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WO2020066270A1 - PROCÉDÉ DE PRODUCTION D'UN ANTICORPS CONTENANT UNE RÉGION VARIABLE DE CHAÎNE κ ET/OU D'UN FRAGMENT D'ANTICORPS - Google Patents

PROCÉDÉ DE PRODUCTION D'UN ANTICORPS CONTENANT UNE RÉGION VARIABLE DE CHAÎNE κ ET/OU D'UN FRAGMENT D'ANTICORPS Download PDF

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WO2020066270A1
WO2020066270A1 PCT/JP2019/029617 JP2019029617W WO2020066270A1 WO 2020066270 A1 WO2020066270 A1 WO 2020066270A1 JP 2019029617 W JP2019029617 W JP 2019029617W WO 2020066270 A1 WO2020066270 A1 WO 2020066270A1
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antibody
fab
antibody fragment
eluate
fraction
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Japanese (ja)
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大 村田
和信 水口
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Kaneka Corp
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Kaneka Corp
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

Definitions

  • the present invention relates to a method for producing an antibody and / or antibody fragment containing a ⁇ chain variable region with high purity by efficiently separating the antibody and / or antibody fragment from by-products.
  • Protein A affinity separation matrix (hereinafter referred to as “SpA” for protein A) used for capturing and purifying an antibody drug from animal cell culture at a time with high purity is used. (Which may be abbreviated as ".”).
  • Patent Document 1 discloses that a fraction of a monomeric monoclonal antibody is collected from an SpA affinity chromatography column to form an SpA product pool, the pH of the product pool is set to about 3.5 to about 4.5, and antibody aggregation is performed.
  • Monoclonal antibodies are basically developed as antibody drugs, and are produced in large quantities using recombinant cultured cell technology and the like.
  • “Monoclonal antibody” refers to an antibody obtained from a clone derived from a single antibody-producing cell. Most of the currently marketed antibody drugs are immunoglobulin G (IgG) subclass in molecular structure.
  • Antibody drugs comprising fragment antibodies, which are antibody derivatives having a molecular structure obtained by fragmenting immunoglobulin, are also being actively developed clinically, and clinical development of various fragment antibody drugs is progressing (Non-Patent Document 3).
  • SpA affinity separation matrix is used in the initial purification step in antibody drug production.
  • SpA is basically a protein that specifically binds to the Fc region of IgG. Therefore, a fragment antibody containing no Fc region, such as Fab, cannot be captured using the SpA affinity separation matrix. Therefore, from the viewpoint of developing a platform for an antibody drug purification process, there is a high industrial need for an affinity separation matrix capable of capturing a fragment antibody that does not contain the Fc region of IgG.
  • Non-Patent Document 4 A plurality of peptides that bind to regions other than the Fc region of IgG are already known (Non-Patent Document 4). Among them, a peptide that can bind to a variable region that is an antigen-binding domain is most preferable from the viewpoint that there are many types of fragment antibody formats that can bind and that it can also bind to IgM, IgA, and the like.
  • protein L hereinafter, protein L may be abbreviated as “PpL”).
  • PpL is a protein containing a plurality of ⁇ -chain variable region-binding domains (hereinafter, the ⁇ -chain variable region may be abbreviated as “VL- ⁇ ”), and the amino acid sequence of each VL- ⁇ binding domain is different.
  • VL- ⁇ ⁇ -chain variable region-binding domains
  • the number of VL- ⁇ binding domains and individual amino acid sequences vary depending on the type of strain. For example, the number of VL- ⁇ binding domains contained in the PpL of Peptostreptococcus magnus 312 strain 512 is 5, and the number of VL- ⁇ binding domains contained in the PpL of Peptostreptococcus magnus strain 3316 is five.
  • Non-Patent Documents 5 to 7, Patent Document 2 and Patent Document 3 are four (Non-Patent Documents 5 to 7, Patent Document 2 and Patent Document 3).
  • Non-Patent Document 8 describes an example in which Fab, which is a fragment antibody expressed and cultured in animal cells, Escherichia coli, and yeast, is purified using an affinity separation matrix containing PpL.
  • JP 2011-530606 A Japanese Patent Publication No. 7-506573 Japanese Patent Publication No. 7-507682
  • the present invention produces an antibody and / or antibody fragment containing a ⁇ chain variable region with high purity by efficiently separating an antibody and / or an antibody fragment containing the ⁇ chain variable region from a by-product derived from the antibody.
  • the aim is to provide a method.
  • the present inventors have intensively studied to solve the above problems. As a result, when an antibody and / or antibody fragment containing VL- ⁇ is purified using an affinity separation matrix containing PpL, the target antibody and / or antibody fragment can be purified by appropriately setting the pH of the eluate. The present inventors have found that they can be purified with high purity and completed the present invention. Hereinafter, the present invention will be described.
  • a method for producing an antibody and / or an antibody fragment contains a ⁇ chain variable region
  • a liquid sample containing at least one of a light chain derivative and a heavy chain derivative in addition to the antibody and / or the antibody fragment is prepared by injecting a protein L, a domain of the protein L, a protein L variant, or a protein L domain variant as a ligand into an insoluble carrier.
  • a target antibody and / or antibody fragment containing VL- ⁇ can be obtained with high purity.
  • the subsequent purification process can be simplified. As a result, the production cost of an antibody and / or antibody fragment containing VL- ⁇ can be reduced.
  • FIG. 1 shows the results of SDS-PAGE of each fraction obtained in the experiment of FIG. 1 under non-reducing conditions.
  • FIG. 3 shows the case where a gradient of pH 5.0 to pH 2.0 was applied with a 50 mM citrate buffer when purifying Fab from a Fab-containing culture supernatant using a commercially available PpL carrier (KANEKA KanCap TM L). It is an elution profile.
  • FIG. 1 shows the results of SDS-PAGE of each fraction obtained in the experiment of FIG. 1 under non-reducing conditions.
  • FIG. 3 shows the case where a gradient of pH 5.0 to pH 2.0 was applied with a 50 mM citrate buffer when purifying Fab from a Fab-containing culture supernatant using a commercially available PpL carrier (KANEKA KanCap TM L). It is an elution profile.
  • FIG. 1 shows the results of SDS-PAGE of each fraction obtained in the experiment of FIG. 1 under non-reducing conditions.
  • FIG. 3 shows the case where
  • FIG. 4 shows the results of SDS-PAGE of each fraction obtained in the experiment of FIG. 3 under non-reducing conditions.
  • FIG. 5 shows elution when a Fab is purified from a culture supernatant containing Fab using a commercially available PpL carrier (KANEKA KanCap TM L) with a gradient from pH 5.0 to pH 3.0 in 50 mM acetate buffer. Profile.
  • FIG. 6 shows the results of SDS-PAGE of each fraction obtained in the experiment of FIG. 5 under non-reducing conditions.
  • FIG. 5 shows elution when a Fab is purified from a culture supernatant containing Fab using a commercially available PpL carrier (KANEKA KanCap TM L) with a gradient from pH 5.0 to pH 3.0 in 50 mM acetate buffer. Profile.
  • FIG. 6 shows the results of SDS-PAGE of each fraction obtained in the experiment of FIG. 5 under non-reducing conditions.
  • FIG. 7 shows a gradient from pH 5.0 to pH 2.2 in 50 mM citrate buffer containing 100 mM NaCl when purifying Fab from a culture supernatant containing Fab using a commercially available PpL carrier (KANEKA KanCap TM L). It is an elution profile when applied.
  • FIG. 8 shows the results of SDS-PAGE of each fraction obtained in the experiment of FIG. 7 under non-reducing conditions.
  • FIG. 9 shows a gradient from pH 5.0 to pH 2.2 in 50 mM citrate buffer containing 100 mM NaCl when purifying Fab from a culture supernatant containing Fab using a commercially available PpL carrier (Capto TM L).
  • 11 is an elution profile obtained when FIG.
  • FIG. 10 shows the results of SDS-PAGE of each fraction obtained in the experiment of FIG. 9 under non-reducing conditions.
  • FIG. 11 shows a gradient from pH 5.0 to pH 2.2 in 50 mM citrate buffer containing 100 mM MgCl 2 when purifying Fab from the culture supernatant containing Fab using a commercially available PpL carrier (KANEKA KanCap TM L). Is an elution profile when multiplied by.
  • FIG. 12 shows the results of SDS-PAGE of each fraction obtained in the experiment of FIG. 11 under non-reducing conditions.
  • FIG. 11 shows a gradient from pH 5.0 to pH 2.2 in 50 mM citrate buffer containing 100 mM MgCl 2 when purifying Fab from the culture supernatant containing Fab using a commercially available PpL carrier (KANEKA KanCap TM L). Is an elution profile when multiplied by.
  • FIG. 12 shows the results of SDS-PAGE of each fraction obtained in the experiment of
  • FIG. 13 shows a case where a two-step elution was carried out at pH 3.1 and pH 2.5 with a 50 mM buffer when purifying Fab from a culture supernatant containing Fab using a commercially available PpL carrier (KANEKA KanCap TM L). It is an elution profile.
  • FIG. 14 shows the results of SDS-PAGE of each fraction in FIG. 13 under non-reducing conditions.
  • FIG. 15 shows an elution profile when using only a 50 mM citrate buffer (pH 2.5) as an eluent when purifying Fab from a culture supernatant containing Fab using a commercially available PpL carrier (KANEKA KanCap TM L). It is.
  • FIG. 1 shows a case where a two-step elution was carried out at pH 3.1 and pH 2.5 with a 50 mM buffer when purifying Fab from a culture supernatant containing Fab using a commercially available PpL carrier (KANEKA KanCap
  • FIG. 16 shows the results of SDS-PAGE of each fraction in FIG. 15 under non-reducing conditions.
  • FIG. 17 shows that a Fab solution purified by the method of the present invention is loaded on a commercially available protein G carrier (KANEKA KanCap TM G) having an affinity for the CH1 region, and then, using a 50 mM citrate buffer at pH 2.5. It is a chromatogram at the time of elution.
  • FIG. 18 shows the results of SDS-PAGE of each fraction in FIG. 17 under non-reducing conditions.
  • FIG. 19 shows that a Fab solution purified by a conventional method is loaded onto a commercially available protein G carrier (KANEKA KanCap TM G) having an affinity for the CH1 region, and then eluted with a 50 mM citrate buffer at pH 2.5. It is a chromatogram at the time of performing.
  • FIG. 20 shows the results of SDS-PAGE of each fraction in FIG. 19 under non-reducing conditions.
  • FIG. 21 shows a 50 mM citrate buffer (pH 2.7) containing 100 mM NaCl and a 50 mM citrate buffer containing no NaCl when purifying Fab from a culture supernatant containing Fab using a commercially available PpL carrier (KANEKA KanCap TM L).
  • FIG. 22 shows the results of SDS-PAGE of each fraction in FIG. 21 under non-reducing conditions.
  • FIG. 23 shows that a Fab solution purified by the method of the present invention is loaded on a commercially available protein G carrier (KANEKA KanCap TM G) having an affinity for the CH1 region, and then a 50 mM citrate buffer (pH 2.5) is used. It is a chromatogram at the time of elution.
  • the method of the present invention is a method for purifying an antibody and / or antibody fragment containing VL- ⁇ with high purity using an affinity separation matrix on which protein L, its domain, or a mutant thereof is immobilized.
  • the term “antibody and / or antibody fragment” means at least one of an antibody and an antibody fragment, and one or more selected from an antibody and an antibody fragment, and is preferably an “antibody or antibody fragment”.
  • antibody and / or antibody fragment may be abbreviated as “antibody / antibody fragment”.
  • Step 1 Step of Producing Antibody / Antibody Fragment
  • Immunoglobulin (Ig) is a glycoprotein produced by B cells of lymphocytes and has a function of recognizing and binding to a molecule such as a specific protein.
  • An immunoglobulin has a function of specifically binding to such a specific molecule called an antigen, and a function of detoxifying or removing a factor having an antigen in cooperation with other biomolecules and cells.
  • Immunoglobulins are generally called "antibodies,” which are names that focus on such functions.
  • All immunoglobulins basically have the same molecular structure, and have a basic structure of a “Y” -shaped four-chain structure.
  • the four-chain structure is composed of two polypeptide chains called light chains and two heavy chains.
  • Immunoglobulin G is a monomeric immunoglobulin composed of two ⁇ chains and two light chains and has two antigen binding sites.
  • the location corresponding to the vertical bar in the lower half of the “Y” of the immunoglobulin is called the Fc region, and the “V” in the upper half is called the Fab region.
  • the Fc region has an effector function to induce a reaction after the antibody has bound to the antigen, and the Fab region has a function to bind to the antigen.
  • the Fab region and the Fc region of the heavy chain are connected by a hinge, and the protease, papain, contained in papaya degrades the hinge to cut into two Fab regions and one Fc region.
  • the portion of the Fab region near the tip of the “Y” character is called a variable region (V region) because various changes are seen in the amino acid sequence so that it can bind to various antigens.
  • variable region of the light chain is called a VL region
  • variable region of a heavy chain is called a 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 called a CL region
  • the constant region of the heavy chain is called a CH region.
  • the CH region is further divided into three, CH1 to CH3.
  • the Fab region of the heavy chain is composed of the VH region and CH1, and the Fc region of the heavy chain is composed of CH2 and CH3.
  • the hinge part is located between CH1 and CH2.
  • PpL binds to a variable region (VL- ⁇ ) in which the light chain is a ⁇ chain (Non-patent Documents 5 to 7).
  • Antibody / antibody fragment to be purified by the method of the present invention contains VL- ⁇ and may further contain a heavy chain variable region.
  • the heavy chain variable region (VH) may or may not include a hinge portion connecting CH1 and CH2 of the heavy chain constant region in the heavy chain, but does not include at least the Fc region.
  • Antibodies comprise a light chain and a heavy chain, wherein the heavy chain comprises a heavy chain variable region and a heavy chain constant region.
  • the antibody / antibody fragment to be purified in the method of the present invention includes, for example, a light chain or a light chain fragment containing VL- ⁇ and a heavy chain or heavy chain fragment containing VH by a covalent bond such as a disulfide bond or a peptide linker. Those that are bound can be mentioned.
  • Such antibody fragments include, for example, Fab; F (ab ') 2 which is a Fab dimer; F (ab') 3 which is a Fab trimer; dsFv in which a VH chain and a VL chain are linked by a disulfide bond.
  • an antibody fragment in which a light chain or light chain fragment containing VL- ⁇ and a heavy chain or heavy chain fragment containing VH are associated without covalent bond may be purified by the method of the present invention depending on conditions. .
  • Such antibody fragments include, for example, Fv in which a VH chain and a VL chain are associated.
  • the peptide linker for binding the light chain or light chain fragment containing VL- ⁇ with the heavy chain or heavy chain fragment containing VH is not particularly limited.
  • a peptide linker containing 5 or more and 25 or less amino acid residues may be used.
  • Examples of such a peptide linker include a GS linker having a repeating sequence of glycine and serine.
  • the antibody / antibody fragment is preferably produced directly by genetic engineering. Specifically, for example, after designing an amino acid sequence of each chain constituting an antibody or an antibody fragment containing VH and VL- ⁇ , a base sequence encoding the amino acid sequence is designed by reverse translation. A DNA encoding the nucleotide sequence is chemically synthesized and inserted into a vector such as a plasmid. The vector is introduced into cells to obtain a transformant, and the transformant is cultured to produce the above antibody or antibody fragment. A solution containing the antibody or antibody fragment is obtained by roughly purifying a culture solution or cell lysate containing the antibody or antibody fragment. The crude purification includes a treatment for removing insoluble components such as cells by filtration or centrifugation.
  • Examples of cells used for producing the antibody / antibody fragment include, for example, E. coli and Bacillus; cerevisiae and P.S. pastoris and other fungi; plant cells; insect cells; non-human animal cells; human cells; and fused cells such as hybridomas.
  • Non-human animal cells include, for example, hamster cells, mouse cells, rat cells and the like.
  • the solution containing the light chain or light chain fragment containing VL- ⁇ and VH by a chemical reaction in a solution containing the light chain or light chain fragment containing VL- ⁇ and the heavy chain or heavy chain fragment containing VH Heavy chains or heavy chain fragments may be linked.
  • Step 2 Adsorption Step
  • the target VL- ⁇ -containing antibody / antibody fragment and, for example, by-products derived from the VL- ⁇ -containing light chain and the VH fragment constituting the target VL- ⁇ -containing antibody / antibody fragment are separated.
  • a liquid sample containing the antibody / protein L domain, a protein L mutant, or a protein L domain mutant is brought into contact with an affinity separation matrix immobilized as a ligand on an insoluble carrier, whereby the antibody / The antibody fragments are adsorbed to the affinity separation matrix.
  • the liquid sample is not particularly limited as long as it contains the VL- ⁇ -containing antibody / antibody fragment to be purified, but it is preferable that the VL- ⁇ -containing antibody / antibody fragment is dissolved in an aqueous solvent.
  • by-products derived from antibodies include monomers of VL- ⁇ -containing light chains or VH fragments, homodimers of VL- ⁇ -containing light chains and VH fragments, and fragments of VL- ⁇ -containing light chains and VH fragments. Is mentioned.
  • the liquid sample include a serum sample containing a VL- ⁇ -containing antibody / antibody fragment, a supernatant of a culture or disrupted cell culture of a VL- ⁇ -containing antibody / antibody fragment, and a reaction solution thereof. Can be.
  • the pH of the liquid sample is around 5.0 to 9.0 and around neutrality.
  • the pH is 5.0 or more, it becomes possible to more reliably adsorb the VL- ⁇ -containing antibody / antibody fragment contained in the liquid sample to the affinity separation matrix according to the present invention.
  • the pH is 9.0 or less, the VL- ⁇ -containing antibody / antibody fragment contained in the liquid sample can be adsorbed to the affinity separation matrix of the present invention in a state where denaturation due to alkaline conditions is suppressed.
  • the solvent of the liquid sample may be water alone, or may be a solvent containing a water-miscible organic solvent such as C 1-4 alcohol as long as it has water as a main component.
  • the buffer may be from about 0 to about 9.0.
  • the affinity separation matrix used in the present invention is one in which protein L, a domain of protein L, a protein L mutant, or a protein L domain mutant is immobilized as a ligand on an insoluble carrier.
  • the ligand of the affinity separation matrix according to the present invention is based on the sequence of protein L (PpL) and binds to the ⁇ chain variable region of immunoglobulin (VL- ⁇ ).
  • protein L, its domain, or a mutant thereof may be collectively referred to as “VL- ⁇ binding peptide”.
  • peptide includes any molecule having a polypeptide structure, and includes not only so-called proteins, but also fragmented ones and those in which other peptides are linked by peptide bonds. Shall be.
  • a “domain” is a unit of higher-order structure of a protein, consisting of a sequence of tens to hundreds of amino acid residues, and is a unit of protein that is sufficient to express some physicochemical or biochemical function.
  • the “protein L domain” in the present invention refers to a protein that exhibits an affinity for VL- ⁇ .
  • the “variant” of protein L or domain is a protein or peptide in which at least one substitution, addition or deletion has been introduced at the amino acid level with respect to the sequence of wild-type protein L or domain. Wherein at least the affinity for VL- ⁇ is maintained and preferably improved.
  • the number of mutations in the amino acid sequence is preferably 20 or less, 15 or less, more preferably 10 or 5 or less, and even more preferably 2 or 1.
  • Protein L is a protein derived from the cell wall of an anaerobic gram-positive coccus belonging to the genus Peptostreptococcus.
  • PpL derived from Peptostreptococcus magnus Peptostreptococcus magnus
  • two types of PpL derived from Peptostreptococcus magnus 312 strain and Peptostreptococcus magnus strain 3316 strain are preferable, but are not limited thereto. Is not performed (Non-Patent Documents 4 to 6).
  • PpL contains multiple VL- ⁇ binding domains consisting of 70-80 residues in the protein.
  • the number of VL- ⁇ binding domains contained in PpL312 is 5, and the number of VL- ⁇ binding domains contained in PpL3316 is 4.
  • the VL- ⁇ binding domain of PpL312 is called B1 to 5 domains in order from the N-terminus
  • the VL- ⁇ binding domain of PpL3316 is called C1 to 4 domains in order from the N-terminus (Non-patent Document 5 and Non-patent Documents). 6).
  • Non-patent Document 7 Studies have also shown that about 20 residues at the N-terminus of the VL- ⁇ binding domain of PpL do not adopt a specific secondary structure. It retains a three-dimensional structure as a sex domain and exhibits VL- ⁇ binding.
  • PpL is a protein containing four or five VL- ⁇ binding domains arranged in tandem as described above. Therefore, in one embodiment of the VL- ⁇ binding peptide according to the present invention, two or more, preferably three or more, more preferably four or more VL- ⁇ binding peptides which are monomers or single domains are used.
  • the multimer may be a multi-domain multi-domain of more than five, more preferably more than five.
  • the upper limit of the number of domains to be linked is 10 or less, preferably 8 or less, more preferably 6 or less.
  • These multimers may be homodimers, such as homodimers and homotrimers, which are conjugates of a single VL- ⁇ binding peptide, or may be conjugates of plural types of VL- ⁇ binding peptides. It may be a heteromultimer such as a heterodimer or a heterotrimer.
  • a method of linking the monomer VL- ⁇ binding peptide includes a method of linking with one or more amino acid residues, but is not limited to this method. From another viewpoint, those which do not destabilize the three-dimensional structure of the monomeric VL- ⁇ binding peptide are preferable.
  • a VL- ⁇ binding peptide or a multimer in which two or more VL- ⁇ binding domains are linked is one component.
  • a fusion peptide characterized by being fused with another peptide having a different function.
  • the fusion peptide include, but are not limited to, peptides fused with albumin and GST (glutathione S-transferase).
  • a nucleic acid such as a DNA aptamer, a drug such as an antibiotic, or a polymer such as PEG (polyethylene glycol) is fused, if the affinity separation matrix obtained by the present invention is useful, Included in the invention.
  • V The VL- ⁇ binding peptide used in the present invention can be prepared by a conventional method. That is, a DNA encoding the amino acid sequence of the desired VL- ⁇ binding peptide or a fragment thereof is chemically synthesized, the DNA encoding the VL- ⁇ binding peptide is amplified by PCR, and incorporated into a vector such as a plasmid. The resulting vector may be infected with Escherichia coli or the like and cultured, and the desired VL- ⁇ binding peptide may be purified from the cultured cells or culture solution by chromatography or the like.
  • the affinity separation matrix used in the present invention is one in which the VL- ⁇ binding peptide is immobilized on an insoluble carrier.
  • the “insoluble carrier” used in the present invention refers to the above-mentioned compound that shows insolubility in an aqueous solvent that is a solvent of a liquid sample containing a VL- ⁇ binding peptide, and specifically binds to a ligand by carrying the ligand. Refers to those that can be used for purification of antibodies / antibody fragments.
  • insoluble carrier used in the present invention examples include inorganic carriers such as glass beads and silica gel; synthetic polymers such as cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked polyacrylamide, and cross-linked polystyrene; and crystalline cellulose, cross-linked cellulose, cross-linked agarose, and cross-linked.
  • GCL2000 which is a porous cellulose gel
  • Sephacryl @ S-1000 in which allyldextran and methylenebisacrylamide are crosslinked by a covalent bond
  • Toyopearl which is an acrylate-based carrier
  • Sepharose @ CL4B which is an agarose-based crosslinked carrier
  • Cellulfine which is a cellulose-based cross-linking carrier
  • the water-insoluble carrier in the present invention is not limited to only these exemplified carriers.
  • the insoluble carrier used in the present invention preferably has a large surface area and is preferably porous having many pores of an appropriate size, in view of the purpose and method of using the affinity separation matrix.
  • the carrier may be in any form such as a bead, a monolith, a fiber, and a membrane (including a hollow fiber), and an arbitrary form can be selected.
  • a VL- ⁇ binding peptide as a ligand may be immobilized on an insoluble carrier by a conventional method.
  • the immobilization is performed using a reactive group present on the surface of the insoluble carrier.
  • a reactive group present on the surface of the insoluble carrier.
  • there are reactive groups such as an amino group, a hydroxyl group, and a carboxy group, which are activated, substituted with another reactive group, or reacted with these.
  • a linker group having a functional group may be introduced.
  • an epoxy group may be introduced onto the surface of a water-insoluble carrier using epichlorohydrin, diglycidyl ether, 1,4-bis (2,3-epoxypropoxy) butane, or the like, or an iodoacetyl group, a bromoacetyl group, or the like.
  • a maleimide group, an N-hydroxysuccinimide ester group, or the like is introduced, the coupling reaction with the reactive group of the VL- ⁇ binding peptide easily proceeds.
  • the linker group is not particularly limited.
  • 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.
  • the VL- ⁇ binding peptide according to the present invention may be chemically modified.
  • the VL- ⁇ -containing antibody / antibody fragment is selectively bound to the VL- ⁇ binding peptide as a ligand by contacting the liquid sample with the affinity separation matrix.
  • the specific embodiment is not particularly limited, and the liquid sample and the affinity separation matrix may be merely mixed.
  • the affinity separation matrix according to the present invention may be filled in a column to form an affinity separation. It is preferred that a liquid sample be passed through the affinity column to selectively adsorb the VL- ⁇ -containing antibody / antibody fragment to the VL- ⁇ binding peptide.
  • the conditions of this step may be appropriately adjusted within a range in which the VL- ⁇ -containing antibody / antibody fragment contained in the liquid sample is sufficiently adsorbed to the affinity separation matrix, and may be other than the target antibody fragment contained in the liquid sample. May be adsorbed to the matrix or not adsorbed. If a by-product containing VL- ⁇ is present, it may be adsorbed to the affinity separation matrix at this stage.
  • Step 3 Washing Affinity Separation Matrix
  • the affinity separation matrix on which the VL- ⁇ -containing antibody / antibody fragment has been adsorbed and retained in Step 1 is washed, and the VL- ⁇ -containing antibody / antibody fragment and VL Remove impurities other than by-products including - ⁇ .
  • by-products containing VL- ⁇ may be removed by washing depending on the affinity with the VL- ⁇ binding peptide. At this point, at least the target VL- ⁇ -containing antibody / antibody fragment has been adsorbed to the affinity separation matrix.
  • washing solution used for washing the affinity separation matrix in this step a solution that does not prevent the interaction between the VL- ⁇ -containing antibody fragment and the VL- ⁇ binding peptide is used.
  • water or a buffer having a pH of 5.0 or more and 9.0 or less can be used as a washing solution.
  • the amount of the washing solution used may be appropriately adjusted within a range in which impurities can be sufficiently removed from the affinity separation matrix. Whether or not the impurities have been sufficiently removed can be easily determined by monitoring the elution profile when using a chromatography system, for example.
  • Step 4 Separation Step of VL- ⁇ -Containing Antibody / Antibody Fragment
  • the VL- ⁇ -containing antibody / antibody fragment is separated from the affinity separation matrix washed in step 3 using an eluate having a lower pH than the washing liquid in step 2.
  • the antibody / antibody fragment and VL- ⁇ -containing by-product are mainly eluted.
  • the pH of the eluate is reduced continuously or stepwise. According to the experimental findings by the present inventors, even antibodies / antibody fragments that are common in that they contain VL- ⁇ have different affinities for VL- ⁇ binding peptides, and can be obtained by changing the pH of the eluate. Can be separated from each other.
  • stepwise refers to using an eluate having the same pH in a predetermined amount and for a predetermined time when lowering the pH of the eluate.
  • the predetermined amount and the predetermined time may be the same or different between eluates having different pHs.
  • the number of pH steps is preferably 2 or more, 5 or less, more preferably 4 or 3 or less, and even more preferably 2.
  • the VL- ⁇ -containing by-product contained in the liquid sample is specified, and the elution pH of the target VL- ⁇ -containing antibody / antibody fragment and the elution pH of the by-product are clear by preliminary experiments or the like, both are determined.
  • the stepwise decrease in eluate pH is particularly useful in industrial mass production of target VL- ⁇ -containing antibody fragments from the viewpoint of reducing the amount of eluate used and the amount of waste liquid.
  • the affinity separation matrix may be washed between the eluates having different pHs by using the washing solution used in the above step 2.
  • the affinity separation matrix may be washed with a washing solution, and the by-product may be eluted with an even lower pH eluate.
  • continuous means that the pH of the eluate decreases linearly with the lapse of time.
  • the elution pH of the target VL- ⁇ -containing antibody / antibody fragment or by-product can be specified, There is an advantage that both can be separated.
  • the amount of the eluate used in this step is preferably 5 times or more and 100 times or less the volume of the affinity separation matrix.
  • the ⁇ volume of the affinity separation matrix '' refers to a gel when the dispersion of the affinity separation matrix is allowed to stand or tap for a sufficient time until the volume of the gel portion containing the affinity separation matrix does not further decrease. Refers to the volume of the part.
  • the volume can be rephrased as “column volume” (CV), and may be represented by “mL-gel”.
  • the pH range at the start and end points of the pH gradient preferably includes 6.0 and 2.0, more preferably 5.0 and 2.0, and more preferably 4.0 and 2. Even more preferably, but is not limited to this range.
  • the eluate is not particularly limited, but a general buffer using, for example, citric acid, acetic acid, glycine, hydrochloric acid, phosphoric acid, formic acid, etc. may be used.
  • ⁇ A salt may be further added to the eluate, since the separation of the target VL- ⁇ -containing antibody / antibody fragment can be improved.
  • Such salts include one or more selected from the group consisting essentially of lithium chloride, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium iodide, potassium iodide, and sodium thiocyanate.
  • the concentration of the salt in the eluate may be appropriately adjusted, and may be, for example, 5 mM or more and 200 mM or less.
  • a solution different from the first eluate may be further flowed in order to elute by-products remaining in the carrier.
  • the type of the solution different from the first eluate may be one or two. Examples of conditions different from the first eluate include, in addition to pH, the type and concentration of the buffer, the presence or absence of addition of a salt, and the like, but are not limited thereto.
  • the volume of the affinity separation matrix serving as a reference is the volume of the gel-state affinity separation matrix that is in a suspended state and is tapped or allowed to stand until the volume does not decrease.
  • the separation efficiency of the target VL- ⁇ -containing antibody / antibody fragment is further increased by reducing the fractionation amount.
  • the amount of one fraction can be not less than 0.1 CV (column volume) and not more than 2.0 CV.
  • the amount is preferably 1.5 CV or less or 1.0 CV or less, more preferably 0.5 CV or less, and even more preferably 0.2 CV or less.
  • Step 5 Regeneration Step of Affinity Separation Matrix
  • the affinity separation matrix from which the VL- ⁇ -containing antibody / antibody fragment has been separated in the above step 3 is regenerated by washing with an alkaline aqueous solution.
  • this step need not necessarily be performed after the above step 3, and may be performed once every three times, once every five times, or once every ten times, by repeating the above steps 1 to 3. Absent. In other words, this step is not necessarily required to be performed when the performance of the affinity separation matrix such as the binding capacity is maintained, and the frequency or frequency of performing the step depends on the liquid sample containing the VL- ⁇ -containing antibody / antibody fragment to be purified. Conditions are different.
  • ⁇ An“ alkaline aqueous solution ”used for regenerating the affinity separation matrix is an aqueous solution exhibiting an alkalinity sufficient to achieve a purpose such as washing or sterilization. More specifically, an aqueous solution of sodium hydroxide having a concentration of 0.01 M or more and 1.0 M or less, or 0.01 N or more and 1.0 N or less corresponds to, but is not limited to, this. When sodium hydroxide is taken as an example, the lower limit of the concentration is preferably 0.01 M, more preferably 0.02 M, and even more preferably 0.05 M.
  • the upper limit of the concentration of sodium hydroxide is preferably 1.0 M, more preferably 0.5 M, still more preferably 0.3 M, even more preferably 0.2 M, and even more preferably 0.1 M.
  • the alkaline aqueous solution does not need to be a sodium hydroxide aqueous solution, but its 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.
  • the upper limit of pH it is preferably 14 or less, more preferably 13.5 or less, and still more preferably 13.0 or less.
  • the time for treating the affinity separation matrix obtained through the above step 3 with an alkaline aqueous solution is not particularly limited, since the damage to the peptide varies depending on the concentration of the alkaline aqueous solution and the temperature during the treatment, and may be appropriately adjusted.
  • the concentration of sodium hydroxide is 0.05 M and the temperature at the time of immersion is room temperature
  • the lower limit of the time of immersion in the alkaline aqueous solution is preferably 10 minutes or 30 minutes, more preferably 1 hour, 2 hours or 4 hours.
  • the time is more preferably 10 hours, but there is no particular limitation as long as the conditions allow regeneration of the affinity separation matrix.
  • the upper limit of the time can be, for example, 20 hours.
  • Example 1 Fab Purification Experiment-Continuous Gradient of pH (1) Preparation of Fab-Containing Supernatant As VL- ⁇ -containing antibody fragment, public sequence information of the sequence of fully humanized anti-TNF- ⁇ antibody (adalimumab) Fab designed based on the above was selected.
  • the Fab gene was prepared by designing a gene encoding the amino acid sequence of the Fd chain and the amino acid sequence of the light chain of the anti-TNF- ⁇ antibody and performing PCR using a chemically synthesized gene as a template.
  • the Fd chain refers to a CH1 region and a VH region obtained by removing a hinge site and an Fc region from a heavy chain of an antibody.
  • the Fab was produced by methanol-assimilating yeast.
  • the production and cultivation of the present Fab fragment-producing yeast were performed according to the methods described in Examples 1, 8, and 9 of WO2012 / 102171.
  • a Fab fragment in which the Fd chain and the light chain are linked by a disulfide bond is generated.
  • the culture solution containing the obtained Fab fragment was centrifuged, and the culture supernatant was collected.
  • the collected culture supernatant was filtered using a sterile filtration filter (“Minisart” Sartorius) having a pore size of 0.22 ⁇ m.
  • adsorbable affinity separation matrix to antibody fragments comprising the preparation kappa chain variable region affinity separation matrix (VL-kappa) containing PpL, of Tosoh Corporation "TOYOPEARL (R) AF-rProtein L -650F " And “KANEKA KanCap TM L” obtained from Kaneka Corporation, and 1 mL-gel was packed in a commercially available column (“Tricorn 5/50” GE Healthcare).
  • “1 mL-gel” means that the volume of the gel-state affinity separation matrix obtained by tapping or standing the suspended affinity separation matrix until the volume does not decrease is 1 mL.
  • Fab was eluted with a 50 mM citrate buffer with a linear pH gradient from pH 5.0 to pH 2.0. More specifically, after the column was equilibrated with 5 CV of eluate A (50 mM citrate, pH 5.0), eluate B (50 mM citrate, pH 2.0 In the step of linearly increasing the concentration of 0) from 0% to 100%, 2 mL fractions were collected. The pH of each fraction (2 mL) was measured with a pH meter, and the elution pH at the peak top position was determined from the pH of the Fab elution peak fraction. In the above operation, the flow rate was 0.33 mL / min. In the purification using any carrier, fractions of sample loading, washing and elution were collected. The collected elution fraction was neutralized with a 2M Tris solution.
  • FIG. 3 shows a dissolution profile obtained by using the KANEKA KanCap TM L.
  • the collected sample loaded fraction, washed fraction, and eluted fraction were analyzed by SDS-PAGE.
  • a non-reducing treatment was carried out using a mini slab electrophoresis tank equipped with a power supply (manufactured by “Pajeran” Atto) and a 15% polyacrylamide precast gel (“e-PAGEL” manufactured by Atto) according to the attached manual. SDS-PAGE was performed under the conditions.
  • FIG. 2 shows the results of confirming each fraction by SDS-PAGE.
  • the elution peak was confirmed for each fraction divided into three parts in order from the first half.
  • the pH of the eluted fractions 1 to 3 decreases in order.
  • the bands of the eluted fraction in FIG. 2 are confirmed, only a band having a molecular weight of about 50 kDa is present in lane 4 (eluted fraction 1), bands of about 50 kDa and about 25 kDa are present in lane 5, and about 25 kDa in lane 6. It was found that the ratio of the band was small.
  • the band at about 25 kDa is considered to be due to the light chain monomer from the molecular weight.
  • FIG. 4 shows the results of confirming each fraction by SDS-PAGE. The elution peak was confirmed for each fraction divided into two parts in order from the first half. When the bands of the eluted fraction in FIG. 4 were confirmed, only a band having a molecular weight of about 50 kDa was present in lane 4, and bands of about 50 kDa and about 25 kDa were present in lane 5.
  • Example 2 Fab Purification Experiment-Acetate Buffer Fab was eluted with a linear gradient from pH 5.0 to pH 3.0 using KANEKA KanCap TM L alone and 50 mM acetate buffer as commercially available Protein L carriers.
  • a Fab purification experiment was performed in the same manner as in Example 1 (3) except for the above.
  • FIG. 5 shows the elution profile
  • FIG. 6 shows the result of SDS-PAGE.
  • FIG. 5 shows the results shown in FIG. 5
  • the Fab culture supernatant was loaded on KANEKA KanCap TM L and subjected to a pH gradient with a 50 mM acetate buffer, two elution peaks were obtained.
  • FIG. 6 shows the results of confirming each fraction by SDS-PAGE.
  • the elution peak was confirmed for each fraction divided into three parts in order from the first half.
  • bands of the eluted fraction in FIG. 6 were confirmed, only a band having a molecular weight of about 50 kDa was present in lane 4, and bands of about 50 kDa and about 25 kDa were present in lane 5. From the above results, it was found that there is a common tendency that Fab elutes at a higher pH than the light chain monomer and light chain dimer regardless of the type of eluate.
  • Example 3 Fab purification experiment-Continuous gradient of pH and combined use of sodium chloride
  • the Fab-containing culture supernatant prepared in Example 1 (1) above was filtered through a filter having a pore size of 0.22 ⁇ m ("Minisart” Sartorius).
  • Fab was purified using the commercially available protein L carrier prepared in Example 1 (2) (“Kaneka KanCap TM L” manufactured by Kaneka Corporation and “Capto L” manufactured by GE Healthcare Company).
  • the column packed with the carrier was used by connecting to a chromatography system AKTAavant25 (GE Healthcare). Specifically, the following operation was performed.
  • FIG. 7 shows an elution profile of KANEKA KanCap TM L
  • FIG. 9 shows an elution profile of Capto L.
  • the collected sample-loaded fraction, washed fraction, and eluted fraction were confirmed by SDS-PAGE.
  • FIG. 8 shows the results of KANEKA KanCap TM L
  • FIG. 10 shows the results of Capto L.
  • the results shown in FIG. 7 when the Fab culture supernatant was loaded on KANEKA KanCap TM L and subjected to a pH gradient with a 50 mM citrate buffer containing 100 mM NaCl, two elution peaks were obtained.
  • FIG. 8 shows the results of confirming each fraction by SDS-PAGE.
  • the fractions of the elution peak are arranged in order from the first half, and the pH of the fraction is lower in order from lane 4 to lane 10.
  • a band of about 50 kDa, a band of about 25 kDa, and a band of about 50 kDa are mainly contained in the fraction, respectively.
  • FIG. 10 shows the results of confirming each fraction by SDS-PAGE. The pH of the fraction decreases in order from lane 4 to lane 7.
  • the band of about 50 kDa, the band of about 25 kDa, and the band of about 50 kDa were mainly contained in the fraction, respectively, as the pH became lower. It was shown that even if NaCl was added to the eluate, the Fab, light chain monomer and light chain dimer could be separated.
  • Example 4 Fab Purification Experiments-Continuous pH Gradient and Combination of Magnesium Chloride Using only Kaneka KanCap TM L as a commercially available Protein L carrier and 50 mM citrate buffer containing 100 mM MgCl 2 , from pH 5.0 A Fab purification experiment was performed in the same manner as in Example 1 (3) except that the Fab was eluted with a linear pH gradient to pH 2.2.
  • FIG. 11 shows the elution profile
  • FIG. 12 shows the result of SDS-PAGE. As shown in the results shown in FIG.
  • FIG. 12 shows the results of confirming each fraction by SDS-PAGE. The elution peak was confirmed for each fraction divided into three parts in order from the first half. When the bands of the eluted fraction in FIG. 12 were confirmed, only the band having a molecular weight of about 50 kDa was present in lanes 4 to 6.
  • the band of about 50 kDa contained in the lane of the strongly eluted fraction shows that two bands having slightly different molecular weights are contained. Since the molecular weight of the Fab containing the hinge portion is larger than that of the light chain dimer, of the band of about 50 kDa, the band with the higher molecular weight is the Fab, and the band with the smaller molecular weight is the light chain dimer.
  • the 50 kDa band is thought to contain mainly light chain dimers. The results showed that Fab could be separated with high purity even when MgCl 2 was added to the eluate.
  • Example 5 Fab purification experiment-stepwise gradient of pH (1)
  • Fab purification experiment The Fab-containing culture supernatant prepared in Example 1 (1) above was filtered with a filter having a pore size of 0.22 ⁇ m ("Minisart” Sartorius). Then, using the commercially available protein L carrier prepared in Example 1 (2) (“KANEKA KanCap TM L” manufactured by Kaneka Corporation), the pH was reduced stepwise to purify the Fab.
  • the pH of the eluate 1 used first was set to “3.1”, which is the pH of the first elution peak in the study using KANEKA KanCap TM L in Example 1 (3).
  • Example 1 (3) the column filled with the carrier was used by being connected to a chromatography system AKTAavant25 (GE Healthcare). Specifically, the following operation was performed. First, 5 CV (column volume) of an equilibration buffer (20 mM Na 2 HPO 4 -NaH 2 PO 4 , 150 mM NaCl, pH 7.4) was passed through the column to equilibrate the carrier. Next, 5 mL of the Fab fragment-containing supernatant prepared in Example 1 (1) was loaded onto the column. Then, the equilibration buffer solution for 5 CV was passed through and washed, and then eluate 1 (50 mM citrate, pH 3.1) for 10 CV was passed.
  • an equilibration buffer (20 mM Na 2 HPO 4 -NaH 2 PO 4 , 150 mM NaCl, pH 7.4
  • 5 mL of the Fab fragment-containing supernatant prepared in Example 1 (1) was loaded onto the column. Then, the equilibration buffer solution for
  • the Fab culture supernatant was loaded on KANEKA KanCap TM L, and only a band of about 50 kDa was present in the fraction eluted with eluate 1 (50 mM citrate, pH 3.1) ( Lanes 4 and 5) and the fraction (lane 6) eluted with eluate 2 (50 mM citrate, pH 2.5) contained bands of about 50 kDa and about 25 kDa. Two bands are included in the vicinity of 50 kDa in lane 6, and it is considered that a lower-molecular light chain dimer is mainly contained. From this result, it can be seen that it is possible to purify and obtain Fab with high purity by using an eluate having an appropriately adjusted pH.
  • FIG. 15 shows the elution profile
  • FIG. 16 shows the results of confirming the collected sample loading fraction, washing fraction, and elution fraction by SDS-PAGE.
  • bands of about 50 kDa and about 25 kDa were present in the eluted fraction, and Fab and light chain monomer could not be separated.
  • the band of about 50 kDa is broad, and it is considered that the Fab and the light chain dimer are contained without being separated at all.
  • an equilibration buffer (20 mM Na 2 HPO 4 -NaH 2 PO 4 , 150 mM NaCl, pH 7.4) for 3 CV (column volume) was passed through the column to equilibrate the carrier.
  • 1 mL of the Fab fragment-containing eluate obtained in Example 5 (1) was loaded on the column.
  • the above-mentioned equilibration buffer solution for 5 CV was passed through and washed, and then the eluate (5OmM citrate, pH 2.5) for 5 CV was passed. Thereafter, an equilibration buffer solution for 3 CV was circulated, and a 1 M acetic acid aqueous solution for 5 CV was further circulated.
  • FIG. 17 shows the chromatogram from the sample loading to the elution
  • FIG. 18 shows the results of the collected sample loading fraction, washing fraction, and elution fraction confirmed by SDS-PAGE.
  • FIG. 19 shows a chromatogram obtained by similarly analyzing the Fab-containing eluate obtained in the comparative experiment of Example 5 (2)
  • FIG. 20 shows the results confirmed by SDS-PAGE.
  • Table 1 shows the ratio of the loaded fraction and the eluted fraction in the total peak area of the chromatograms in FIGS. It is considered that Fab is included in the eluted fraction because it is adsorbed to protein G, whereas light chain monomers and dimers that do not have a CH1 region are not adsorbed to protein G and thus are included in the loaded fraction.
  • the ratio of the peak area of the loaded fraction and the ratio of the peak area of the eluted fraction are largely different, and there is a clear difference when the ratio of each of the total areas in Table 1 is compared.
  • the Fab has a CH1 region, it is included in the eluted fraction because it is adsorbed to KANEKA KanCap TM G containing protein G as a ligand, while the light chain monomer and the light chain dimer having no CH1 region are KANEKA KanCap TM. Since G is not adsorbed, it is considered to be included in the load fraction.
  • the higher the ratio of the eluted fraction the higher the Fab purity in the Fab-containing eluate obtained by purification.
  • the loaded fraction contains bands of about 50 kDa and about 25 kDa of light chain monomer and light chain dimer, whereas the eluted fraction contains Fab. It was confirmed that only the 50 kDa band was present. From these results, it was found that by appropriately setting the pH of the eluate, it was possible to purify and obtain Fab with higher purity.
  • Example 6 Fab purification experiment-stepwise gradient of pH using salt
  • the Fab-containing culture supernatant prepared in Example 1 (1) above was filtered through a filter having a pore size of 0.22 ⁇ m ("Minisart” Sartorius). Thereafter, using the commercially available protein L carrier prepared in Example 1 (2) (“Kaneka KanCap TM L” manufactured by Kaneka Corporation), the Fab was eluted and purified while stepwise decreasing the pH.
  • the pH of the eluate 1 used first was set to “2.7”, which is the pH of the first elution peak in the study using KANEKA KanCap TM L in Example 3 above.
  • the pH of the eluate 2 used next was “2.5”.
  • Example 1 (3) the column filled with the carrier was used by being connected to a chromatography system AKTAavant25 (GE Healthcare). Specifically, the following operation was performed. First, 5 CV (column volume) of an equilibration buffer (20 mM Na 2 HPO 4 -NaH 2 PO 4 , 150 mM NaCl, pH 7.4) was passed through the column to equilibrate the carrier. Next, 5 mL of the Fab fragment-containing supernatant prepared in Example 1 (1) was loaded onto the column. Then, the equilibration buffer solution for 5 CV was passed through and washed, and then eluate 1 (50 mM citrate, 100 mM NaCl, pH 2.7) for 10 CV was passed.
  • an equilibration buffer (20 mM Na 2 HPO 4 -NaH 2 PO 4 , 150 mM NaCl, pH 7.4
  • 5 mL of the Fab fragment-containing supernatant prepared in Example 1 (1) was loaded onto the column. Then, the
  • the Fab culture supernatant was loaded on KANEKA KanCap TM L and the fraction eluted with eluate 1 (50 mM citrate, 100 mM NaCl, pH 2.7) had only a band of about 50 kDa. Then, the fraction (lane 7) eluted with the eluate 2 (50 mM citrate, pH 2.5) contained bands of about 50 kDa and about 25 kDa. Two bands are included in the vicinity of 50 kDa in lane 7, and it is considered that a light chain dimer having a lower molecular weight is mainly contained.
  • the ratio of Fab in the eluate obtained by purification was the same as in Example 5 except that a 50 mM citrate buffer (pH 2.7) containing 100 mM NaCl was used as the first-stage eluate. The method was evaluated. The results are shown in FIG.

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Abstract

L'objectif de la présente invention est de fournir un procédé de production d'un anticorps contenant une région variable de chaîne κ et/ou d'un fragment d'anticorps avec une pureté élevée par une séparation efficace de l'anticorps contenant la région variable de chaîne κ et/ou d'un fragment d'anticorps d'un sous-produit dérivé de l'anticorps. Le procédé fourni par la présente invention permet de produire un anticorps et/ou un fragment d'anticorps, ledit anticorps et/ou fragment d'anticorps contenant une région variable de chaîne κ, ledit procédé comprenant : une étape qui consiste à mettre en contact un échantillon liquide, contenant l'anticorps et/ou le fragment d'anticorps mentionné ci-dessus conjointement avec un dérivé de chaîne légère et/ou un dérivé de chaîne lourde, avec une matrice de séparation d'affinité, la protéine L, etc. étant immobilisé comme ligand sur un support insoluble, et adsorbant ainsi l'anticorps et/ou le fragment d'anticorps sur le ligand ; une étape qui consiste à laver une matrice de séparation par affinité sur laquelle l'anticorps et/ou le fragment d'anticorps sont adsorbés ; et une étape qui consiste à séparer l'anticorps et/ou le fragment d'anticorps de la matrice de séparation par affinité par élution avec un éluant, ledit procédé étant caractérisé en ce que, dans l'étape d'élution, le pH de l'éluant étant diminué de manière continue ou de manière progressive.
PCT/JP2019/029617 2018-09-28 2019-07-29 PROCÉDÉ DE PRODUCTION D'UN ANTICORPS CONTENANT UNE RÉGION VARIABLE DE CHAÎNE κ ET/OU D'UN FRAGMENT D'ANTICORPS Ceased WO2020066270A1 (fr)

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JP7620456B2 (ja) 2021-03-15 2025-01-23 株式会社カネカ κ鎖を含む抗体の製造方法

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