Method for producing an antibody containing a κ chain

Abstract

To provide a method for producing a κ chain containing antibody, which has high avidity to an antigen, at high purity and simply.SOLUTION: A method for producing an antibody including a κ chain includes: a process of allowing liquid sample including the antibody to pass through a column into which a carrier carrying protein L as ligand is filled; and a process of allowing eluate to pass through the column and eluting the antibody adsorbed by the carrier, where the liquid sample includes an isomer having the same or approximately the same molecular weight as molecular weight of the antibody and / or an aggregate of the antibody or the isomer, and the liquid sample in which the total amount of the antibody, isomer and aggregate exceeds an adsorption capacity of the column is allowed to pass through the column.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for conveniently producing a highly pure κ chain-containing antibody having high antigen-binding activity. [Background technology]

[0002] Compared to small molecule drugs, antibody drugs can pinpoint and attack abnormal cells, so there is less risk of damaging normal cells, and they are expected to have high therapeutic effects and reduced side effects. In addition, antibody drugs have a relatively high in vivo stability, so they remain effective in the body for a long time. For this reason, the development of antibody drugs has become more active in recent years.

[0003] Among antibody drugs, monoclonal antibodies are generally produced from animal cells such as Chinese hamster ovarian (CHO) cells and human embryonic kidney (HEK) cells, or from hybridomas. Minibodies that can be produced using transformed microbial cells have also been developed.

[0004] When producing antibody drugs using cells, the culture medium containing the antibody drug contains not only medium components but also a large amount of impurities such as cell-derived proteins (HCPs). Therefore, affinity column chromatography and ion exchange chromatography are used in combination to purify the target antibody from the cell culture medium.

[0005] The ligands used in affinity column chromatography are mainly Protein A, which specifically binds to the Fc region of immunoglobulin, Protein G, which specifically binds to the Fc region of immunoglobulin and weakly binds to Fab fragments, and Protein L, which specifically binds to the κ chain of immunoglobulin. However, as described above, cell culture fluid contains a large amount of impurities, so studies have been conducted to improve the efficiency of purification by column chromatography. For example, Patent Document 1 describes an invention in which a protein is adsorbed to Protein A or the like and then washed with a washing buffer containing an aliphatic carboxylate. In addition, the present inventors have developed an invention in which an antibody adsorbed to Protein L is efficiently eluted by using an acetate buffer or by continuously or stepwise lowering the pH (Patent Documents 2 and 3). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2020-19795 A [Patent Document 2] International Publication No. 2017 / 195638 Brochure [Patent Document 3] International Publication No. 2020 / 066270 Brochure Summary of the Invention [Problem to be solved by the invention]

[0007] As mentioned above, Protein L is used as a ligand for affinity columns for purifying antibodies, and various conditions for efficiently purifying antibodies by affinity chromatography using Protein L have been investigated.

[0008] However, the inventors have found that when producing antibodies using cells, substances with low binding activity to the antigen are produced as by-products, even though they have the same or nearly the same molecular weight as the target antibody, and even if the target antibody is purified by affinity chromatography, these substances may become contaminated with the target antibody and reduce the binding activity of the target antibody to the antigen.

[0009] Therefore, an object of the present invention is to provide a method for easily producing a highly pure κ chain-containing antibody having high antigen-binding activity. [Means for solving the problem]

[0010] The present inventors have conducted extensive research to solve the above problems. As a result, they have found that a substance having the same or nearly the same molecular weight as a target antibody containing a κ chain but low antigen-binding activity has a relatively low affinity for Protein L compared to the target antibody. Therefore, the present inventors have found that, while a crude composition in an amount exceeding the adsorption capacity of the column is not usually loaded onto the column in order to suppress loss of the target antibody when purifying the target antibody by affinity chromatography, if a crude product in an amount exceeding the adsorption capacity of the column is loaded onto the Protein L column, the substance will flow out of the column first, and a target antibody with higher binding activity can be obtained in high purity, thereby completing the present invention. The present invention will now be described.

[0011] [1] A method for producing an antibody containing a κ chain, comprising the steps of: passing a liquid sample containing the antibody through a column packed with a carrier carrying Protein L as a ligand; and passing an elution solution through the column to elute the antibody adsorbed to the carrier; the liquid sample contains an isomer having a molecular weight identical or substantially identical to the molecular weight of the antibody and / or an aggregate of the antibody or the isomer; The method comprises passing the liquid sample through the column, the liquid sample being such that the combined amount of the antibody, the isomer and the aggregate exceeds the adsorption capacity of the column. [2] The method according to [1], wherein the amount of the antibody contained in the liquid sample is 80% or more of the adsorption capacity of the column. [3] The method according to [1] or [2] above, wherein the binding activity of the antibody to the antigen is higher than the binding activity of the isomer and the aggregate to the antigen. [4] The method according to any one of [1] to [3] above, wherein the pH of the liquid sample is 5.5 or more and 9 or less, and the pH of the elution solution is 5 or less. [5] The method according to any one of [1] to [4] above, wherein the antibody is a minibody. [6] The method according to [5] above, wherein the minibody is a single-chain antibody. Effect of the Invention

[0012] According to the method of the present invention, a κ chain-containing antibody having high antigen-binding activity can be purified by a simple procedure of loading a liquid sample containing the antibody in an amount exceeding the binding capacity of a Protein L column, thereby making it possible to efficiently produce a highly pure and highly active target antibody. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a chromatograph obtained when 50 mL of a liquid sample containing scFv was added to a column packed with 1 mL-gel of Protein L carrier. [Diagram 2] FIG. 2 is a photograph of a gel showing the results of SDS-PAGE of each fraction obtained by adding 50 mL of an scFv-containing liquid sample to a column packed with 1 mL-gel of Protein L carrier. [Diagram 3] FIG. 3 is a graph showing the binding activity to Protein L of the flow-through fraction and the eluted fraction obtained by adding 50 mL of an scFv-containing liquid sample to a column packed with 1 mL-gel of Protein L carrier. [Figure 4]FIG. 4 is a graph showing the antigen-binding activity of the flow-through fraction and the eluted fraction obtained by adding 50 mL of an scFv-containing liquid sample to a column packed with 1 mL-gel of Protein L carrier. [Diagram 5] FIG. 5 is a chromatograph obtained when 50 mL of a liquid sample containing scFv was added to a column packed with 1 mL-gel of Protein L carrier. [Figure 6] FIG. 6 is a photograph of a gel showing the results of SDS-PAGE of each fraction obtained by adding 50 mL of an scFv-containing liquid sample to a column packed with 1 mL-gel of Protein L carrier. [Figure 7] FIG. 7 is a graph showing the binding activity to Protein L of the flow-through fraction and the eluted fraction obtained by adding 50 mL of an scFv-containing liquid sample to a column packed with 1 mL-gel of Protein L carrier. [Figure 8] FIG. 8 is a graph showing the antigen-binding activity of the flow-through fraction and the eluted fraction obtained by adding 50 mL of an scFv-containing liquid sample to a column packed with 1 mL-gel of Protein L carrier. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] In the method of the present invention, an antibody containing a κ chain is purified to a high purity using a column packed with a carrier carrying protein L as a ligand. Each step of the method of the present invention will be described below.

[0015] 1. Manufacturing process for antibodies containing κ chains In this step, an antibody containing a κ chain is produced mainly using cells. However, the implementation of this step is optional, and if a liquid sample containing the antibody is already available, this step does not need to be performed.

[0016] The method of the present invention produces antibodies containing a κ chain. In the present disclosure, antibodies containing a κ chain may be abbreviated as "κ chain-containing antibodies". Antibody light chains include κ chains and λ chains, and the variable region genes of the κ chains are a combination of genes selected one each from approximately 40 types of light chain variable region genes V and approximately 5 types of light chain variable region genes J, with the ratio of κ chains to λ chains differing depending on the organism species.

[0017] The subject of the production by the method of the present invention is not particularly limited as long as it is an antibody containing a κ chain, and may be a minibody. Examples of the antibody include IgM, IgD, IgG, IgA, and IgE; Fab; and F(ab') which is a Fab dimer. 2 Fab trimer, F(ab') 3 dsFv in which the VH chain and the VL chain are linked by a disulfide bond; single-chain antibody (scFv) in which the VH chain and the VL chain are linked by a peptide linker; multimers of single-chain antibodies such as diabody, which is a dimer of scFv, triabody, which is a trimer of scFv, and tetrabody, which is a tetramer of scFv; fusion proteins in which different low molecular weight antibodies are linked, such as scFv-Fc in which scFv and Fc are linked, scFv-Fab in which scFv and Fab are linked, and minibody in which scFv and CH3 are linked. In addition, antibody fragments in which a light chain or a light chain fragment containing a κ chain and a heavy chain or a heavy chain fragment containing a heavy chain variable region are associated with each other without a covalent bond may also be purified by the method of the present invention under certain conditions. Examples of such antibody fragments include Fv in which a heavy chain variable region is associated with a κ chain variable region. The target of production by the method of the present invention is preferably a single-chain antibody or a multimer of single-chain antibodies.

[0018] The peptide linker for linking the antibody fragment is not particularly limited, and examples thereof include peptide linkers containing 5 to 25 amino acid residues. Examples of such peptide linkers include a GS linker having a repeating sequence of glycine and serine.

[0019] Antibodies can be produced using standard methods. For example, B cells are extracted from the spleen or lymph nodes of an animal immunized with an antigen, and the B cells are fused with myeloma to obtain hybridomas, from which cells that produce antibodies that react with the antigen are selected and cultured, to obtain the desired monoclonal antibody. Minibodies can also be produced using bacterial cells or yeast cells. Specifically, for example, the amino acid sequences of each chain constituting a minibodies including a κ chain are designed, and then a base sequence encoding the amino acid sequence is designed by reverse translation. At this time, a base sequence encoding the amino acid sequence of a minibodies in which each chain is covalently bonded with a peptide linker may be designed. DNA encoding the base sequence is chemically synthesized and inserted into a vector such as a plasmid. The vector is introduced into a cell to obtain a transformant, and the transformant is cultured to produce the desired minibodies. Alternatively, minibodies may be produced by an enzymatic reaction using papain, pepsin, or the like after producing IgG or the like.

[0020] The liquid sample containing the κ chain-containing antibody is not particularly limited as long as it contains the κ chain-containing antibody and is liquid, and examples thereof include the culture solution of the above-mentioned hybridoma or transformant, its homogenate, or its extract, from which insoluble components such as bacteria cells have been removed by filtration or centrifugation. When the above-mentioned enzyme reaction is performed, the reaction solution itself after the reaction, or a solution obtained by removing insoluble components from the reaction solution, may be used as the liquid sample.

[0021] When each chain constituting a minibody is produced by a cell, the chains may be bonded by chemical reaction in the culture supernatant to produce a minibody. In this case, the reaction solution itself after the reaction, or a solution obtained by removing insoluble components from the reaction solution, may be used as a liquid sample.

[0022] 2.Adsorption process In this step, a liquid sample containing the antibody is passed through a column packed with a carrier that supports Protein L as a ligand, thereby selectively adsorbing the target antibody to Protein L. In the present invention, the liquid sample contains an isomer having the same or approximately the same molecular weight as the molecular weight of the antibody and / or an aggregate of the antibody or the isomer, and the liquid sample is passed through the column in such a manner that the total amount of the antibody, the isomer, and the aggregate exceeds the adsorption capacity of the column.

[0023] The present inventors have found that when cells are made to produce an antibody, isomers or aggregates that have the same or nearly the same molecular weight as the target antibody but have inferior antigen-binding activity to the target antibody are produced as by-products, and such isomers or aggregates contaminate the target antibody, reducing the binding activity of the target antibody. "Nearly the same molecular weight" refers to a molecular weight that differs from the molecular weight of the target antibody to such an extent that it cannot be clearly distinguished by SDA-PAGE analysis, and is not particularly limited to a molecular weight that differs from the molecular weight of the target antibody by within ±10%.

[0024] The isomer is not particularly limited, and may be, for example, a misfolded, modified, or unmodified form of the target antibody. In addition, the aggregates can be removed by filtration or centrifugation if they are clearly insoluble, but the aggregates in this case are considered to be so fine that they cannot be separated from a liquid sample by filtration or centrifugation.

[0025] The isomers and aggregates are difficult to separate from the target antibody by normal affinity chromatography or ion exchange chromatography. However, the present inventors found that the affinity of the isomers and aggregates for Protein L is lower than the affinity of the target antibody for Protein L. Therefore, in normal affinity chromatography, a sample exceeding the adsorption capacity of the column is not loaded from the viewpoint of production efficiency. In contrast, in the present invention, a liquid sample containing the antibody, isomers, and aggregates in total exceeding the adsorption capacity of the column is loaded onto the column, so that the isomers and aggregates are eluted from the column first, and a target antibody of higher purity is obtained.

[0026] In the present invention, a column is used that is packed with a carrier that carries as a ligand Protein L. Protein L is a 35.8 kDa protein derived from Peptostreptococcus magnus, and specifically binds to immunoglobulins, particularly the κ light chain of IgG.

[0027] The material of the column carrier is not particularly limited as long as it is insoluble in the solvent of the liquid sample such as water, and examples thereof include polysaccharides such as cellulose, agarose, dextran, starch, pullulan, chitosan, and chitin; synthetic polymers such as poly(meth)acrylic acid, poly(meth)acrylic acid esters, polyacrylamide, and polyvinyl alcohol; and glasses such as silica glass, borosilicate glass, optical glass, and soda glass. In addition, the surface of a substrate made of a synthetic polymer having no functional group, such as polystyrene or a styrene-divinylbenzene copolymer, may be coated with a polymer material having a reactive functional group such as a hydroxyl group. Examples of such polymer materials for coating include graft copolymers such as hydroxyethyl methacrylate and copolymers of a monomer having a polyethylene oxide chain and another polymerizable monomer having a reactive functional group. Among the above materials, polysaccharides and polyvinyl alcohol are preferably used because active groups can be easily introduced to the substrate surface. Protein L can be supported on the column carrier via a functional group possessed by the column carrier material, or further via a linker group such as an epoxy group or a formyl group.

[0028] The adsorption capacity of a column refers to the amount of the protein L-supported carrier packed in the column that can adsorb the adsorption target. For example, it refers to the amount obtained by multiplying the amount of the adsorption target per tapping volume of the protein L-supported carrier packed in the column by the tapping volume of the protein L-supported carrier packed in the column. The tapping volume refers to the volume of the carrier in a state in which a slurry containing the carrier and a dispersion medium such as water is placed in a measuring container and allowed to settle while being vibrated until the volume no longer decreases.

[0029] If a catalog value of the dynamic binding capacity of the column product used is available, the catalog value may be used as the adsorption capacity of the column. If no catalog value is available, the adsorption capacity of the same Protein L carrier varies depending on the target, so the dynamic binding capacity may be measured as the adsorption capacity. For example, a column packed with a Protein L-supported carrier is equilibrated, a liquid sample containing a κ-chain-containing antibody is passed through at a constant flow rate, and the amount of the liquid sample passed through until the absorbance of the κ-chain-containing antibody in the eluate exceeds 5% of the absorbance of the κ-chain-containing antibody in the passed liquid sample is determined, and the total amount of the κ-chain-containing antibody contained in that amount of liquid sample can be used as the dynamic binding capacity of the column.

[0030] The liquid sample used in the present invention contains, in addition to the target antibody, an isomer having the same or approximately the same molecular weight as the target antibody and / or an aggregate of the antibody or the isomer, which has a lower binding activity to an antigen than the target antibody.

[0031] The term "approximately the same molecular weight as that of the target antibody" is not particularly limited, but refers to a molecular weight that cannot be clearly distinguished from the molecular weight of the target antibody in SDS-PAGE analysis, and examples of such molecular weights include a molecular weight within the range of ±10% of the molecular weight of the target antibody. In addition, in SDS-PAGE, the aggregates are also considered to be dissociated into the target antibody or isomers by SDS. In addition, while general aggregates are insoluble, insoluble aggregates can be separated from liquid samples by filtration or centrifugation, so the aggregates are considered to be soluble or fine aggregates that cannot be removed by filtration or centrifugation.

[0032] In the present invention, a liquid sample containing the antibody, isomers and aggregates in a total amount exceeding the adsorption capacity of the column is passed through the column. The binding activity of the isomers and aggregates to the antigen is lower than that of the antibody, and the binding activity of the isomers and aggregates to protein L is also lower than that of the antibody. Therefore, by passing a sufficient amount of liquid sample through the column, the target antibody is preferentially adsorbed to the protein L in the column, and the isomers and aggregates are discharged, making it possible to obtain an antibody with high purity and high binding activity to the antigen. Preferably, a liquid sample containing the antibody in an amount exceeding the adsorption capacity of the column is passed through the column. As a result, although the production efficiency of the antibody is somewhat reduced, an antibody with higher purity and binding activity is obtained. More specifically, a liquid sample containing the antibody, isomers and aggregates in a total amount exceeding 100% and not exceeding 200% of the adsorption capacity of the column (100%) is passed through the column. The ratio is preferably 105% or more, more preferably 110% or more. It is also preferable that the liquid sample containing the antibody is passed through the column in an amount greater than 80% and not more than 110% of the adsorption capacity of the column (100%), and the proportion is preferably not more than 105%.

[0033] The loading volume of the liquid sample onto the column can be, for example, 2 to 2000 times the tapping volume of the Protein L-loaded monolayer packed into the column. The ratio is preferably 5 times or more, more preferably 10 times or more, even more preferably 20 times or more, and preferably 1000 times or less, more preferably 500 times or less, and even more preferably 200 times or less. The higher the ratio, the easier it is to obtain an antibody with high purity and binding activity, and the lower the ratio, the higher the production efficiency.

[0034] The total amount and concentration of the antibody, isomers and aggregates in a liquid sample can be roughly determined, for example, by comparing the band intensity of the target antibody with that of a molecular weight marker in SDS-PAGE using image analysis software, or from the peak intensity in HPLC or mass spectrometry.

[0035] The pH of the liquid sample is preferably near neutral, about 5.5 or more and 9 or less. If the pH is 5.5 or more, the target antibody contained in the liquid sample can be more reliably adsorbed to Protein L. If the pH is 9 or less, the target antibody contained in the liquid sample can be adsorbed to Protein L in a state in which denaturation due to alkaline conditions is suppressed. The solvent of the liquid sample may be water alone, or if it is mainly composed of water, C 1-4 The solvent may contain a water-miscible organic solvent such as an alcohol, or may be a buffer solution having a pH of about 5.5 or more and about 9 or less.

[0036] 3. Cleaning process In this step, the column to which the target antibody has been preferentially adsorbed to Protein L in step 2 above is washed, and impurities that have no or weak binding activity to Protein L are selectively eluted and removed. Note that in this step, at least the target antibody is basically adsorbed to Protein L, but a portion of it may be eluted depending on the amount of liquid sample loaded.

[0037] The washing solution used to wash the column in this step should be one that does not interfere with the interaction between the target antibody and Protein L. For example, water or a buffer solution with a pH of 5.5 or more and 9 or less can be used as the washing solution. The amount of washing solution used may be appropriately adjusted within a range in which impurities can be sufficiently removed from the column. Whether or not impurities have been sufficiently removed can be easily determined by monitoring the elution profile, for example, when a chromatography system is used.

[0038] 4.Elution process In this step, an elution solution having a lower pH than the liquid sample and the washing solution is used to elute mainly the target antibody from the column to which the target antibody has been adsorbed.

[0039] The pH of the elution solution may be appropriately adjusted, and may be, for example, 2 or more and 5 or less. If the pH is 2 or more, damage to the target antibody can be more reliably suppressed, and if the pH is less than 5, the target antibody can be more efficiently eluted from the column. The pH is preferably 2.5 or more, more preferably 3 or more, and preferably 4.5 or less, more preferably 4 or less. The elution solution is not particularly limited, and may be, for example, a general buffer solution using citric acid, acetic acid, glycine, hydrochloric acid, phosphoric acid, formic acid, or the like.

[0040] A salt may be further added to the elution solution, since this may improve the separation of the target antibody. Such salts may include one or more essential salts selected from the group consisting 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 elution solution may be appropriately adjusted, and may be, for example, 5 mM or more and 200 mM or less.

[0041] 5. Column regeneration process In this step, the column after elution of the target antibody in step 4 is regenerated by washing with a washing solution. However, this step does not necessarily have to be performed after step 4, and may be performed once every three, five, or ten times the steps 2 to 4 are repeated. In other words, this step does not necessarily have to be performed as long as the adsorption performance of the column is maintained, and the frequency and conditions of its execution vary depending on the liquid sample containing the target antibody to be purified. The column regenerated through this step can be used again in steps 2 to 4.

[0042] As the cleaning liquid, for example, an alkaline aqueous solution can be used. The alkaline aqueous solution is an aqueous solution that exhibits an alkalinity to a degree that can achieve the purpose of cleaning, sterilization, etc. More specifically, it is an aqueous sodium hydroxide solution of 0.01M to 1.0M or 0.01N to 1.0N, but is not limited thereto. In the case of sodium hydroxide as an example, the lower limit of the concentration is preferably 0.01M, more preferably 0.02M. On the other hand, the upper limit of the concentration of sodium hydroxide is preferably 0.2M, more preferably 0.1M or 0.05M. The alkaline aqueous solution does not need to be an aqueous sodium hydroxide solution, but its pH is preferably 11 or more and 12 or less. EXAMPLES

[0043] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples, and it is possible to carry out the present invention by making appropriate modifications within the scope of the above and below-mentioned aims, and all of these modifications are included in the technical scope of the present invention.

[0044] Example 1 (1) Preparation of CHO cell culture medium containing anti-CD3 scFv A protein (SEQ ID NO: 1) was designed in which a His-tag was added to the C-terminus of a single-chain antibody (scFv) in which the heavy and light chain variable regions of the anti-CD3 antibody Muromonab were linked by a GS linker, and a signal sequence of human-derived Azurocidin preproprotein was added to the N-terminus. The codons of the gene encoding this protein were optimized for Cricetulus griseus, and an artificial gene with the Kozak sequence GCCGCCGCC (SEQ ID NO: 2) added before the start codon was created by GeneArt. TMThe gene was designed and synthesized by an artificial gene synthesis service (Thermofisher Scientific) (SEQ ID NO: 3). This gene was introduced into a mammalian expression vector pcDNA3.4 (Thermofisher Scientific) to construct an anti-CD3 scFV expression plasmid. E. coli DH5α (Takara Bio) was transformed using the anti-CD3 scFV expression plasmid, the transformant was cultured, and a plasmid for transfection was extracted from the resulting cells using a high-speed open column for plasmid DNA purification ("NucleoBond Xtra Midi EF" Takara Bio). A CHO cell culture solution containing anti-CD3 scFv was prepared using this plasmid and a CHO cell transient expression kit ("ExpiCHOTM Expression System" Thermofisher Scientific). The host cells, medium, and transfection reagents included in the kit were used, and the culture and transfection were also performed according to the method included in the kit. After transfection, the culture was terminated on the 7th day of culture. At the end of the culture, the total number of cells in 200 mL of culture medium was 2.2 × 10 9 The viability was 65%. The cells were separated from the culture medium by centrifugation to obtain a culture supernatant. This culture supernatant was used as a CHO cell culture medium containing anti-CD3 scFv.

[0045] (2) Purification of scFv from CHO cell culture medium using Protein L support A commercial column ("Tricorn 5 / 50" manufactured by Cytiva) was filled with 1 mL of protein L support ("KANEKA KanCap TM The anti-CD3 scFv antibody was purified from the CHO cell culture medium (pH 7.0) containing the anti-CD3 scFv using a column packed with ELISA kit (manufactured by Kaneka Corporation). Specifically, the column was connected to a chromatography system ("AKTA avant 25" manufactured by Cytiva). The column was equilibrated with an equilibration buffer (20 mM sodium phosphate + 150 mM sodium chloride, pH 6.0), and then 50 mL of the CHO cell culture medium was loaded onto the column. After washing with 10 CV (column volume) of the equilibration buffer, the scFv antibody was eluted by passing 10 CV of 50 mM sodium acetate buffer (pH 3.6) as an eluent. In the above operations, the flow rate was adjusted to 0.5 mL / min in all cases. The flow-through fraction, washing fraction, and elution fraction obtained by the above operations were collected. Figure 1 shows a chromatogram at a wavelength of 280 nm when 50 mL of culture medium was loaded. As shown in Figure 1, a main peak was confirmed in the elution fraction. Figure 2 shows the results of SDS-PAGE of each fraction when 50 mL of culture medium was loaded. As shown in the SDS-PAGE results in Figure 2, an scFv band was confirmed in the elution fraction, and scFv bands were also confirmed in the flow-through fraction and wash fraction.

[0046] (3) Evaluation of binding activity of scFv to Protein L The binding activity of the flow-through fraction and elution fraction obtained in (2) above to Protein L, the ligand of the carrier used in purification, was evaluated using a molecular interaction analyzer that utilizes biolayer interference method ("OctetRED 384" manufactured by MOLECULAR DEVICE). Commercially available affinity chromatography support (KANEKA KanCap TM Protein L ligand for leak quantification (Kaneka Corp.) was dissolved in phosphate buffer (20 mM NaCl) at a concentration of 2 mg / mL. 2 HPO 4 -NaH 2 PO 4 + 150 mM NaCl, pH 7.4) and add 20 mg / mL biotinylation reagent ("EZ-Lin TM20 μL of "NHS-PEG4-Biotin" (ThermoFisher Scientific) was added and stirred at 4° C. for 2 hours. Then, a 5 mL HiTrap Desalting column (Cytiva) was connected to a chromatography system ("AKTA avant 25" (Cytiva)), and unreacted biotin was removed using the column to obtain biotinylated protein L. Phosphate buffer (10 mM Na 2 HPO 4 -NaH 2 PO 4 The concentration of biotinylated Protein L was adjusted to 25μg / mL using a dilution buffer consisting of 10% 10xKINETICS BUFFER (ForteBio part No.18-5032) added to 10% 10xKINETICS BUFFER (pH6.0). Using a molecular interaction analyzer ("OctetRed 384" manufactured by MOLECULAR DEVICE) that utilizes biolayer interference, a biosensor SA (manufactured by MOLECULAR DEVICE) was immersed in this solution, and Protein L was immobilized on the sensor by performing the following operations: shake speed 1000rpm, loading 3min, and washing 1min. In the SDS-PAGE results of the flow-through fraction and the eluted fraction obtained in (2) above, the intensity of the band having the molecular weight of scFv was analyzed using Western Blot Analysis software ("Image Studio TM Each fraction was diluted with phosphate buffer (20 mM NaCl) so that the concentration of the component having the same or nearly the same molecular weight as the scFv contained in each fraction was the same. 2 HPO 4 -NaH 2 PO 4 +150 mM NaCl, pH 7.4). Using a sensor with immobilized Protein L, the binding ability of each of the diluted solutions of the flow-through fraction and the eluted fraction obtained in (2) above to Protein L was evaluated. The shake speed was adjusted to 1000 rpm, the association time to 1 min, and the dissociation time to 2 min. Phosphate buffer (10 mM Na 2 HPO 4 -NaH 2 PO 4 , pH 6.0) was used. Each buffer solution was prepared so that it contained 10% 10×KINETICS BUFFER (ForteBio part No. 18-5032). The results are shown in Figure 3.

[0047] As shown in Figure 3, both the flow-through fraction and the eluted fraction contained components that had binding activity to Protein L. Therefore, it is considered that the 50 mL of loaded culture solution contained scFvs and the like that exceeded the adsorption capacity of the column.

[0048] (4) Evaluation of the binding activity of scFv to the CD3 antigen The binding activity of the flow-through fraction and eluted fraction obtained in (2) above to the antigen CD3 was evaluated using a molecular interaction analyzer ("OctetRED 384" manufactured by MOLECULAR DEVICE) that utilizes biolayer interference method. The antigen was an Fc fusion product of the CD3εγ complex subunit, and the activated recombinant human CD3 protein (Abcam) was adjusted to a concentration of 1 mg / mL with ultrapure water. 2 HPO 4 -NaH 2 PO 4+150 mM NaCl, pH 7.4) to a concentration of 100 nM or 200 nM to prepare an antigen solution to be used for immobilization on biosensor ProA (MOLECULAR DEVICE). Using a molecular interaction analyzer ("OctetRed 384" MOLECULAR DEVICE) and this antigen solution, the antigen was immobilized on biosensor ProA under the conditions of a shake speed of 1000 rpm, a loading time of 10 min, and a wash time of 1 min. In order to mask Protein A on the sensor, the sensor on which CD3 was immobilized was immersed in a solution containing the Fc region of an antibody that binds to Protein A, and treated under the conditions of a shake speed of 1000 rpm, a loading time of 10 min, and a wash time of 1 min. The solution was prepared by decomposing the humanized antibody Omalizumab ("Xolair" Novartis Pharma) with papain and immobilizing it on an affinity carrier for antibody drug purification ("KANEKA KanCapA TM The Fc concentration of the Fc-containing solution was adjusted to 3 μM by purification using Fc-containing antibody (Kaneka Corporation). In the SDS-PAGE results of the flow-through fraction and the eluted fraction obtained in (2) above, the intensity of the band having the molecular weight of scFv was analyzed using Western Blot Analysis software ("Image Studio TM Each fraction was diluted with phosphate buffer (20 mM NaCl) so that the concentration of the component having the same or nearly the same molecular weight as the scFv contained in each fraction was the same. 2 HPO 4 -NaH 2 PO 4 +150 mM NaCl, pH 7.4). Using the obtained CD3 immobilized sensor, the binding ability to CD3 was evaluated for each dilution of the flow-through fraction and elution fraction obtained in (2) above. The shake speed was adjusted to 1000 rpm, the association time to 1 min, and the dissociation time to 2 min. The results are shown in Figure 4.

[0049] As shown in the results in Figures 3 and 4, the flow-through fraction contained components that had binding activity to protein L, but it was found that these components had almost no binding activity to CD antigens. In contrast, the eluted fraction had higher binding activity to both Protein L and the CD3 antigen than the flow-through fraction. Since the concentrations of components having the same or nearly the same molecular weight as the scFv in each fraction sample were adjusted to be the same, the purity of the scFv in the eluted fraction was higher than that of the flow-through fraction, and as a result, it can be said that the binding activity of the eluted fraction to the antigen is high. From the above results, it was demonstrated that by loading a column containing a carrier carrying Protein L with a liquid containing scFv in an amount exceeding its adsorption capacity, it is possible to obtain scFv that is highly pure and has high antigen-binding activity.

[0050] Example 2 (1) Preparation of Pichia yeast culture containing anti-CD3 scFv Using the anti-CD3-scFv gene prepared in Example 1(1), scFv was produced in Pichia yeast. The scFv-producing yeast was obtained and cultured according to the method described in Examples 1, 8, and 9 of WO2012 / 102171. The resulting culture solution containing the scFv was centrifuged to recover the culture solution. The recovered culture solution was filtered using a sterile filtration filter with a pore size of 0.22 μm ("Minisart", Sartorius).

[0051] (2) Purification of scFv from Pichia pastoris cultures using Protein L support Using the Pichia yeast culture solution containing the scFv obtained in Example 2(1) above, scFv was purified in the same manner as in Example 1(2), except that the pH of the loading solution was adjusted to 6.0. The chromatogram when 50 mL of culture medium was loaded is shown in Figure 5. As shown in Figure 5, a main peak was confirmed in the eluted fraction. The results of SDS-PAGE of each fraction are shown in Figure 6. As shown in the SDS-PAGE results in Figure 6, bands of components with molecular weights similar to those of scFv were confirmed in the flow-through fraction and elution fraction. Therefore, it is believed that the 50 mL of loaded culture solution contained scFv and the like in excess of the adsorption capacity of the column.

[0052] (3) Evaluation of binding activity of scFv purified from Pichia yeast cultures The binding activity of the flow-through fraction and the eluted fraction obtained in Example 2(1) to Protein L, which is the ligand of the carrier used in purification, and to CD3, which is the antigen, were evaluated using a molecular interaction analyzer ("OctetRED 384" manufactured by MOLECULAR DEVICE). The binding activity of scFv to Protein L is shown in FIG. 7, and the binding activity of scFv to the CD3 antigen is shown in FIG. 8.

[0053] As shown in the results in Figures 7 and 8, the flow-through fraction also contained a component that had binding activity to Protein L, but the binding activity of that component to CD antigens was very low. In contrast, the eluted fraction had higher binding activity to both Protein L and the CD3 antigen than the flow-through fraction. Since the concentrations of components having the same or nearly the same molecular weight as the scFv in each fraction sample were adjusted to be the same, the purity of the scFv in the eluted fraction was higher than that of the flow-through fraction, and as a result, it can be said that the binding activity of the eluted fraction to the antigen is high. From the above results, it was revealed that, similarly to scFv produced in CHO cells, scFv produced in Pichia yeast can also be obtained with high purity and high antigen-binding activity by loading a column containing a carrier carrying Protein L with an scFv-containing solution in an amount that exceeds its adsorption capacity.

Claims

1. 1. A method for producing an antibody comprising a κ chain, comprising the steps of: A step of passing a liquid sample containing the antibody through a column packed with a carrier carrying Protein L as a ligand; and passing an elution solution through the column to elute the antibody adsorbed to the carrier; the liquid sample contains an isomer having a molecular weight identical or substantially identical to the molecular weight of the antibody and / or an aggregate of the antibody or the isomer; the binding activity of the antibody to an antigen is higher than the binding activity of the isomer and the aggregate to the antigen; The method comprises passing the liquid sample through the column, the liquid sample being such that the combined amount of the antibody, the isomer and the aggregate exceeds the adsorption capacity of the column.

2. The method according to claim 1, wherein the amount of the antibody contained in the liquid sample is 80% or more of the adsorption capacity of the column.

3. 3. The method according to claim 1, wherein the pH of the liquid sample is 5.5 or more and 9 or less, and the pH of the elution solution is 5 or less.

4. The method according to any one of claims 1 to 3, wherein the antibody is a minibody.

5. The method according to claim 4 , wherein the minibody is a single-chain antibody.

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