JP2020117454A - Method for producing immunoglobulin - Google Patents

Abstract

To provide methods capable of efficiently producing immunoglobulin G and the like by separating a liquid sample containing immunoglobulin G and the like derived from an animal belonging to subgenus Capra and immunoglobulin G and the like derived from an animal other than animals belonging to subgenus Capra.SOLUTION: The method for producing immunoglobulin G and the like according to the present invention comprises: by contacting to an affinity separation matrix in which a ligand is immobilized on a carrier, a liquid sample that contains immunoglobulin G and the like derived from an animal belonging to subgenus Capra and immunoglobulin G and the like derived from an animal other than animals belonging to subgenus Capra which binds to wild-type protein A, adsorbing, to the ligand, the immunoglobulin G or a derivative thereof derived from an animal other than animals belonging to subgenus Capra; washing the affinity separation matrix; and separating, from the ligand using an eluent, the immunoglobulin G or a derivative thereof derived from an animal other than subgenus Capra, adsorbed into the ligand, the ligand being a domain of protein A with a specific mutation.SELECTED DRAWING: None

Description

The present invention separates immunoglobulin G or a derivative thereof from a goat subgenus and immunoglobulin G or a derivative thereof from an animal other than a goat, and efficiently produces immunoglobulin G or a derivative thereof. It is about how you can do it.

Antibodies have a function of specifically binding to a substance called an antigen and a function of detoxifying and removing a factor having an antigen in cooperation with other biomolecules and cells. The name of antibody is a name that attaches importance to the function of binding to such an antigen, and is called "immunoglobulin (Ig)" as a substance.

In recent years, with the development of genetic engineering, protein engineering, and cell engineering, a drug called an antibody drug, which utilizes the function of an antibody, has been actively developed. Since antibody drugs work more specifically to target molecules than conventional drugs, it is expected that side effects will be reduced and a high therapeutic effect will be obtained. It contributes to the improvement.

On the other hand, since antibody medicines are administered in large quantities to the living body, it is said that their purity has a greater effect on quality than other recombinant protein medicines. Therefore, in order to produce a highly pure antibody, a technique such as affinity chromatography using an adsorbent material using a molecule that specifically binds to the antibody as a ligand is generally used.

Most of the antibody drugs currently on the market are immunoglobulin G (IgG) subclasses in terms of molecular structure. Protein A is well known as an immunoglobulin-binding protein having affinity for IgG antibodies. Protein A is a kind of cell wall protein produced by the gram-positive bacterium Staphylococcus aureus, and has a signal sequence S and five immunoglobulin-binding domains (E domain, D domain, A domain, B Domain, C domain), and an XM region that is a cell wall binding domain (Non-Patent Document 1). A column for affinity chromatography in which protein A is immobilized as a ligand on a water-insoluble carrier (hereinafter referred to as “protein A column”) is generally used in the initial purification step in the antibody drug manufacturing step (non-patent document). References 1-3).

Various technical developments have been made to improve the performance of protein A columns. Technology development from the aspect of ligands is also in progress. Initially, natural protein A was used as a ligand, but many techniques for improving the performance of the column have also been found using recombinant protein A modified by protein engineering as a ligand.

Although many studies have been conducted on the introduction of mutagenesis of protein A, the introduction of amino acid substitution mutations at the antibody-binding site of protein A, particularly the Fc-binding site, may cause a marked decrease in antibody-binding activity. Among them, hydrophobic amino acid residues such as Phe at the 13th position, Leu at the 17th position, and Ile at the 31st position are considered to be essential amino acid residues for binding to Fc (Non-Patent Documents 4 and 5).

In many cases, antibody pharmaceuticals are generally produced by culturing using CHO cells as a production host, but studies on antibody production using transgenic animals as hosts are also underway. For example, a system for producing a human antibody using a mouse or a rat as a host is useful as a screening tool for an antibody that is a drug candidate (Non-Patent Document 6). In addition, transgenic goats and transgenic sheep can highly express the target recombinant protein by introducing the target gene downstream of the milk protein promoter (Patent Document 1). However, in the production of human antibodies using such transgenic animals, separation and purification of the host-derived antibody and the desired human antibody poses a problem. It is known that existing protein A carriers bind to goat IgG2 and sheep IgG2, and attempts have been made to separate them from human IgG by devising the combination of a plurality of chromatography and elution conditions. For example, there has been a report of reducing goat-derived antibodies from human antibodies produced by transgenic goats by combining protein A chromatography, protein G chromatography, ion exchange chromatography, and hydrophobic interaction chromatography (non-patent document). Reference 7). There is also an example in which a human antibody is separated from a goat antibody or a sheep antibody by pH linear gradient elution using protein A chromatography (Patent Document 2).

In antibody production, transformed CHO cells are generally cultivated in a serum-free medium, but in the field of research, it is common to cultivate cells in a medium supplemented with serum. Serum is an important factor for promoting cell growth, but serum components including antibodies may become impurities, making antibody purification difficult. Therefore, from the viewpoint of antibody purification, it is desirable to culture CHO cells in a serum-free medium, but in that case, it is necessary to acclimate the cells so that they can be cultured in a serum-free medium. On the other hand, if it is possible to remove the antibody derived from the serum of a specific animal, it can be said that it becomes easy to culture the hybridoma or the like in a serum medium to produce the antibody.

Japanese Patent Publication No. 2016-513105 International Publication No. 2004/087761 Pamphlet

Huber S. J. J. et al. Chromatogr. B, 2007, 848, pp. 40-47. Low D. J. J. et al. Chromatogr. B, 2007, 848, 48-63. Roque A. C. A. J. J. et al. Chromatogr. A, 2007, 1160, pp. 44-55. O'Seaghha M. FEBS J, 2006, 273, 4831-4841. Tsukamoto M. J. J. et al. Biol. Eng. , 2014, Volume 8, Page 15 Bruggemann M.D. Arch. Immunol. Ther. Exp. , 2015, 63, 101-108 Pollock D. J. J. et al. Immunol. Methods, 1999, 231, 147-157.

As described above, if the antibodies derived from different animals can be separated from each other, the production efficiency of the antibodies may be further improved.
Therefore, the present invention separates a liquid sample containing immunoglobulin G or a derivative thereof from a goat subgenus and immunoglobulin G or a derivative thereof from an animal other than a goat subgenus to obtain immunoglobulin G or a derivative thereof. It is an object of the present invention to provide a method capable of efficiently producing

The present inventors have conducted extensive studies to solve the above problems. As a result, the domain of mutated protein A having a specific mutation has a relatively low affinity for immunoglobulin G derived from the subfamily Goat and its derivatives, while immunity derived from other animals. The present invention has been completed by finding that immunoglobulin G or a derivative thereof can be efficiently produced by using such a mutant protein A because the affinity for globulin G or a derivative thereof is maintained.
The present invention will be described below.

[1] A method for producing immunoglobulin G or a derivative thereof, comprising:
A ligand immobilizes a liquid sample containing immunoglobulin G or a derivative thereof from a goat subgenus and immunoglobulin G or a derivative thereof from a non-goat animal that binds to wild-type protein A on a carrier. Adsorbing an immunoglobulin G or a derivative thereof derived from an animal other than a goat subfamily on a ligand by contacting with the affinity separation matrix prepared above,
Washing the affinity separation matrix, and
A step of separating immunoglobulin G or a derivative thereof derived from an animal other than a sub-goat animal adsorbed to the ligand from the ligand using an eluent,
The ligand is one or more hydrophobic amino acid residues in the Fc binding site of one or more domains selected from E domain, D domain, A domain, B domain or C domain of protein A A method comprising having an amino acid sequence substituted with a polar uncharged amino acid residue.

[2] The hydrophobic amino acid residue to be replaced is Phe at position 5 of the C domain of SEQ ID NO: 5, Phe at position 13, Leu at position 17, or Ile at position 31. [1]. the method of.

[3] The method according to [1] or [2] above, wherein the hydrophobic amino acid residue to be replaced is Ile at position 31 of the B domain of SEQ ID NO: 4.

[4] The method according to any one of [1] to [3] above, wherein the other hydrophobic amino acid residue is Ala, Leu, or Ile.

[5] The method according to any one of [1] to [4] above, wherein the polar uncharged amino acid residue is Thr.

[6] In the amino acid sequence of the ligand, Gln at the 9th position, Gln at the 10th position, Tyr at the 14th position, Pro at the 20th position, Asn at the 21st position, and 22th position in the C domain of SEQ ID NO: 5 Leu, No. 26 Gln, No. 27 Arg, No. 30 Phe, No. 34 Leu, No. 38 Pro, No. 39 Ser, No. 45 Leu, No. 51 Leu The method according to any one of [1] to [5] above, wherein 90% or more of amino acid residues corresponding to Asn at the 52nd position, Gln at the 55th position, and Pro at the 57th position are retained.

[7] The method according to any one of [1] to [6] above, wherein the ligand comprises a plurality of domains obtained by linking two or more of the domains.

[8] The method according to any one of [1] to [7] above, wherein the liquid sample is milk of a goat subfamily.

According to the method of the present invention, a liquid sample containing immunoglobulin G or a derivative thereof derived from a goat subgenus and immunoglobulin G or a derivative thereof derived from an animal other than a goat subfamily is separated to obtain an immunoglobulin G or The derivative can be efficiently produced. Therefore, the present invention is industrially very useful because it can contribute to efficient production of immunoglobulin G or its derivative.

FIG. 1 is an alignment of amino acid sequences of E domain, D domain, A domain, B domain and C domain of protein A.

Hereinafter, the method of the present invention will be described step by step.
1. Adsorption Step In this step, a liquid sample containing an immunoglobulin G or a derivative thereof derived from a subfamily of goats and an immunoglobulin G or a derivative thereof derived from an animal other than a subfamily of a goat that binds to wild-type protein A, By contacting the affinity separation matrix in which the ligand is immobilized on a carrier, immunoglobulin G or its derivative derived from an animal other than Goat subfamily is adsorbed to the ligand. Hereinafter, immunoglobulin G or a derivative thereof may be collectively referred to as “IgG”.

Goat subgenus is easier to handle than large livestock, has multiple fetuses, has a short generation change, and can efficiently obtain a target IgG from milk or the like by transformation. Examples of the goat subgenus include goat animals and sheep animals.

Animals other than Goats are not particularly limited as long as they produce a useful antibody. For example, humans; mice such as mice and rats; rabbits such as rabbits; bovines such as cows; equids such as horses; dogs such as dogs; cats such as cats; Examples include boar subspecies such as pigs; camelids such as camels, llamas, and alpaca.

The liquid sample used in this step is not particularly limited as long as it contains IgG derived from a caprine subgenus animal and IgG derived from an animal other than a caprine subgenus animal that binds to wild-type protein A. For example, a liquid sample such as blood, milk or egg white derived from a goat subfamily animal transformed to produce a target IgG derived from an animal other than a goat subfamily animal can be mentioned. In particular, when the target IgG gene is introduced by utilizing the promoter of milk protein gene such as casein gene, the target IgG is effectively secreted into milk. These may be pre-purified prior to contact with the ligand. For example, serum or plasma may be used as the blood sample.

Transformation of the above-mentioned animals may be carried out using a conventional method. For example, a method of microinjecting a target gene into the pronucleus of a fertilized egg of the above animal; a method of co-culturing a target IgG gene and sperm for in vitro fertilization or microfertilization; A method of obtaining a transformed animal via production; a method of introducing a target IgG gene into somatic cells or primordial germ cells (PGC), and further producing a cloned individual by somatic cell nuclear transfer can be mentioned.

A liquid sample may be a culture medium obtained by culturing cells transformed to produce a target IgG derived from an animal other than Goat subgenus in a medium containing serum of Goat subgenus. Transformation of cells can be performed by a conventional method.

In the present invention, the ligand for selectively adsorbing IgG derived from animals other than Goat subgenus is one or more selected from the E domain, D domain, A domain, B domain or C domain of protein A. It has an amino acid sequence in which one or more hydrophobic amino acid residues in the Fc binding site of the domain have been replaced with other hydrophobic amino acid residues or polar uncharged amino acid residues. The ligand has at least an affinity for a wild-type protein A or an IgG derived from a non-Goat subfamily animal that binds to the wild-type protein A, as compared with the wild-type protein A ligand. The affinity for IgG derived from the family gene is reduced. Hereinafter, Protein A may be abbreviated as “SpA”.

SpA is one of the cell wall proteins produced by the Gram-positive bacterium Staphylococcus aureus, and has a signal sequence S and five immunoglobulin-binding domains (E domain, D domain, A domain, B domain). , C domain) and an XM region that is a cell wall binding domain (Non-patent document 1). The E, D, A, B, and C domains of SpA are immunoglobulin binding domains capable of binding to regions of the immunoglobulin other than the complementarity determining regions (CDRs), and both domains are immunoglobulin It has an activity of binding to the Fc region, the Fab region, and particularly the Fv region in the Fab region. In the mutant SpA used in the present invention, a hydrophobic amino acid residue in the Fc binding site of one or more of the immunoglobulin binding domains is replaced with another hydrophobic amino acid residue or a polar uncharged amino acid residue. It has an amino acid sequence.

Further, the mutant SpA domain used in the present invention may be one domain selected from the above immunoglobulin-binding domains, or may be a multimer in which two or more domains are bound. The multimer may be a homo-multimer having one immunoglobulin-binding domain bound thereto or a hetero-multimer having two or more immunoglobulin-binding domains bound thereto. The number of domains in the immunoglobulin-binding domain multimer may be 2 or more, preferably 3 or more, more preferably 4 or more, more preferably 5 or more, and 10 or less. It is preferably 8 or less, more preferably 6 or less.

Examples of the method of linking each domain in the immunoglobulin-binding domain multimer include a method of linking with one or more amino acid residues and a method of directly linking without interposing amino acid residues. The number of amino acid residues for linking is not particularly limited, but is preferably 20 residues or less, more preferably 15 residues or less, even more preferably 10 residues or less, and still more preferably 5 residues or less. It is less than or equal to the residue, and even more preferably less than or equal to 2 residues. It is preferable that these amino acid sequences do not destabilize the three-dimensional structure of each domain.

The amino acid sequence of the mutant SpA or the mutant SpA domain having an amino acid sequence in which the hydrophobic amino acid residue in the Fc binding site is replaced with another hydrophobic amino acid residue or a polar uncharged amino acid residue is E of wild-type SpA. Domain, D domain, A domain, B domain or C domain, that is, an amino acid sequence partially modified by amino acid substitution, insertion, deletion and chemical modification without being limited to the amino acid sequences of SEQ ID NOS: 1 to 5. However, as long as it is a domain capable of binding to the Fc region, it is included in this. The number of such mutations (substitution, insertion, deletion and chemical modification) is preferably 10 or less, more preferably 5 or less, still more preferably 1 or 2. As the modified amino acid sequence, for example, from an amino acid sequence obtained by introducing a mutation for substituting Aly for Gly corresponding to position 29 of the C domain to the E, D, A, B, and C domains of wild-type SpA Is a protein. Further, the Z domain in which the mutations A1V and G29A are introduced into the B domain also has the ability to bind to the Fc region, and thus corresponds to the amino acid sequence derived from the domain. The amino acid sequence derived from the domain is preferably a domain having high chemical stability, or a variant thereof.

The mutant SpA and mutant SpA domain used in the present invention have the ability to bind to the Fc region. The amino acid sequences of these mutant SpA and mutant SpA domain preferably have 85% or more sequence identity with the amino acid sequences of wild-type SpA or wild-type SpA domain (SEQ ID NOs: 1 to 5). The sequence identity is preferably 90% or more, 92% or more, 93% or more, 94% or more or 95% or more, more preferably 96% or more, 97% or more, 98% or more or 99% or more, and 99. It is even more preferably 5% or more or 99.8% or more.

Amino acid substitution means a mutation that deletes the original amino acid and adds another amino acid of a different type at that position. Regarding the notation of a mutation that substitutes an amino acid, the wild-type or non-mutated amino acid is added before the number of the substitution position, and the mutated amino acid is added after the number of the substitution position. For example, the mutation that replaces Gly at position 29 with Ala is described as G29A.

The alignment of the amino acid sequences of E domain, D domain, A domain, B domain and C domain of wild-type SpA is shown in FIG. In FIG. 1, “−” indicates the same amino acid residue as the amino acid residue in the same row of the C domain, and the portion surrounded by a square frame indicates the Fc binding site. In the E domain (SEQ ID NO: 1), the hydrophobic amino acid residues at the Fc binding site are His at position 3, Phe at position 11, Leu at position 15, and Ile at position 29. In the D domain (SEQ ID NO: 2), the hydrophobic amino acid residues at the Fc binding site are Phe at position 8, Phe at position 16, Leu at position 20, and Ile at position 34. In the A domain (SEQ ID NO: 3), the hydrophobic amino acid residues at the Fc binding site are Phe at position 5, Phe at position 13, Leu at position 17, and Ile at position 31. In the B domain (SEQ ID NO: 4), the hydrophobic amino acid residues at the Fc binding site are Phe at position 5, Phe at position 13, Leu at position 17, and Ile at position 31. In the C domain (SEQ ID NO: 5), the hydrophobic amino acid residues at the Fc binding site are Phe at position 5, Phe at position 13, Leu at position 17, and Ile at position 31. In the present invention, one or more of the hydrophobic amino acid residues in the Fc binding site of the domain contained in the ligand are replaced with another hydrophobic amino acid residue or a polar uncharged amino acid residue.

Examples of the other hydrophobic amino acid residue that replaces the hydrophobic amino acid residue in the Fc binding site of SpA include Gly, Ala, Val, Leu, Ile, Met, Phe, and Trp, and among them, Ala, Val, Leu, Ile and Phe are preferable, and Ala, Leu, and Ile are more preferable. Here, the other hydrophobic amino acid residue means a hydrophobic amino acid residue different from the original hydrophobic amino acid residue to be replaced. For example, when the original amino acid residue to be replaced is Phe at the 5th or 13th position of the C domain, the other hydrophobic amino acid residue may be any other amino acid residue other than Phe among the other amino acid residues described above. Amino acid residues of. When the original amino acid residue to be replaced is Leu at position 17 of the C domain, other hydrophobic amino acid residues include amino acid residues other than Leu among the other amino acid residues described above. Can be mentioned. When the original amino acid residue to be replaced is Ile at position 31 of the C domain, the other hydrophobic amino acid residue may be an amino acid residue other than Ile among the above-mentioned other amino acid residues. Can be mentioned.

Examples of the polar uncharged amino acid residue that replaces the hydrophobic amino acid residue in the Fc binding site of SpA include Ser, Thr, Gln, Asn, Tyr, Cys, and among them, Ser, Thr, Gln, Asn, Tyr are preferable. , Thr are more preferable.

More specific substitution modes include substitution of Phe at position 5 of the C domain with Ala or Tyr, substitution of Phe at position 13 of the C domain with Tyr, and Ile of Leu at position 17 of the C domain, Val or Thr. To Leu, Ser, Thr or Val of Ile at position 31 of the C domain. Among these, F5A, F5Y in the C domain, F13Y in the C domain, L17I, L17V, L17T in the C domain, I31L, I31S, I31T, I31V in the C domain, and I31L, I31T in the B domain are preferable.

Further, as a more specific substitution mode, substitution of Ile at position 31 of the B domain with Leu can be mentioned. In addition, in the E domain, D domain, A domain and B domain, substitution at a position corresponding to the above substitution position in the C domain is also preferable. Corresponding positions between the domains can be grasped by the alignment shown in FIG.

The number of amino acid substitutions at the Fc-binding site is not particularly limited as long as the affinity for the Fc region is maintained or improved as compared with that before the substitution, but the three-dimensional structure of the protein before mutation introduction and the neutral region can be maintained. From the viewpoint of maintaining the antibody binding ability, 4 or less or 3 or less is preferable, and 2 or less or 1 is more preferable. Examples of the two amino acid substitution include, for example, substitution of L17I and I31L in the C domain, and substitution of amino acids at positions corresponding to positions 17 and 31 of the C domain in the E, D, A, or B domain in the same manner. There is a method of doing.

As long as the affinity for the Fc region is maintained or improved as compared with that before substitution, in addition to the substitution of the hydrophobic amino acid residue of the Fc binding site with another hydrophobic amino acid residue or a polar uncharged amino acid residue, It may contain arbitrary amino acid substitutions. Examples of such amino acid substitution include substitution of G29A in the C domain. Also, in the E, D, A, or B domain, substitution at a position corresponding to the position corresponding to position 29 of the C domain can be mentioned. Furthermore, examples of the arbitrary amino acid substitution include substitution of Val corresponding to the 40th position of the C domain with an amino acid residue other than Val, and specifically, substitution of V40Q in the C domain can be mentioned. Moreover, the substitution of a hydrophobic amino acid residue, an acidic amino acid residue, or a polar uncharged amino acid residue with a basic amino acid residue is mentioned, and specifically, A12R, L19R, L22R, Q26R, Q32R in the C domain, Substitutions at S33H, V40H or at corresponding positions in the E, D, A, or B domains.

An amino acid sequence obtained by substituting the hydrophobic amino acid residue at the Fc binding site with another hydrophobic amino acid residue or a polar uncharged amino acid residue, and E, D, and A of protein A described in SEQ ID NOS: 1 to 5. The sequence identity with the B, C, or C domain is preferably 85% or more. The sequence identity is preferably 90% or more, 92% or more, 93% or more, 94% or more or 95% or more, more preferably 96% or more, 97% or more, 98% or more or 99% or more, and 99. It is even more preferably 5% or more or 99.8% or more.

In the mutant SpA and mutant SpA domain used in the present invention, 90% or more of the amino acid residues at the positions of the C domain shown below and at the positions corresponding to the following positions of the E, D, A, and B domains are 92, % Or more, 93% or more, 94% or more or 95% or more is preferable, 96% or more, 97% or more, 98% or more or 99% or more is more preferable, 99.5% Or more or 99.8% or more is still more preferable. : 9th Gln, 10th Gln, 14th Tyr, 20th Pro, 21st Asn, 22nd Leu, 26th Gln, 27th Arg, 30th Phe, 34th Leu, 38th Pro, 39th Ser, 45 Leu, 51 Leu, 52 Asn, 55 Gln, and 57 Pro.

The mutant SpA and mutant SpA domain used in the present invention are preferably immobilized on a water-insoluble carrier as a ligand to form an affinity separation matrix. Examples of the water-insoluble carrier used in the present invention 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; crystalline cellulose, cross-linked cellulose, cross-linked agarose, Examples include organic carriers composed of polysaccharides such as cross-linked dextran; and composite carriers such as organic-organic and organic-inorganic obtained by combining these. As commercial products, GCL2000 which is a porous cellulose gel, Sephacryl S-1000 obtained by covalently cross-linking allyl dextran and methylene bisacrylamide, Toyopearl which is an acrylate carrier, Sepharose CL4B which is an agarose cross-linking carrier, and Cellulofine, which is a cellulosic cross-linking carrier, can be exemplified. However, 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 present affinity separation matrix, and is preferably porous 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 is immobilized on the water-insoluble carrier by a covalent bond, either directly or via a linker group. Examples of the linker group include a C _1-6 alkylene group, an amino group (-NH-), an ether group (-O-), a carbonyl group (-C(=O)-), an ester group (-C(= O)O- or -OC(=O)-), an amide group (-C(=O)NH- or -NHC(=O)-), a urea group (-NHC(=O)NH-); C _{1 -6 a} group in which 2 to 10 groups selected from the group consisting of an alkylene group, an amino group, an ether group, a carbonyl group, an ester group, an amide group and a urea group are linked; an amino group, an ether group, a carbonyl group, A C _1-6 alkylene group having a group selected from the group consisting of an ester group, an amide group and a urea group at one end or both ends can be mentioned. The number of connections is preferably 8 or less, or 6 or less, more preferably 5 or less, still more preferably 4 or less. The C _1-6 alkylene group may be substituted with a substituent such as a hydroxyl group.

Regarding the method of immobilizing the ligand, for example, the amino group, the carboxy group or the thiol group existing in the ligand may be used to bind to the carrier by a conventional coupling method. As the coupling method, cyanogen bromide, epichlorohydrin, diglycidyl ether, tosyl chloride, tresyl chloride, hydrazine or sodium periodate is reacted with the carrier to activate the carrier, or the carrier surface is used. A method of immobilizing a reactive functional group into a compound by immobilizing it with a compound to be immobilized as a ligand, a condensation reagent such as carbodiimide in a system in which a compound to be immobilized as a carrier and a ligand is present, or, An immobilization method may be mentioned in which a reagent having a plurality of functional groups in a molecule such as glutaraldehyde is added and condensed and crosslinked.

Further, 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 immobilization, the VL-κ binding peptide according to the present invention may be chemically modified, or amino acid residues useful for immobilization may be added. Examples of amino acids useful for immobilization include amino acids having a functional group useful for the chemical reaction of immobilization on the side chain, for example, Lys containing an amino group in the side chain, or a thiol group in the side chain. Cys including is mentioned. The essence of the present invention is that the VL-κ binding property imparted to the peptide in the present invention is also imparted to the matrix in which the peptide is immobilized as a ligand. Modifications are included in the scope of the present invention.

As a method for bringing the liquid sample and the affinity separation matrix into contact with each other, a conventional method can be used. For example, the liquid sample and the affinity separation matrix may be simply mixed, but from the viewpoint of efficiency, it is preferable to prepare an affinity column by packing the affinity separation matrix into the column and to flow the immunoglobulin-containing sample into the column. ..

The ligand used in the present invention has a relatively low affinity for IgG derived from a sub-subfamily of goats, while having a relatively high affinity for an IgG derived from an animal other than the sub-genus of Goats. Therefore, by contacting an affinity separation matrix containing such a ligand with a liquid sample containing IgG derived from a sub-subfamily of goats and IgG derived from a non-subfamily of goats, IgG of 1) can be selectively adsorbed to the ligand, and IgG derived from a goat subfamily and IgG derived from an animal other than a goat can be separated.

The liquid sample when contacting the liquid sample with the affinity separation matrix is preferably adjusted to be neutral. Specifically, the pH of the liquid sample is preferably 6.5 or more and 8.5 or less, more preferably 7 or more and 8 or less. The temperature at which the liquid sample is adsorbed on the affinity separation matrix is preferably 1°C or higher and 40°C or lower, more preferably 4°C or higher and 25°C or lower.

2. Washing step The affinity separation matrix in which IgG derived from an animal other than Goat subgenus is adsorbed and retained by the above selective adsorption step is washed, and in addition to IgG derived from Goat subgenus, Impurities other than IgG derived from animals are removed. At this point, IgG derived from an animal other than a sub-goat is adsorbed to the ligand.

In this washing step, the washing solution used for washing the affinity separation matrix is one that does not interfere with the interaction between the above-mentioned ligand and IgG derived from an animal other than Goats. For example, only a buffer solution having a pH of 6 or more and 8 or less can be used as the washing solution.

3. Elution step In this step, the acidic buffer is used to separate the IgG from the affinity separation matrix on which the IgG derived from an animal other than the subgenus Goat has been adsorbed. By this elution step, purified IgG derived from animals other than the subgenus Goat can be obtained.

In this elution, the pH of the acidic buffer solution used for separating IgG derived from animals other than Goat subgenus from the ligand may be appropriately adjusted, but, for example, should be about 2.0 or more and 4.0 or less. You can In addition, a substance that promotes dissociation from a ligand may be added to the acidic buffer solution used for eluting IgG derived from animals other than Goats.

4. Regeneration Step of Affinity Separation Matrix In this step, the affinity separation matrix is regenerated by washing the affinity separation matrix in which IgG derived from animals other than Goat subgenus has been separated in the elution step with an alkaline aqueous solution. However, this regeneration step does not necessarily have to be carried out after the elution step, and is carried out once every 3 times, once every 5 times, or once every 10 times of the selective adsorption step to the elution step. But it doesn't matter.

The "alkaline aqueous solution" used for the regeneration of the affinity separation matrix is an aqueous solution that is alkaline enough to achieve the purpose of washing or sterilization. More specifically, 0.01 M or more and 1.0 M or less, or 0.01 N or more and 1.0 N or less sodium hydroxide aqueous solution or the like is applicable, but is not limited thereto. When sodium hydroxide is taken as an example, the lower limit of the concentration is preferably 0.01M, more preferably 0.02M, and even more preferably 0.05M. On the other hand, the upper limit of the concentration of sodium hydroxide is preferably 1.0 M, more preferably 0.5 M, even more preferably 0.3 M, even more preferably 0.2 M, even more preferably 0.1 M. The alkaline aqueous solution need not be an aqueous sodium hydroxide 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. With respect to the upper limit of pH, 14 or less is preferable, 13.5 or less is still more preferable, and 13.0 or less is even more preferable.

The time for treating the affinity separation matrix that has undergone the elution step with the alkaline aqueous solution is not particularly limited and may be appropriately adjusted, because the peptide damage is different depending on the concentration of the alkaline aqueous solution and the temperature during the treatment. For example, when 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 for immersion in the alkaline aqueous solution is preferably 1 hour, more preferably 2 hours, more preferably 4 hours, and more preferably 10 hours. The time is more preferable, and the upper limit is preferably 20 hours, but it is not particularly limited.

The ligand immobilized on the affinity separation matrix according to the present invention has a relatively high affinity for IgG derived from animals other than Goat and also to IgG derived from Goat. Show a relatively low affinity, for example, from a sample obtained from a goat subfamily transformed to produce IgG derived from an animal other than a goat subfamily, a goat subfamily Other animal-derived IgG can be selectively adsorbed. Therefore, by the selective adsorption step, washing step, and elution step, it is possible to effectively purify IgG derived from an animal other than the subfamily Goat from the transformed animal.

Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.

In the following examples, the production and manipulation of recombinant DNA were carried out according to the following experimental manuals unless otherwise specified.
Reference 1: T. Maniatis et al., "Molecular Cloning/A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory (USA), 1989.
Reference 2: Masamitsu Muramatsu, "Lab Manual, Genetic Engineering, 3rd Edition", Maruzen Co., Ltd., 1996

Further, with respect to the reagents and restriction enzymes used in the following examples, commercially available products were used unless otherwise specified.

The various proteins obtained in the examples are described in the form of "alphabet indicating domain-introduced mutation". The wild-type protein in which the mutation has not been introduced is denoted by “Wild” instead of “introduced mutation”. For example, the wild-type C domain of protein A is referred to as “C-wild”, and the C domain mutant introduced with the mutation G29E is referred to as “C-G29E”. The notation of a mutant in which two kinds of mutations are introduced at the same time is described together using a slash. For example, a C domain mutant into which the mutation G29E and the mutation S13L are introduced is referred to as "C-G29E/S13L". In addition, a protein in which a plurality of single domains are linked is described by adding a "d" to the linked number after a period. For example, a protein in which a C domain mutant having a mutation G29E and a mutation S13L introduced therein is linked by 5 is referred to as “C-G29E/S13L.5d”.

Example 1: Evaluation of antibody binding ability of protein A ligand Using a biosensor utilizing surface plasmon resonance ("Biacore 3000" manufactured by GE Healthcare), affinity between protein A ligand and immunoglobulin was analyzed. In the present Example 1, a protein A ligand standard for leak measurement (“KANEKA KanCapA ^™ 3G” manufactured by Kaneka) and commercially available antibodies derived from various animals were used. In addition, according to the method described in JP-A-2006-320220, the portion obtained by removing a part of the signal sequence (S domain) and the cell wall binding domain (X domain) from the protein A derived from Staphylococcus aureus ATCC6538P strain is used. It was produced by gene recombination and purified by chromatography to obtain wild type protein A for use. Although the amino acid sequences of KANEKA KanCapA ^™ and KANEKA KanCapA ^™ 3G have not been published, at least KANEKA KanCapA ^™ 3G has the amino acid sequence of the characteristics described in WO2017/014257, Amino acid sequence derived from the E, D, A, B or C domain of protein A of A., in which the hydrophobic amino acid residue at the Fc binding site is replaced with another hydrophobic amino acid residue or a polar uncharged amino acid residue Including protein A having In addition, a control protein A without the above characteristics was also used.
Various proteins A were immobilized on the sensor chip, and various antibodies were flown on the chip to detect the interaction between the two. The protein A ligand standard was immobilized on the sensor chip CM5 by amine coupling using N-hydroxysuccinimide (NHS) and N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). And blocking was performed using ethanolamine according to the protocol attached to Biacore 3000. The sensor chip and the immobilization reagent used were all manufactured by GE Healthcare. A reference cell as a negative control was also prepared by performing a treatment of immobilizing ethanolamine on another flow cell on the chip after activation with EDC/NHS.
Specifically, various antibodies are dissolved in a running buffer solution (20 mM NaH ₂ PO ₄ —Na ₂ HPO ₄ , 150 mM NaCl, 0.005% P-20, pH 7.4), and the concentration of the antibody is adjusted within the range of 10 to 1000 nM. Three different solutions were prepared, and each antibody solution was added to the sensor chip for 30 seconds at a flow rate of 20 μL/min. At a measurement temperature of 25° C., binding reaction curves during addition (binding phase, 30 seconds) and after addition (dissociation phase, 60 seconds) were sequentially observed. After completion of each observation, 10 mM glycine hydrochloride solution (GE Healthcare, pH 2.0) was added for 30 seconds to remove the added antibody remaining on the sensor chip and regenerate the sensor chip. It was confirmed that the binding activity of the immobilized human IgG was almost completely restored.
The obtained binding reaction curve (binding reaction curve obtained by subtracting the binding reaction curve of the reference cell) was subjected to fitting analysis using a binding model of 1:1 using system-attached software BIA evaluation, and the binding rate constant (k _on ), dissociation rate constant (k _off ), and affinity constant (K _A =k _on /k _off ). The results are shown in Table 1.

As shown in the results shown in Table 1, the protein A ligand of KANEKA KanCapA ^™ 3G according to the present invention had a markedly reduced antibody binding ability to Goat IgG and Sheep IgG. On the other hand, the wild type protein A and the control protein A ligand were confirmed to have the binding ability to Goat IgG and Sheep IgG. These results indicate that antibodies against Goat IgG and Sheep IgG are mutated by substitution of the hydrophobic amino acid residue in the Fc binding site introduced into KANEKA KanCapA ^™ 3G with another hydrophobic amino acid residue or a polar uncharged amino acid residue. It was shown that the binding ability was reduced.

Example 2: Evaluation of antibody binding ability of C domain mutant of protein A C-G29A. A modified form of the DNA encoding 2d (SEQ ID NO: 6) having the PstI recognition site at the 5′ end and the XbaI recognition site at the 3′ end (SEQ ID NO: 7) having the amino acid substitution mutations shown in Table 2 introduced therein. C-G29A. The artificial synthetic gene of 2d was totally synthesized by outsourcing to Eurofin Genomics. The expression plasmid after subcloning into which each artificially synthesized gene is introduced is digested with restriction enzymes PstI and XbaI (manufactured by Takara Bio Inc.), and the obtained DNA fragment is digested with the same restriction enzyme to express Brevibacillus expression vector pNCMO2 (Takara Bio). (Manufactured by K.K.) and modified C-G29A. An expression plasmid in which a DNA encoding the amino acid sequence of 2d was inserted was prepared. The Escherichia coli JM109 strain was used for plasmid preparation.
Brevibacillus choshinensis SP3 strain (Takara Bio Inc.) was transformed with the obtained plasmid, and modified C-G29A. A gene recombinant that secretes and produces 2d was bred. 30 mL of A medium containing 60 μg/mL neomycin (polypeptone 3.0%, yeast extract 0.5%, glucose 3%, magnesium sulfate 0.01%, iron sulfate 0.001%, Manganese chloride (0.001%, zinc chloride 0.0001%) was shake-cultured at 30°C for 3 days. After culturing, the culture solution was centrifuged at 15,000 rpm at 25° C. for 5 minutes to remove the cells, and then the modified C-G29A. 2d and C-G29A. 2d purified product was obtained. The purified product was analyzed by high performance liquid chromatography to measure the concentration of each C domain mutant.
Next, using a biosensor utilizing surface plasmon resonance ("Biacore 3000" manufactured by GE Healthcare), the affinity of each obtained C domain mutant for immunoglobulin was analyzed. Human IgG, Goat IgG, and Sheep IgG were each immobilized on a sensor chip, and each C domain mutant was flown on the chip to detect the interaction between the two. Immobilization of IgG on the sensor chip CM5 was performed by an amine coupling method using N-hydroxysuccinimide (NHS) and N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). For blocking, ethanolamine was used. The sensor chip and the immobilizing reagent used were all manufactured by GE Healthcare. The IgG solution was prepared by dissolving it in a standard buffer solution (20 mM NaH ₂ PO ₄ —Na ₂ HPO ₄ , 150 mM NaCl, pH 7.4) at 1.0 mg/mL. The IgG solution was diluted 100 times with an immobilization buffer (10 mM CH ₃ COOH-CH ₃ COONa, pH 5.0), and IgG was immobilized on the sensor chip according to the protocol attached to Biacore 3000. A reference cell as a negative control was also prepared by performing a treatment of immobilizing ethanolamine on another flow cell on the chip after activation with EDC/NHS.
Each C domain mutant was dissolved in a running buffer (20 mM NaH ₂ PO ₄ —Na ₂ HPO ₄ , 150 mM NaCl, 0.005% P-20, pH 7.4), and the concentration was varied in the range of 10 to 1000 nM. A seed solution was prepared, and each C domain mutant solution was added to the sensor chip for 30 seconds at a flow rate of 20 μL/min. At a measurement temperature of 25° C., binding reaction curves during addition (bonding phase, 30 seconds) and after addition (dissociation phase, 60 seconds) were sequentially observed. After the completion of each observation, a 10 mM glycine hydrochloride solution (GE Healthcare, pH 3.0) was added for 30 seconds to remove the added antibody remaining on the sensor chip and regenerate the sensor chip. It was confirmed that the binding activity of the immobilized human IgG was almost completely restored.
The obtained binding reaction curve (binding reaction curve obtained by subtracting the binding reaction curve of the reference cell) was subjected to fitting analysis using a 1:1 binding model using system-attached software BIA evaluation, and the binding rate constant (k _on ), a dissociation rate constant (k _off ), and an affinity constant (K _A =k _on /k _off ). The results are shown in Table 2.

As shown in the results shown in Table 2, the modified C-G29A. The binding parameters for 2d human IgG are C-G29A. It was comparable to 2d (control). The 29th position glycine in the C domain of protein A (SEQ ID NO: 5) is contained in the Fab binding site.
On the other hand, in addition to the mutation G29A, the hydrophobic amino acid residue at position 5, 29 or 31 of SEQ ID NO: 5 contained in the Fc binding site was replaced with another hydrophobic amino acid residue or a polar uncharged amino acid residue. To the modified C-G29A. The binding capacity of 2d to Goat IgG and Sheep IgG was found to be undetectably reduced.

Example 3: Evaluation of antibody binding ability of B domain mutant B-G29A. Modified form of the DNA encoding 2d (SEQ ID NO: 8) having the PstI recognition site at the 5'end and the XbaI recognition site at the 3'end and having the amino acid substitution mutations shown in Table 3 introduced therein. B-G29A. The artificial synthetic gene of 2d was totally synthesized by outsourcing to Eurofin Genomics. Then, in the same manner as in Example 2, the modified B-G29A. 2d and B-G29A. 2d purified product was obtained. The purified product was analyzed by high performance liquid chromatography to measure the concentration of each B domain mutant. The affinity of the B domain mutant for immunoglobulin was analyzed in the same manner as in Example 2. The results are shown in Table 3.

As shown in the results shown in Table 3, the modified B-G29A. The binding parameters for 2d human IgG are B-G29A. It was comparable to 2d (control). The glycine at the 29th position in the B domain of protein A (SEQ ID NO: 4) is contained in the Fab binding site.
On the other hand, in addition to the mutated G29A, the modified B-G29A.I according to the present invention in which isoleucine at position 31 of SEQ ID NO: 4 contained in the Fc binding site was replaced with leucine. It was found that the binding ability of 2d to Goat IgG and Sheep IgG was undetectably decreased as in the case of Example 2.

Example 4: Antibody purification experiment from serum using a commercially available carrier Assuming production of an antibody using a transgenic goat or transgenic sheep, the affinity of the mutant according to the present invention with a goat-derived antibody or a sheep-derived antibody Was measured. In the amino acid sequences derived from the E, D, A, B or C domain of protein A set forth in SEQ ID NOs: 1 to 5, the hydrophobic amino acid residue at the Fc binding site is replaced with another hydrophobic amino acid residue or a polar uncharged amino acid. Antibody purification from goat serum (Gibco) and sheep serum (Sigma) using KANEKA KanCapA ^™ 3G (Kaneka), which is a carrier carrying protein A having an amino acid sequence substituted with a residue. Experiments were conducted. As a control, a protein A chromatographic carrier KANEKA KanCapA ^™ (manufactured by Kaneka) without the above mutation was also tested.
Specifically, a column (“Tricorn” GE Healthcare, 0.5 cm diameter×5 cm height) was filled with 1 mL of each carrier, and 3 column volumes (CV) of Dulbecco's phosphate buffered saline (Sigma· Aldrich) was flowed at a flow rate of 1 mL/min to equilibrate the column. Each serum was diluted 5 times with Dulbecco's phosphate-buffered saline (manufactured by Sigma-Aldrich), and 5 mL was flown at a flow rate of 0.25 mL/min. Then, the flow rate is returned to 1 mL/min, 3 CV of equilibration buffer is flown, then 5 CV of 50 mM sodium citrate buffer (pH 6.0) is flown to wash the column, and further 5 CV of 50 mM sodium citrate buffer. The antibody was eluted by flowing a liquid (pH 3.0). The antibody recovery rate was calculated by measuring the absorbance of the eluate. Table 4 shows the results of the dynamic binding capacity of each protein A to human IgG described in the product catalog and the amount of antibody recovered from each serum. In addition, when the product catalog value of the dynamic binding capacity for human IgG is 100%, the recovery rate of each antibody is shown in parentheses as a relative value.

As shown in the results shown in Table 4, KANEKA KanCapA ^™ 3G in which a specific mutation was introduced at the Fc binding site of protein A was compared to KANEKA KanCapA ^™ in which such a mutation was not introduced, from goat serum and sheep serum. The recovery rate of goat IgG and sheep IgG was low, which was 20% or less as compared with the dynamic binding capacity for human IgG. From this result, it was found that the affinity separation matrix loaded with KANEKA KanCapA ^™ 3G is a protein A carrier capable of efficiently separating human IgG from goat-derived IgG or sheep-derived IgG.

Claims (8)

A method for producing immunoglobulin G or a derivative thereof, comprising:
A ligand immobilizes a liquid sample containing immunoglobulin G or a derivative thereof from a goat subgenus and immunoglobulin G or a derivative thereof from a non-goat animal that binds to wild-type protein A on a carrier. Adsorbing an immunoglobulin G or a derivative thereof derived from an animal other than a goat subfamily on a ligand by contacting with the affinity separation matrix prepared above,
Washing the affinity separation matrix, and
A step of separating immunoglobulin G or a derivative thereof derived from an animal other than a sub-goat animal adsorbed to the ligand from the ligand using an eluent,
The ligand is one or more hydrophobic amino acid residues in the Fc binding site of one or more domains selected from E domain, D domain, A domain, B domain or C domain of protein A A method comprising having an amino acid sequence substituted with a polar uncharged amino acid residue. The method according to claim 1, wherein the hydrophobic amino acid residue to be replaced is Phe at position 5, Phe at position 13, Leu at position 17, or Ile at position 31 of the C domain of SEQ ID NO: 5. The method according to claim 1 or 2, wherein the hydrophobic amino acid residue to be replaced is Ile at position 31 of the B domain of SEQ ID NO:4. The method according to claim 1, wherein the other hydrophobic amino acid residue is Ala, Leu, or Ile. The method according to claim 1, wherein the polar uncharged amino acid residue is Thr. In the amino acid sequence of the ligand, Gln at the 9th position, Gln at the 10th position, Tyr at the 14th position, Pro at the 20th position, Asn at the 21st position, and Leu at the 22nd position in the C domain of SEQ ID NO:5, 26th Gln, 27th Arg, 30th Phe, 34th Leu, 38th Pro, 39th Ser, 45th Leu, 51st Leu, 52nd The method according to claim 1, wherein 90% or more of the amino acid residues corresponding to Asn at the position, Gln at the 55th position, and Pro at the 57th position are retained. The method according to any one of claims 1 to 6, wherein the ligand comprises a plurality of domains obtained by linking two or more of the domains. The method according to any one of claims 1 to 7, wherein the liquid sample is milk of a subfamily of Goats.

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