited to these exemplified carriers.
- the water-insoluble carrier used in the present invention preferably has a large surface area in view of the purpose and method of use of the affinity separation matrix of the present invention, and is preferably a porous material having a large number of pores of an appropriate size.
- the form of the carrier can be any of beads, monoliths, fibers, membranes (including hollow fibers), and any form can be selected.
- the ligand may be bound to the carrier by a conventional coupling method using an amino group, a carboxy group or a thiol group present in the ligand.
- the carrier is activated by reacting the carrier with cyanogen bromide, epichlorohydrin, diglycidyl ether, tosyl chloride, tresyl chloride, hydrazine or sodium periodate, or the surface of the carrier.
- the immobilization method include addition of a reagent having a plurality of functional groups in the molecule such as glutaraldehyde, condensation, and crosslinking.
- a spacer molecule composed of a plurality of atoms may be introduced between the ligand and the carrier, or the ligand may be directly immobilized on the carrier. Therefore, for the immobilization, the immunoglobulin-binding peptide used in the affinity separation matrix according to the present invention may be chemically modified, or an amino acid residue useful for immobilization may be added.
- amino acids useful for immobilization include amino acids having functional groups useful for immobilization chemical reactions in the side chain, such as Lys containing an amino group in the side chain, and thiol groups in the side chain. Cys containing is mentioned.
- the essence of the present invention is that the immunoglobulin binding property imparted to a peptide in the present invention is similarly imparted to a matrix in which the peptide is immobilized as a ligand, and how it is modified and altered for immobilization. Even within the scope of the present invention.
- an immunoglobulin-binding peptide comprising SEQ ID NOs: 2 to 16 or an amino acid sequence showing 94% or more sequence identity with those amino acid sequences is immobilized as a ligand on a water-insoluble carrier. More preferably.
- the sequence identity is preferably 95% or more or 96% or more, more preferably 98% or more or 99% or more, and still more preferably 99.5% or more or 99.8% or more.
- An affinity separation matrix for purifying a modified immunoglobulin containing only the Fab region but not the Fc region is SEQ ID NO: 8 to 13, or immunoglobulin binding showing 94% or more sequence identity with the amino acid sequence thereof. More preferably, the peptide is immobilized on a water-insoluble carrier as a ligand.
- the sequence identity is preferably 95% or more or 96% or more, more preferably 98% or more or 99% or more, and still more preferably 99.5% or more or 99.8% or more.
- immunoglobulin-binding peptide of the affinity separation matrix In the case of the immunoglobulin-binding peptide of the affinity separation matrix according to the present invention, a case where one or several amino acids contain an amino acid sequence deleted, substituted and / or added is also included in the present invention.
- the range of “one to several” is not particularly limited as long as an immunoglobulin-binding peptide having a deletion or the like has a high binding force to immunoglobulin.
- the range of “1 to several” can be, for example, 30 or less, preferably 20 or less, more preferably 10 or less, still more preferably 7 or less, even more preferably 5 or less, especially Preferably, it can be 3 or less, 1 or more, 2 or less, and 1 or so.
- examples of the positions for deletion, substitution and / or addition of amino acid residues include the N-terminus and / or C-terminus. These sites are particularly preferred as deletion and / or addition sites.
- An embodiment of the amino acid sequence to be added includes adding an amino acid sequence containing Lys or Cys useful for immobilizing the peptide to a matrix to the C-terminal side.
- an immunoglobulin-binding peptide having the sequence homology and the mutation is also excellent in chemical stability.
- the chemical stability of the immunoglobulin-binding peptide having the sequence homology and the mutation with respect to an alkaline aqueous solution is preferably superior to the chemical stability of wild-type SpG. More preferably, it is equivalent to or better than the chemical stability of the immunoglobulin-binding peptide having a sequence.
- the binding activity to the Fc region and / or Fab region of the immunoglobulin-binding peptide having the sequence homology and the mutation is preferably superior to the binding activity of wild-type SpG, and the amino acid sequence of SEQ ID NO: 1 More preferably, it is equivalent or superior to the binding activity of the immunoglobulin-binding peptide.
- the position of the amino acid residue after mutagenesis corresponding to the position of the amino acid residue before mutagenesis is the amino acid before and after mutagenesis. It can be easily searched by performing alignment analysis of sequences. Such alignment analysis techniques are widely known to those skilled in the art.
- the affinity separation matrix obtained by the present invention is characterized by high chemical stability against an alkaline aqueous solution. That is, even if it processes with alkaline aqueous solution, there is little damage by an alkali and the immunoglobulin binding performance is maintained at the high level.
- Alkaline aqueous solution refers to alkalinity that can achieve the purpose of cleaning or sterilization. More specifically, an aqueous solution of sodium hydroxide of 0.01M to 1.0M or 0.01N to 1.0N is applicable, but not limited thereto.
- the lower limit of the concentration is preferably 10 mM, more preferably 15 mM, more preferably 20 mM, and even more preferably 25 mM.
- the upper limit of the concentration of sodium hydroxide is preferably 1.0M, more preferably 0.5M, even more preferably 0.3M, still more preferably 0.2M, and even more preferably 0.1M.
- the alkaline aqueous solution is not necessarily a sodium hydroxide aqueous solution, but the pH is preferably 12 or more and 14 or less. Regarding the lower limit of pH, 12.0 or more is preferable, and 12.5 or more is more preferable. Regarding the upper limit of the pH, it is preferably 14 or less, more preferably 13.5 or less, and even more preferably 13.0 or less.
- “Chemical stability” refers to the property that a peptide retains the function of the peptide against chemical modifications such as chemical changes of amino acid residues and chemical modifications such as amide bond transfer and cleavage.
- function retention of a peptide refers to binding activity to immunoglobulin.
- “binding activity to immunoglobulin” refers to the proportion of a polypeptide that retains affinity for immunoglobulin without undergoing chemical modification. That is, the higher the “chemical stability” is, the smaller the degree to which the binding activity to the immunoglobulin decreases after the immersion treatment in the alkaline aqueous solution.
- the term “alkali resistance” is also synonymous with “chemical stability under alkaline conditions”.
- the time for immersing the peptide in the alkali is not particularly limited because the damage to the peptide varies greatly depending on the concentration of the alkali and the temperature at the time of immersion.
- the concentration of sodium hydroxide is 15 mM and the temperature during immersion is room temperature
- the lower limit of the time for immersion in alkali is preferably 30 minutes, more preferably 1 hour, more preferably 2 hours, and more than 4 hours.
- 10 hours is more preferable, and 20 hours is more preferable, but there is no particular limitation.
- the binding activity to the antibody or fragment thereof can be tested by a biosensor such as Biacore system (GE Healthcare) using the surface plasmon resonance principle and Octet (Pall ForteBio) using biolayer interferometry.
- a biosensor such as Biacore system (GE Healthcare) using the surface plasmon resonance principle and Octet (Pall ForteBio) using biolayer interferometry.
- the present invention is not limited to this.
- the temperature is a constant temperature of 20 ° C. or more and 40 ° C. or less
- the pH when the bonding state is seen is a neutral condition of 5 or more and 8 or less.
- the buffer component include, but are not limited to, phosphoric acid, tris, bistris, and the like when neutral.
- the sodium chloride concentration of the buffer solution is not particularly limited, but is preferably about 0 M or more and 0.15 M or less.
- an affinity constant (K A ) or dissociation constant (K D ) can be used as a parameter indicating that it is bound to an antibody or a fragment thereof (Nagata et al., “Real-time analysis of biological substance interaction” Law “, Springer Fairlark Tokyo, 1998, page 41).
- the affinity constant for the antibody of the peptide of the present invention or a fragment thereof can be determined by, for example, using the Octet system, immobilizing human IgG or a fragment thereof on a biosensor, under the conditions of a temperature of 25 ° C. and a pH of 7.4. It can be determined in an experimental system in which a domain mutant is added to the channel.
- the immunoglobulin-binding peptide used in the affinity separation matrix according to the present invention has an affinity constant (K A ) for human immunoglobulin of 1 ⁇ 10 5 (M ⁇ 1 ) or more, more preferably 1 ⁇ 10 6 (M ⁇ 1 ) It is preferable that it is above.
- K A affinity constant
- the affinity constant varies depending on the type of immunoglobulin and the number of domains of the immunoglobulin-binding peptide, and is not limited to this.
- K A and the K D is inappropriate. This is because, even when the ratio of molecules capable of binding to the antibody or fragment thereof is changed by alkali treatment, if the binding ability of one peptide molecule to the antibody or fragment thereof does not change, no change is seen as a parameter.
- the antibody or fragment thereof is immobilized on a biosensor, and the binding signal when the same concentration of antibody or fragment thereof is added before and after chemical treatment of the peptide. It is preferable to use a binding parameter in units of binding response (nm), which is the size or the theoretical maximum binding capacity (R max ), but is not limited thereto.
- the magnitude of the binding signal may be compared by immobilizing the peptide and adding the same concentration of antibody or fragment thereof before and after alkali treatment of the immobilized chip.
- the residual binding activity is a comparison before and after alkali treatment, it can be basically expressed as a ratio (percentage) in which the binding activity before alkali treatment is the denominator and the binding activity after alkali treatment is the molecule.
- the numerical value is not particularly limited as long as it is higher than that of the peptide in which the mutation of the present invention treated with alkali under the same conditions is not introduced, but the ratio is preferably 10% or more, and more preferably 20% or more. Is more preferably 30% or more, still more preferably 40% or more, and even more preferably 50% or more.
- the immunoglobulin-binding peptide used in the affinity separation matrix of the present invention has, as one embodiment, two or more, preferably three or more, more preferably the immunoglobulin-binding peptide that is a monomer or a single domain. May be a multimer of multiple domains linked by 4 or more, more preferably 5 or more. The upper limit of the number of domains connected is, for example, 10, preferably 8, and more preferably 6. These multimers may be homopolymers such as homodimers and homotrimers that are linked to a single immunoglobulin-binding peptide, heterodimers that are linked to multiple types of immunoglobulin-binding peptides, It may be a heteropolymer such as a heterotrimer.
- Examples of the way of linking the immunoglobulin-binding peptides include a method of linking with one or a plurality of amino acid residues, and a method of directly linking without interposing amino acid residues, but are limited to these methods. Is not to be done.
- the number of amino acid residues to be linked is not particularly limited, but is preferably 1 to 20 residues, more preferably 15 residues or less, still more preferably 10 residues or less, and even more preferably. Is 5 residues or less, and even more preferably 2 residues or less. In linking, those that do not destabilize the three-dimensional structure of the monomeric immunoglobulin-binding peptide are preferred.
- the immunoglobulin-binding peptide may be bound with other peptides or compounds.
- the immunoglobulin-binding peptide or a multimer in which two or more of the peptides are linked is fused as a component with another peptide having different functions.
- An affinity separation matrix using a fusion peptide characterized by Examples of fusion peptides include, but are not limited to, peptides fused with albumin or GST (glutathione S-transferase).
- the utility of the peptide used in the affinity separation matrix of the present invention may be utilized. For example, it is included in the present invention.
- proteins containing immunoglobulin Fc region and / or Fab region can be separated and purified by affinity column chromatography purification method.
- These protein purification methods can be achieved by a procedure according to the affinity column chromatography purification method of immunoglobulin (Non-patent Document 1). That is, after the buffer containing the protein is prepared (pH is near neutral), the solution is contacted by passing it through an affinity column packed with the affinity separation matrix of the present invention to adsorb the protein. Next, an appropriate amount of pure buffer is passed through the affinity column, and the inside of the column is washed. At this point, the desired protein is adsorbed to the affinity separation matrix of the present invention in the column.
- the affinity separation matrix in which the peptide of the present invention is immobilized as a ligand is excellent in the ability to adsorb and retain the target protein from the sample addition step to the matrix washing step. Then, an acidic buffer adjusted to an appropriate pH is passed through the column to elute the desired protein, thereby achieving high purity purification. A substance that promotes dissociation from the matrix may be added to the acidic buffer used for elution.
- the protein may be an immunoglobulin itself or an antibody fragment such as an Fc fragment or Fab fragment.
- the affinity separation matrix of the present invention can be reused by washing with a suitable strong acid or strong alkaline buffer that does not completely impair the function of the ligand compound or carrier substrate. It is.
- An appropriate denaturing agent or an organic solvent may be added to the regeneration buffer.
- the affinity separation matrix of the present invention is particularly excellent in chemical stability against an alkaline aqueous solution, it is preferably reused by passing it through a strong alkaline pure buffer and washing.
- the timing of regeneration with a strong alkaline pure buffer solution need not be every time after use, and may be, for example, once every five times or once every ten times.
- the DNA encoding the peptide used in the affinity separation matrix of the present invention may be any DNA as long as the amino acid sequence translated from the base sequence constitutes the peptide.
- a base sequence can be obtained by using a commonly used known method, for example, a polymerase chain reaction (hereinafter abbreviated as “PCR”) method. It can also be synthesized by a known chemical synthesis method, and can also be obtained from a DNA library.
- the base sequence may not be the same as the original base sequence as long as the codon may be substituted with a degenerate codon and it encodes the same amino acid when translated.
- Recombinant DNA having one or more of the nucleotide sequences vectors such as plasmids and phages containing the recombinant DNA, and transformants transformed with vectors having the DNA, and introducing the DNA And a cell-free protein synthesis system using the DNA as a template DNA for transcription.
- the immunoglobulin-binding peptide used in the affinity separation matrix according to the present invention can be obtained as a fusion peptide with a known protein having an advantage of assisting protein expression or facilitating purification. That is, a microorganism or cell containing at least one recombinant DNA encoding the fusion peptide can be obtained.
- the protein include maltose binding protein (MBP) and glutathione-S-transferase (GST), but are not limited to these proteins.
- the introduction of site-specific mutations for modifying the DNA encoding the peptide can be carried out using recombinant DNA techniques, PCR methods and the like as follows. That is, the introduction of mutations by recombinant DNA technology is performed, for example, when there are appropriate restriction enzyme recognition sequences on both sides of the target site where mutations are desired in the gene encoding the peptide of the present invention.
- the recognition sequence can be cleaved with the restriction enzyme, and after removing the region containing the site desired to be mutated, the cassette mutation method can be used in which a DNA fragment mutated only at the desired site is inserted by chemical synthesis or the like. .
- the introduction of site-specific mutation by PCR is performed by, for example, using a double-stranded plasmid encoding the above peptide as a template and PCR using two synthetic oligo primers containing mutations complementary to the + strand and the ⁇ strand.
- the double primer method can be used.
- a DNA encoding a multimeric peptide can be prepared by linking in a desired number of DNAs encoding the monomer peptide (one domain) in series.
- an appropriate restriction enzyme site can be introduced into the DNA sequence, and double-stranded DNA fragmented with the restriction enzyme can be ligated with DNA ligase.
- restriction enzyme site There may be one type of restriction enzyme site, but a plurality of different types of restriction enzyme sites may be introduced.
- the sequence identity between the nucleotide sequences of DNA encoding the peptide is 90% or less, preferably 85% or less, more preferably 80% or less, and even more preferably 75% or less.
- the identity of the base sequence can be determined by a conventional method as in the case of the amino acid sequence.
- the vector includes a base sequence encoding the above-mentioned peptide or a partial amino acid sequence thereof, and a promoter that can function in a host operably linked to the base sequence.
- the gene encoding the above peptide can be obtained by linking or inserting into a suitable vector, and the vector for inserting the gene is not particularly limited as long as it can replicate autonomously in the host.
- Plasmid DNA or phage DNA can be used as a vector.
- vectors such as pQE vectors (Qiagen), pET vectors (Merck), and pGEX vectors (GE Healthcare Bioscience) may be mentioned.
- the transformant can be obtained by introducing the recombinant vector of the present invention into a host cell.
- methods for introducing recombinant DNA into a host include a method using calcium ions, an electroporation method, a spheroplast method, a lithium acetate method, an Agrobacterium infection method, a particle gun method, and a polyethylene glycol method. However, it is not limited to these.
- a method for expressing the function of the obtained gene in a host include a method for incorporating the obtained gene into a genome (chromosome).
- the host cell is not particularly limited, but for mass production at low cost, Escherichia coli, Bacillus subtilis, Brevibacillus genus, Staphylococcus genus, Streptococcus genus, Streptomyces genus (Streptomyces), Corynebacterium Bacteria (Eubacteria) such as Corynebacterium can be preferably used.
- the above-mentioned immunoglobulin-binding peptide is obtained by culturing the above-described transformant in a medium, and generating and accumulating the peptide of the present invention in the culture (including the bacterial periplasm region) or in the culture solution (extracellular). It can be produced by collecting a desired peptide from the culture.
- the above-mentioned peptide is obtained by culturing the above-described transformed cells in a medium, and generating and accumulating a fusion protein containing the peptide in the culture (including the bacterial periplasm region) or in the culture solution (extracellular). It can be produced by collecting the fusion peptide from the culture, cleaving the fusion peptide with an appropriate protease, and collecting the desired peptide.
- the method of culturing the transformant in a medium is performed according to a normal method used for host culture.
- the medium used for culturing the obtained transformant is not particularly limited as long as the peptide can be produced with high efficiency and high yield.
- carbon sources and nitrogen sources such as glucose, sucrose, glycerol, polypeptone, meat extract, yeast extract, and casamino acid can be used.
- inorganic salts such as potassium salt, sodium salt, phosphate, magnesium salt, manganese salt, zinc salt and iron salt are added as necessary.
- an auxotrophic host cell a nutrient substance required for growth may be added. If necessary, antibiotics such as penicillin, erythromycin, chloramphenicol, neomycin may be added.
- protease inhibitors ie, phenylmethanesulfonylfluoride (PMSF), benzamideline, 4- (2-aminoethyl)- Benzenesulfonyl fluoride (AEBSF), Antipain, Chymostatin, Leupeptin, Pepstatin A, Phosphoramidon, Aprotinin, Ethylenediamine etietic or other inhibitors (EDTA)
- molecular chaperones such as GroEL / ES, Hsp70 / DnaK, Hsp90, and Hsp104 / ClpB may be used to correctly fold the immunoglobulin-binding peptide.
- Such molecular chaperone is allowed to coexist with the peptide of the present invention by a technique such as co-expression or fusion proteinization.
- LB medium tryptone 1%, yeast extract 0.5%, NaCl 1%) or 2 ⁇ YT medium (tryptone 1.6%, yeast extract) 1.0%, NaCl 0.5%) and the like.
- the culture temperature is, for example, 15 to 42 ° C., preferably 20 to 37 ° C., and aerobically cultivated for several hours to several days under aeration and stirring conditions, whereby the peptide is cultured in cultured cells (in the periplasmic region). Contained) or accumulated in the culture solution (extracellular) and collected. In some cases, the culture may be performed anaerobically by blocking aeration.
- the replacement peptide can be recovered.
- the cells are collected from the culture solution by a method such as centrifugation or filtration, and then the cells are sonicated.
- the peptide accumulated and produced in the cells can be recovered by crushing by a French press method and / or solubilizing by adding a surfactant or the like.
- the above-mentioned peptide can be purified by affinity chromatography, cation or anion exchange chromatography, gel filtration chromatography or the like alone or in appropriate combination. Confirmation that the obtained purified substance is the target protein can be performed by usual methods such as SDS polyacrylamide gel electrophoresis, N-terminal amino acid sequence analysis, Western blotting and the like.
- the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.
- the SpG mutation with enhanced binding to the Fab region with reference to the amino acid sequences of three known wild-type SpG immunoglobulin binding domains (SEQ ID NOs: 17 to 19) and Patent Documents 2 to 4 A body amino acid sequence (SEQ ID NO: 20) was used.
- Example 1 Preparation of various immunoglobulin-binding peptides (1) Preparation of expression plasmids of various immunoglobulin-binding peptides Back translation was performed from the amino acid sequences of various immunoglobulin-binding peptides, and DNA sequences encoding the peptides were designed. . The coding DNA was subjected to PCR using three kinds of single-stranded oligo DNAs (f31 / f32 / f33), then digested with the restriction enzymes BamHI / EcoRI and treated with the same restriction enzymes pGEX-6P-1. It was incorporated into the multiple cloning site.
- f32 0.2 ⁇ M, reading
- f33 10 ⁇ M, lagging
- the double-stranded DNA formed by overlapping PCR with f33 (lagging) becomes a coding DNA sequence having restriction enzyme sites at both ends.
- Various oligo DNA sequences corresponding to f31 to f33 of each PG analog were designed so that such a reaction proceeds and synthesized by outsourcing to Eurofin.
- DoubleTaq Plus (TOYOBO) as a polymerase
- PCR reaction is performed, agarose electrophoresis is performed, and the double-stranded DNA extracted by cutting out the target band is subjected to restriction enzymes BamHI and EcoRI ( Cut by Takara Bio Inc.).
- plasmid vector pGEX-6P-1 (GE Healthcare Bioscience) was similarly treated with BamHI / EcoRI and then dephosphorylated with dephosphorylation enzyme CIAP (Takara Bio). Then, a ligation reaction using Ligation high (TOYOBO) was performed.
- Table 1 shows combinations of coding DNA sequences corresponding to amino acid sequences of various immunoglobulin-binding peptides and oligo DNA sequences (f31 to f33) used for preparing expression plasmids.
- plasmid vector pGEX-6P-1 transformation of competent cells (Takara Bio Inc. “E. coli HB101”) was performed according to the protocol attached to this competent cell product.
- GST glutathione-S-transferase
- plasmid DNA was amplified and extracted using a plasmid purification kit (“Wizard Plus SV Minipreps DNA Purification System” manufactured by Promega) according to the standard protocol attached to the kit.
- the cells were collected by centrifugation and resuspended in 5 mL of PBS buffer.
- the cells were disrupted by ultrasonic disruption, centrifuged, and fractionated into a supernatant fraction (cell-free extract) and an insoluble fraction.
- GST is expressed as a fusion peptide attached to the N-terminus.
- SDS electrophoresis all of the various cell-free extracts prepared from the respective transformed cell cultures were found to have peptides that were thought to have been induced by IPTG at a molecular weight of about 25,000 or more. I confirmed the band. The molecular weight was almost the same, but the position of the band was different depending on the type of immunoglobulin-binding peptide.
- the GST fusion peptide was roughly purified from each cell-free extract containing the GST fusion peptide by affinity chromatography using a GSTrap FF column (GE Healthcare Bioscience) having affinity for GST. Each cell-free extract is added to the GSTRap FF column, and the column is washed with a standard buffer (20 mM NaH 2 PO 4 -Na 2 HPO 4 , 150 mM NaCl, pH 7.4), followed by an elution buffer ( The target GST fusion peptide was eluted with 50 mM Tris-HCl, 20 mM glutathione, pH 8.0).
- the sample used in the assay with the GST fused was concentrated using the Amicon (Merck Millipore), which is a centrifugal filter unit, while concentrating the elution buffer with the standard buffer solution.
- the peptide solution substituted with was used.
- the amino acid sequence that can cleave GST with the sequence-specific protease PreScission Protease is between GST and the target peptide.
- GST cleavage reaction was performed using PreScience Protease according to the attached protocol.
- the target peptide was purified by gel filtration chromatography using Superdex 75 10/300 GL column (GE Healthcare Biosciences) from the sample thus cut GST. Each reaction solution was added to a Superdex 75 10/300 GL column equilibrated with a standard buffer, and the target peptide was separated and purified from cleaved GST and PreScission Protease.
- each peptide after GST cleavage obtained in this example has an amino acid sequence in which Gly-Pro-Leu-Gly-Ser derived from the vector pGEX-6P-1 is added to the N-terminal side on the N-terminal side. .
- Example 2 Evaluation of binding ability of various immunoglobulin-binding peptides (1) Preparation of various immunoglobulins An IgG solution was prepared using human blood-derived IgG preparation as a raw material, and this was further converted into Fab fragments (hereinafter simply referred to as Fab) by papain. ) And Fc fragment (hereinafter simply abbreviated as Fc), and Fab and Fc were separated and purified to prepare Fab solution and Fc solution.
- Fab fragments
- Fc Fc fragment
- the plasma fraction preparation gamma globulin muscle injection 1500 mg / 10 mL was dissolved in papain digestion buffer (0.1 M AcOH-AcONa, 2 mM EDTA, 1 mM cysteine, pH 5.5), and Papain Agarose.
- papain digestion buffer 0.1 M AcOH-AcONa, 2 mM EDTA, 1 mM cysteine, pH 5.5
- Papain Agarose From papapa lateax papain-immobilized agarose (SIGMA) was added and incubated at 37 ° C. for about 8 hours while mixing with a rotator. From the reaction solution separated from papain-immobilized agarose (Fab and Fc coexisting), the Fab was collected in a flow-through fraction by affinity chromatography using KanCapA (Kaneka), and separated and purified from Fc.
- Fc adsorbed on the column was eluted with 0.05 M acetic acid / sodium acetate buffer (pH 3.5) and immediately neutralized with 0.1 M Tris-HCl buffer (pH 8.0).
- a solution containing IgG was prepared by collecting the plasma fraction preparation gamma globulin that had not been digested with papain by the same chromatographic operation using KanCapA in the same manner as Fc.
- Each solution containing IgG, Fab, and Fc is purified by gel filtration chromatography using Superdex 75 10/300 GL column (GE Healthcare) (standard buffer is used for equilibration and separation) And various immunoglobulin solutions were obtained.
- purification of the peptide by chromatography was performed using the AKTAprime plus system.
- the amino group biotin-labeled reagent EZ-link-NHS-PEG4-Biotin was used for 1 mL of each solution adjusted to a concentration of 3 mg / mL for IgG obtained in (1) and 1 mg / mL for Fab and Fc. 20 ⁇ L of 1 mM aqueous solution was added and incubated overnight at room temperature to prepare biotinylated IgG, biotinylated Fab and biotinylated Fc.
- Biotinylated IgG, Fab and Fc are diluted with a running buffer (20 mM NaH 2 PO 4 -Na 2 HPO 4 , 150 mM NaCl, pH 7.4) so that the final concentration is about 0.1 mg / mL.
- Eight biosensors were similarly contacted for 600 seconds. All the rotation speeds at the time of contact were adjusted to 1000 rpm. Thereafter, a cycle of contact with 50 mM Citrate, pH 2.4 for 10 seconds ⁇ contact with running buffer for 10 seconds was repeated three times. This is the cleaning / regeneration step. Under these conditions, it has been confirmed that the streptavidin-biotin bond does not dissociate.
- a measurement system was set up to use the same SA biosensor, and data on a binding phase in contact with various peptide solutions for 120 seconds and a dissociation phase in contact with a standard buffer solution for 120 seconds were obtained.
- data on a binding phase in contact with various peptide solutions for 120 seconds and a dissociation phase in contact with a standard buffer solution for 120 seconds were obtained.
- the same type of peptide was repeatedly measured using the same SA biosensor through the above-described washing / regeneration step. Table 2 shows the above measurement results.
- FIG. 1 A graph relating to IgG binding parameters of various immunoglobulin binding peptides shown in SEQ ID NOs: 2 to 7 is shown in FIG. 1 in comparison with a peptide consisting of an IgG binding domain of SpG shown in SEQ ID NOs: 17 to 19 (Comparative Example 1). It was. It was confirmed that all of the various immunoglobulin-binding peptides obtained in the present invention showed IgG binding strength comparable to that of the SpG IgG-binding domain. By using a peptide having a higher numerical value than SpG as a ligand, it may lead to creation of an affinity separation matrix with improved IgG retention performance.
- FIG. 2 is a graph comparing the Fab binding parameters of various immunoglobulin-binding peptides shown in SEQ ID NOs: 8 to 16 with the Fab binding-enhanced mutant of the IgG binding domain of SpG shown in SEQ ID NO: 20 (Comparative Example 1). It was shown to. Similarly to the above, it was confirmed that all the immunoglobulin-binding peptides obtained in the present invention showed sufficient binding strength as immunoglobulin Fab-binding peptides.
- the peptides of SEQ ID NOs: 8 to 16 have amino acid sequences obtained by mutating a part of SEQ ID NOs: 2 to 7 with reference to Patent Documents 2 to 4. These can be said to be data showing that amino acid sequences of various variations as shown in SEQ ID NO: 1 are basically contained in the present invention.
- Example 3 Evaluation of Alkali Resistance of Immunoglobulin Binding Peptides (1) Alkali Treatment of Various Peptides Various peptides dialyzed using ultrapure water were adjusted in concentration to obtain a 4 ⁇ M aqueous solution. A half amount of 45 mM sodium hydroxide aqueous solution (0.1 mL) was added to 0.2 mL of the aqueous solution, incubated at 25 ° C. for 2 hours, and then neutralized with 0.1 mL of 50 mM acetic acid (pH 3.0). The neutralization was confirmed with a pH test paper, and the concentration was adjusted to 100 nM by diluting 20 times with a standard buffer.
- aqueous sodium hydroxide solution 0.1 mL
- 50 mM acetic acid pH 3.0
- the final concentration of peptide in this solution is 100 nM.
- FIG. 3 (2) shows a comparison with 1). Similarly, it was confirmed that the Fab resistance-enhanced peptide obtained in the present invention has enhanced alkali resistance.
- Comparative Example 1 For the proteins shown in SEQ ID NOs: 17 to 20 used as comparative controls in this Example, expression plasmids were prepared using the oligo DNAs shown in Table 3 in the same manner as in Example 1 (1). And the protein sample was prepared by the method similar to Example 1 (2), and in Example 2 and Example 3, it evaluated by the same method simultaneously with the various peptides obtained by this invention.
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Abstract
Description
以下、本発明を示す。
上記[1]~[4]のいずれかに記載のアフィニティー分離マトリックスと、該タンパク質を含む液体試料とを接触させる工程と
アフィニティー分離マトリックスに結合した該タンパク質を、アフィニティー分離マトリックスから分離する工程を含むことを特徴とする方法。
配列番号1: Xaa01-Xaa02-Tyr-Lys-Leu-Xaa06-Val-Lys-Gly-Xaa10-Thr-Xaa12-Xaa13-Gly-Xaa15-Thr-Xaa17-Thr-Xaa19-Ala-Xaa21-Asp-Xaa23-Xaa24-Xaa25-Ala-Glu-Xaa28-Xaa29-Xaa30-Xaa31-Xaa32-Xaa33-Ala-Xaa35-Xaa36-Asn-Xaa38-Xaa39-Xaa40-Gly-Xaa42-Trp-Xaa44-Tyr-Asp-Xaa47-Ala-Thr-Lys-Thr-Phe-Thr-Val-Thr-Glu
Xaa01=Asp/Thr,Xaa02=Thr/Ser,Xaa06=Val/Ile,Xaa10=Ala/Val/Asn,Xaa12=Phe/Leu,Xaa13=Ser/Thr,Xaa15=Glu/Tyr,Xaa17=Thr/Ala,Xaa19=Lys/Ile,Xaa21=Val/Ala,Xaa23=Ala/Thr,Xaa24=Ala/Glu,Xaa25=Thr/Val,Xaa28=Lys/Gln,Xaa29=Ala/Glu/Thr,Xaa30=Phe/Leu,Xaa31=Lys/Arg,Xaa32=Gln/Asp,Xaa33=Tyr/Phe,Xaa35=Thr/Asn/Phe,Xaa36=Ala/Asp/Glu/Lys,Xaa38=Asn/Gly,Xaa39=Val/Thr,Xaa40=Asp/Tyr/Thr,Xaa42=Glu/Val,Xaa44=Ser/Ala,Xaa47=Asp/Ala/Thr
上記定義中、「/」は「または」を示す。また、上記配列同一性は95%以上または96%以上がより好ましく、98%以上または99%以上が更に好ましく、99.5%以上または99.8%以上がより更に好ましい。
(1) 各種免疫グロブリン結合性ペプチドの発現プラスミド調製
各種免疫グロブリン結合性ペプチドのアミノ酸配列から逆翻訳を行い、当該ペプチドをコードするDNA配列を設計した。コードDNAは、3種の一本鎖オリゴDNA(f31/f32/f33)を用いたPCRを行い、その後制限酵素BamHI/EcoRIで消化して、同じ制限酵素で処理した発現ベクターpGEX-6P-1のマルチクローニングサイトに組み込んだ。コードDNA調製時のPCRでは、まず少量のf32(0.2μM、リーディング)とf33(10μM、ラギング)とがオーバーラップPCRで伸張され、次に、f31(10μM、リーディング)と先の反応で伸張したf33(ラギング)とのオーバーラップPCRによって形成される二本鎖DNAが、制限酵素サイトを両端に有したコードDNA配列となる。各々のPG類縁体のf31~f33に該当する各種オリゴDNA配列をこのような反応が進むよう設計し、ユーロフィン社への外注によって合成した。具体的な実験操作については、ポリメラーゼとしてBlendTaq Plus(TOYOBO社)を用い、PCR反応を行い、アガロース電気泳動にかけ、目的のバンドを切り出すことで抽出した二本鎖DNAを、制限酵素BamHIとEcoRI(タカラバイオ社)により切断した。次に、プラスミドベクターpGEX-6P-1(GEヘルスケア・バイオサイエンス社)も、同様にBamHI/EcoRI処理し、次に脱リン酸化酵素CIAP(タカラバイオ社)による脱リン酸化処理を行った後で、Ligation high(TOYOBO社)を用いたライゲーション反応を行った。
上記(1)で得られた、各種免疫グロブリン結合性ペプチドを導入した各形質転換細胞を、アンピシリン含有2×YT培地にて、37℃で終夜培養した。これらの培養液を、100倍量程度のアンピシリン含有2×YT培地に接種し、37℃で約1時間、その後25℃で約1時間培養した後で、終濃度0.1mMになるようイソプロピル1-チオ-β-D-ガラクシド(IPTG)を添加し、さらに25℃にて約18時間培養した。
(1) 各種免疫グロブリンの調製
ヒト血液由来のIgG製剤を原料としてIgG溶液を調製し、さらにこれをパパインによってFabフラグメント(以下単にFabと略す)とFcフラグメント(以下単にFcと略す)に断片化し、FabとFcを分離精製することで、Fab溶液およびFc溶液を調製した。具体的には、血漿分画製剤ガンマグロブリン筋注1500mg/10mL(ニチヤク)を、パパイン消化用緩衝液(0.1M AcOH-AcONa,2mM EDTA,1mMシステイン,pH5.5)に溶解し、Papain Agarose from papaya latexパパイン固定化アガロース(SIGMA社)を添加し、ローテーターで混和させながら、37℃で約8時間インキュベートした。パパイン固定化アガロースから分離した反応溶液(FabとFcが混在)から、KanCapA(カネカ)を利用したアフィニティークロマトグラフィーにより、素通り画分でFabを回収することでFcと分離精製した。カラムに吸着したFcは、0.05M 酢酸/酢酸ナトリウム緩衝液(pH3.5)で溶出し、即座に0.1M Tris-塩酸緩衝液(pH8.0)で中和した。またIgGを含む溶液は、パパイン消化処理しなかった血漿分画製剤ガンマグロブリンを、KanCapAを利用した同様のクロマトグラフィー操作によって、Fcと同様の手法にて回収することで調製した。IgG、Fab、および、Fcを含む各々の溶液を、Superdex 75 10/300 GLカラム(GEヘルスケア社)を用いた、ゲルろ過クロマトグラフィーにて精製(平衡化および分離には標準緩衝液を使用)し、各種免疫グロブリン類の溶液を得た。なお、実施例1と同様に、クロマトグラフィーによるペプチド精製は、AKTAprime plusシステムを利用して実施した。
バイオセンサーOctet(日本ポール社)を用い、本装置で最も標準的な評価システムとして推奨されている、ストレプトアビジン-ビオチン結合を利用してリガンドを固定化するSAバイオセンサーを用いた評価系にて、各種免疫グロブリン結合性ペプチドと免疫グロブリン類との結合力を測定した。
(1) 各種ペプチドのアルカリ処理
超純水を用いて透析した各種ペプチドを濃度調整し、4μM水溶液を得た。当該水溶液0.2mLに半量の45mM水酸化ナトリウム水溶液0.1mLを添加し、25℃で2時間インキュベ―トした後、50mM酢酸(pH3.0)0.1mLで中和した。中和されていることをpH試験紙にて確認し、標準緩衝液で20倍希釈して濃度を100nMに調整した。
上記(1)で調整したアルカリ処理前後の各種ペプチド溶液を用いて、実施例2(2)と同様の方法で、Octetにて免疫グロブリンへの結合を測定した。本実験では、結合の様式を単純化するため、IgGの代わりにFcを用いた。また、結合定数が同じオーダーであることを確認したので、濃度変化に伴う結合シグナルの大きさの変化もほぼ同様と仮定できる。よって、アルカリ処理前の結合シグナル値に対する、アルカリ処理後(N=3)の結合シグナル値の比率を結合残存活性として算出し、その値をグラフにした。
本実施例の比較対照として用いた配列番号17~20に示すタンパク質は、実施例1(1)と同様の方法で、表3に示す配列番号のオリゴDNAを用いて、発現プラスミドを調製した。そして、実施例1(2)と同様の方法でタンパク質サンプルを調製し、実施例2、および、実施例3において、同様の方法で、本発明で得られた各種ペプチドと同時に評価を実施した。
Claims (5)
- 配列番号1で規定されるアミノ酸配列、または、配列番号1で規定されるアミノ酸配列と94%以上の配列同一性を示すアミノ酸配列を含む免疫グロブリン結合性ペプチドと水不溶性担体を含み、免疫グロブリン結合性ペプチドがリガンドとして水不溶性担体に固定化されていることを特徴とするアフィニティー分離マトリックス。
- 上記配列番号1で規定されるアミノ酸配列が配列番号2~16のいずれかによって規定されているアミノ酸配列であることを特徴とする、請求項1に記載のアフィニティー分離マトリックス。
- 上記免疫グロブリン結合性ペプチドが、上記配列番号1で規定されるアミノ酸配列において、N末端および/またはC末端に位置する1個または数個のアミノ酸が、欠失、置換および/または付加されたアミノ酸配列を含むことを特徴とする、請求項1に記載のアフィニティー分離マトリックス。
- 2以上の上記免疫グロブリン結合性ペプチドが連結した免疫グロブリン結合性ペプチド多量体がリガンドとして水不溶性担体に固定化されていることを特徴とする、請求項1~3のいずれかに記載のアフィニティー分離マトリックス。
- 免疫グロブリンのFc領域および/またはFab領域を含むタンパク質を製造する方法であって、
請求項1~4のいずれかに記載のアフィニティー分離マトリックスと、該タンパク質を含む液体試料とを接触させる工程と
アフィニティー分離マトリックスに結合した該タンパク質を、アフィニティー分離マトリックスから分離する工程を含むことを特徴とする方法。
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EP18776444.4A EP3604341A4 (en) | 2017-03-31 | 2018-03-02 | AFFINITY DISSOCIATION MATRIX FOR IMMUNOLOGICAL PURIFICATION PURPOSES |
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