Purification method for anti-4-1BB / anti-HER2 bispecific antibodies

Abstract

The present invention provides a method for purifying an anti-4-1BB / anti-HER2 bispecific antibody, which comprises a step of performing affinity chromatography using a sodium acetate buffer containing a specific inorganic salt as an elution buffer. The purification method of the present invention can provide an intact anti-4-1BB / anti-HER2 bispecific antibody with high purity and high yield by increasing the elution of the intact antibody.

Description

[Technical Field] 【0001】 The present invention relates to a method for purifying an anti-4-1BB / anti-HER2 bispecific antibody. More specifically, the present invention relates to a method for purifying an anti-4-1BB / anti-HER2 bispecific antibody, comprising the step of performing affinity chromatography using a sodium acetate buffer containing a specific inorganic salt as an elution buffer. [Background technology] 【0002】 The 4-1BB protein is a member of the TNF receptor superfamily (TNFRSF) and a co-stimulatory molecule expressed after the activation of both innate and adaptive immune cells. 4-1BB plays a crucial role in regulating the activity of various immune cells. 4-1BB agonists enhance immune cell proliferation and survival, cytokine secretion, and CD8 T cell cytolytic activity. Therefore, 4-1BB may be a promising target molecule in cancer immunology. While anti-4-1BB antibodies possess antitumor effects, they have been shown to cause severe hepatotoxicity in clinical applications. 【0003】 HER2 is a receptor tyrosine kinase (RTK) present on the cell surface that induces cancer cell proliferation, invasion, and angiogenesis. 【0004】 Multispecific antibodies that target two or more antigens are expected to be novel drugs with superior therapeutic effects compared to monoclonal antibodies. Multispecific antibodies that can recognize two antigens, one present on cancer cells and the other on immune cells, can induce a more potent cancer-specific immune response. The applicant has developed various antibodies that specifically bind to both 4-1BB and HER2, namely anti-4-1BB / anti-HER2 bispecific antibodies (International Patent Application No. PCT / KR2020 / 009871). These anti-4-1BB / anti-HER2 bispecific antibodies activate 4-1BB signaling only in the presence of HER2-expressing cells, thereby enhancing potent immune cells. Therefore, since these anti-4-1BB / anti-HER2 bispecific antibodies induce a specific immune response via HER2, it is expected that using these anti-4-1BB / anti-HER2 bispecific antibodies will result in significantly lower hepatotoxicity compared to 4-1BB monoclonal antibodies. International Patent Application No. PCT / KR2020 / 009871 is incorporated herein by reference. [Overview of the Initiative] [Problems that the invention aims to solve] 【0005】 The inventors, in accordance with the disclosure in international patent application PCT / KR2020 / 009871, noticed a problem when attempting to isolate anti-4-1BB / anti-HER2 bispecific antibodies from cells that produce anti-4-1BB / anti-HER2 bispecific antibodies, which are cultured and isolated from these cells, that the majority of the obtained antibodies exist in aggregate form. The inventors conducted various studies to develop a purification method that can increase the purity of intact anti-4-1BB / anti-HER2 bispecific antibodies. As a result, they found that intact anti-4-1BB / anti-HER2 bispecific antibodies can be obtained in high purity and high yield by performing affinity chromatography using a sodium acetate buffer containing a specific inorganic salt as the elution buffer. 【0006】 Therefore, an object of the present invention is to provide a method for purifying an anti-4-1BB / anti-HER2 bispecific antibody, comprising the step of performing affinity chromatography using a sodium acetate buffer containing a specific inorganic salt as an elution buffer. [Means for solving the problem] 【0007】 According to one aspect of the present invention, a method for purifying an anti-4-1BB / anti-HER2 bispecific antibody is provided, comprising the steps of: (a) filtering the culture supernatant of a cell line producing an anti-4-1BB / anti-HER2 bispecific antibody to obtain a filtrate containing the crude antibody; (b) loading the filtrate onto a protein A affinity chromatography column; and (c) eluting the antibody from the column in step (b) using a sodium acetate buffer containing CaCl2. 【0008】 In the purification method of the present invention, the heavy chain of the anti-4-1BB / anti-HER2 bispecific antibody comprises (i) a heavy chain of an anti-HER2 antibody consisting of the amino acid sequence of SEQ ID NO: 9, and (ii) a light chain variable region of an anti-4-1BB antibody comprising the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region of an anti-4-1BB antibody comprising the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, and the amino acid sequence of SEQ ID NO: 7, and the light chain of the anti-4-1BB / anti-HER2 bispecific antibody comprises a light chain of an anti-HER2 antibody consisting of the amino acid sequence of SEQ ID NO: 11. 【0009】 In one embodiment, the light chain variable region of the anti-4-1BB antibody may consist of the amino acid sequence of SEQ ID NO: 4; the heavy chain variable region of the anti-4-1BB antibody may consist of the amino acid sequence of SEQ ID NO: 8. In another embodiment, the scFv of the anti-4-1BB antibody may further include a linker consisting of the amino acid sequence of SEQ ID NO: 13. In yet another embodiment, the heavy chain of the anti-4-1BB / anti-HER2 bispecific antibody may further include a linker consisting of the amino acid sequence of SEQ ID NO: 12. In yet another embodiment, the heavy chain of the anti-4-1BB / anti-HER2 bispecific antibody may consist of the amino acid sequence of SEQ ID NO: 10. 【0010】 In the purification method of the present invention, the concentration of CaCl2 in the sodium acetate buffer may be 30 to 500 mM, preferably 75 to 125 mM. The concentration of sodium acetate in the sodium acetate buffer may be 20 to 100 mM. Furthermore, the pH of the sodium acetate buffer may be pH 3.5 to pH 4.0. [Effects of the Invention] 【0011】 According to the present invention, affinity chromatography can be performed using a sodium acetate buffer containing a specific inorganic salt, namely CaCl2, as the elution buffer, thereby significantly increasing the elution of intact antibodies. This allows for the provision of intact anti-4-1BB / anti-HER2 bispecific antibodies in high purity and high yield. Therefore, the purification method of the present invention can provide anti-4-1BB / anti-HER2 bispecific antibodies in high purity and high yield. [Modes for carrying out the invention] 【0012】 The present invention provides a method for purifying an anti-4-1BB / anti-HER2 bispecific antibody, comprising the steps of: (a) filtering the culture supernatant of a cell line producing an anti-4-1BB / anti-HER2 bispecific antibody to obtain a filtrate containing the crude antibody; (b) loading the filtrate onto a protein A affinity chromatography column; and (c) eluting the antibody from the column in step (b) using a sodium acetate buffer containing CaCl2. 【0013】 In the purification method of the present invention, the anti-4-1BB / anti-HER2 bispecific antibody consists of a heavy chain and a light chain and can be manufactured in accordance with the disclosure of International Patent Application PCT / KR2020 / 009871. Specifically, in the purification method of the present invention, the heavy chain of the anti-4-1BB / anti-HER2 bispecific antibody comprises (i) a heavy chain of an anti-HER2 antibody consisting of the amino acid sequence of SEQ ID NO: 9, and (ii) a light chain variable region of the anti-4-1BB antibody including the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 3; and a heavy chain variable region of the anti-4-1BB antibody including the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, and the amino acid sequence of SEQ ID NO: 7. The light chain of the anti-4-1BB / anti-HER2 bispecific antibody comprises a light chain of an anti-HER2 antibody consisting of the amino acid sequence of SEQ ID NO: 11. 【0014】 In one embodiment, the light chain variable region of the anti-4-1BB antibody may consist of the amino acid sequence of SEQ ID NO: 4; and the heavy chain variable region of the anti-4-1BB antibody may consist of the amino acid sequence of SEQ ID NO: 8. In another embodiment, the scFv of the anti-4-1BB antibody may further include a linker consisting of the amino acid sequence of SEQ ID NO: 13. For example, the scFv of the anti-4-1BB antibody may be a polypeptide in which the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 13, and SEQ ID NO: 8 are linked in order (i.e., N'→C'). 【0015】 In one embodiment, the heavy chain of the anti-4-1BB / anti-HER2 bispecific antibody may further include a linker consisting of the amino acid sequence of SEQ ID NO: 12. For example, the heavy chain of the anti-4-1BB / anti-HER2 bispecific antibody may be a polypeptide in which the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 4, SEQ ID NO: 13, and SEQ ID NO: 8 are linked in order (i.e., N'→C'), i.e., a polypeptide consisting of the amino acid sequence of SEQ ID NO: 10. 【0016】 The purification method of the present invention comprises the step [step (a)] of filtering the culture supernatant of a cell line producing an anti-4-1BB / anti-HER2 bispecific antibody to obtain a filtrate containing the crude antibody. The cell line producing the anti-4-1BB / anti-HER2 bispecific antibody may be cells (e.g., CHO cells) transfected with a plasmid into which a polynucleotide encoding the heavy chain of the anti-4-1BB / anti-HER2 bispecific antibody and a polynucleotide encoding the light chain of the anti-4-1BB / anti-HER2 bispecific antibody are incorporated, respectively. These transfected cells can be obtained in accordance with the disclosure of International Patent Application No. PCT / KR2020 / 009871. These transfected cells may be cultured in accordance with conventional methods using media commonly used in the field of biotechnology. The culture supernatant can be obtained, for example, by centrifugation of a culture of a cell line producing an anti-4-1BB / anti-HER2 bispecific antibody. Filtration may be performed by conventional sterile filtration, for example, sterile filtration using a 0.2 μm membrane filter. The crude antibody-containing filtrate obtained as described above contains approximately 40-45% antibody aggregates. 【0017】 The purification method of the present invention includes the step of loading the filtrate obtained in step (a) onto a protein A affinity chromatography column [step (b)]. Protein A affinity chromatography involves the use of various agarose resins. As resins, known agarose resins, for example, MabSelect SuRe TM LX (Cytiva), MabSelect TMPrismA (Cytiva), Praesto TM Jetted A50 (Purolite Life Sciences), preferably MabSelect. TM Affinity chromatography resins from PrismA (Cytiva) may also be used. For example, MabSelect TM Loading the filtrate obtained in step (a) onto the protein A affinity chromatography column packed with PrismA (Cytiva) resin may be carried out according to conventional methods. For example, the filtrate may be loaded at a rate of 20 to 40 mg per 1 ml of resin, but is not limited to this rate. 【0018】 The purification method of the present invention includes step (c) of eluting an antibody (i.e., an anti-4-1BB / anti-HER2 bispecific antibody) from the column of step (b) using a sodium acetate buffer containing CaCl2 as an elution buffer. The concentration of CaCl2 in the sodium acetate buffer is preferably 30 to 500 mM, more preferably 50 to 500 mM, even more preferably 75 to 125 mM, and particularly preferably about 100 mM. The concentration of sodium acetate in the sodium acetate buffer is preferably 20 to 100 mM. In one embodiment of the present invention, the concentration of CaCl2 in the sodium acetate buffer is 75 to 125 mM, and the concentration of sodium acetate in the sodium acetate buffer is 20 to 100 mM. 【0019】 The pH of the sodium acetate buffer solution may preferably be pH 3.5 to pH 4.0, and more preferably about pH 3.7. In one embodiment of the present invention, the concentration of CaCl2 in the sodium acetate buffer solution may be 75 to 125 mM, the concentration of sodium acetate in the sodium acetate buffer solution may be 20 to 100 mM, and the pH of the sodium acetate buffer solution may be pH 3.5 to pH 4.0. 【0020】 In one embodiment of the purification method according to the present invention, the sodium acetate buffer may be a 100 mM sodium acetate buffer containing about 100 mM of CaCl₂ and having a pH of about 3.7. 【0021】 In another embodiment of the purification method according to the present invention, the sodium acetate buffer may be a 20 mM sodium acetate buffer containing about 100 mM of CaCl₂ and having a pH of about 3.7. 【0022】 The anti-4-1BB / anti-HER2 bispecific antibody obtained by the purification method of the present invention may be isolated in the form of an antibody-containing solution from the eluate of step (c) by performing virus inactivation treatment and neutralization treatment by a conventional method. The virus inactivation treatment may be performed, for example, by adjusting the pH to 3.4 - 3.6, and the neutralization may be performed, for example, by using a 1 M tromethamine solution, but is not limited thereto. If necessary, after performing the step of step (c), one or more chromatographies selected from the group consisting of cation exchange chromatography, anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography may be further performed. 【0023】 Hereinafter, the present invention will be described in more detail by referring to production examples and examples. However, these production examples and examples are for explaining the present invention and do not limit the scope of the present invention. 【Example】 【0024】 Manufacturing example: Production of anti-4-1BB / anti-HER2 bispecific antibody An anti-4-1BB / anti-HER2 bispecific antibody was produced as follows according to the method disclosed in International Patent Application No. PCT / KR2020 / 009871. (1) Anti-4-1BB scFv antibody Screening was performed by phage library panning against 4-1BB using immunotubes. For panning of phage libraries against target molecules (obtained from KBio Health and CUREBIO), a total of four panning operations were performed using immunotubes coated with human 4-1BB (NCBI Accession No. NP_001552.2). 【0025】 The bacterial colonies obtained by three pannings were grown in SB-carbenicillin (Biomatik, catalog No. A2311, 5g) in a 96-deep-well plate until turbidity was reached, and then 10 11 pfu-containing VCSM13 helper phage (K-Bio Health) was added to each well. The cells were infected at 37°C for 1 hour with gentle shaking (80 rpm), then 70 μg / mL of kanamycin was added, and the cells were cultured overnight at 30°C with shaking at 200 rpm. 【0026】 The following day, the plate was centrifuged, and the supernatant containing the phages was added to a 4-1BB antigen-coated ELISA plate blocked with 3% (v / v) BSA (bovine serum albumin) in PBST (phosphate-buffered saline containing Tween 20). After incubation at room temperature for 1 hour, the plate was washed three times with PBST, and anti-M13 antibody (Sino Biological, catalog no. 11973-MM05) was added. The plate was incubated for 1 hour, washed three times with PBST, and the binding activity was measured using tetramethylbenzidine (TMB). 【0027】 A 4-1BB-specific binder was amplified for plasmid DNA sequencing. The sequences of the light chain variable region and heavy chain variable region (VL and VH) were analyzed to identify unique sequences and investigate sequence diversity. For comparison of agonist activity, an anti-4-1BB antibody designated as BMUR (Urelumab, BMS, U.S. Patent No. 7,288,638) was used. The sequences of the resulting full-length human monoclonal antibodies against 4-1BB are shown below. 【0028】 [Table 1] 【0029】 Using (GGGGS)4 from SEQ ID NO: 13 as a linker, an anti-4-1BB scFv antibody with the structure (N')-VL-linker-VH-(C') shown in Table 2 below was prepared. To stabilize scFv by forming disulfide crosslinks, the amino acid residue "G" at position 103 of the light chain variable region was replaced with "C" (SEQ ID NO: 4), and the amino acid residue "G" at position 44 of the heavy chain variable region was replaced with "C" (SEQ ID NO: 8). Specifically, a polynucleotide consisting of the DNA encoding the light chain variable region of SEQ ID NO: 4, the DNA encoding the linker of SEQ ID NO: 13, and the DNA encoding the heavy chain variable region of SEQ ID NO: 8 was inserted into pcDNA 3.4 (Invitrogen, A14697). The resulting vector was processed using ExpiFectamine. TM ExpiCHO cells (Gibco, A29127) were transfected at 37°C using the CHO Kit (Gibco, A29133) and cultured for 8 days. The culture supernatant was filtered through a 0.2 μm filter to obtain an anti-4-1BB scFv antibody having the structure (N')-VL-linker-VH-(C'). 【0030】 [Table 2] 【0031】 (2) Anti-HER2 antibody The heavy and light chains of trastuzumab (Genentech; hereafter referred to as "HER2(WT)"; DrugBank Accession No. DB00072; human IgG1κ monoclonal antibody) were used as the HER2 target portion of the anti-4-1BB / anti-HER2 bispecific antibody. The amino acid sequences of the heavy and light chains of HER2(WT) are shown in Table 3 below. 【0032】 [Table 3] 【0033】 (3) Cell line producing anti-4-1BB / anti-HER2 bispecific antibody Using the anti-4-1BB scFv antibody and the heavy and light chains of HER2 (WT) obtained in the above (1) and (2), an IgG-scFv fusion antibody (a fusion of the scFv antibody fragment to the C-terminus of IgG), that is, an anti-4-1BB / anti-HER2 bispecific antibody having a heavy chain portion consisting of SEQ ID NO: 10 and a light chain portion consisting of SEQ ID NO: 11 shown in Table 4 below was produced. 【0034】 Specifically, DNA segment 2 encoding the GS linker of SEQ ID NO: 12 was fused to the end of DNA segment 1 (SEQ ID NO: 9) encoding the heavy chain of the anti-HER2 antibody, and then DNA segment 3 encoding the scFv of the anti-4-1BB antibody was fused to create a DNA segment (SEQ ID NO: 10) encoding the heavy chain of the anti-HER2 / anti-4-1BB bispecific antibody, and this DNA segment was inserted into plasmid 1 (Invitrogen, A14697). Further, DNA segment 4 (SEQ ID NO: 11) encoding the light chain of the anti-HER2 / anti-4-1BB bispecific antibody was inserted into plasmid 2 (Invitrogen, A14697). 【0035】 The obtained plasmid 1 and plasmid 2 were transfected into ExpiCHO TM Expression System (Gibco, A29133) using ExpiCHO cells (Gibco, A29127). Specifically, the obtained plasmid 1 (250 μg) and plasmid 2 (250 μg) were mixed with OptiPRO TM SFM (12309-050) (800 μL) containing ExpiFectamine CHO reagent (A29129) (final volume 20 mL) and allowed to stand for 3 minutes. For transfection, the obtained mixed solution and 6×10 6ExpiCHO cells were added to ExpiCHO expression medium (A29100-01) and cultured at 37°C in an 8% CO2 humidified shaking incubator. After 18 hours of culture, 1.5 mL of ExpiFectamine was added. TM CHO Transfection Enhancer 1 and ExpiFectamine TM CHO Feed was added and the mixture was incubated at 37°C for 4 days. After adjusting the temperature to 32°C, ExpiFectamine was added. TM CHO Feed was added, and the culture was continued for 8 days. 【0036】 [Table 4] 【0037】 (4) Fed-batch culture for antibody expression The cell line producing the anti-4-1BB / anti-HER2 bispecific antibody obtained in (3) was cultured for 3 days in glutamine-free Actipro medium (Hyclone, SH31037.02), and then subjected to 14 days of fed-batch culture (working volume: 700 ml / 2000 ml flask, 37°C, 5% CO2, 130 rpm), during which Cell Boost was performed. TM Supplement 7a (Hyclone, SH31026.01) / 7b (Hyclone, SH31027.01KR) was added every other day. The culture supernatant obtained by centrifugation at approximately 3000 rpm was filtered through a 0.2 μm membrane filter to obtain a filtrate containing crude antibody. 【0038】 Example 1: Purification of anti-4-1BB / anti-HER2 bispecific antibody by protein A affinity chromatography A protein A affinity chromatography column was prepared by packing a 1.6 cm diameter, 20 cm resin layer height column with MabSelect PrismA affinity chromatography resin (Cytiva) and equilibrated with 1 × phosphate-buffered saline (PBS) (pH 7.4). The filtrate containing the crude antibody obtained in Preparation Example (4) was loaded onto the protein A affinity chromatography column at a rate of 30 mg per 1 mL of resin. After washing the column with 1 × PBS (pH 7.4), the antibody was eluted using either sodium acetate buffer containing an additive (CaCl2) or sodium acetate buffer without the additive as the elution buffer. The concentrations of the additive and sodium acetate in the elution buffer and the pH of the elution buffer are shown in Table 5 below. 【0039】 The yield of the antibody (i.e., anti-4-1BB / anti-HER2 bispecific antibody) was analyzed by measuring the absorbance of the pools obtained using each of the following elution buffers under 280 nm UV light, and the purity of the antibody (i.e., anti-4-1BB / anti-HER2 bispecific antibody) was analyzed by measuring the content (%) of high molecular weight aggregates using size exclusion chromatography (SEC-HPLC). The SEC-HPLC analysis was performed using a Tosoh Corporation column (model number: TSK-GEL G3000SW). XL The analysis was performed using the following method. 1× ​​phosphate-buffered saline (PBS) (pH 7.4) was flowed through the column at a flow rate of 1 ml / min to equilibrate it, and then each eluted fraction (100 μg) was injected for purity analysis. Purity was expressed as intact purity (%) and calculated using the following formula. Purity (%) = 100 - High molecular weight aggregate content (%) 【0040】 As described above, the results obtained by purification using protein A affinity chromatography are shown in Table 5 below. 【0041】 [Table 5] 【0042】 As can be seen from the results in Table 5 above, using a sodium acetate buffer containing CaCl2 as an additive can significantly increase the purity of the anti-4-1BB / anti-HER2 bispecific antibody with a relatively high yield. Furthermore, from the results in Table 5, it can be seen that the concentration of CaCl2 in the sodium acetate buffer is preferably in the range of 30 to 500 mM, more preferably in the range of 50 to 500 mM, even more preferably in the range of 75 to 125 mM, and particularly preferably around 100 mM. In addition, it can be seen that the concentration of sodium acetate in the sodium acetate buffer is preferably in the range of 20 to 100 mM. 【0043】 Comparative Example 1: Purification of anti-4-1BB / anti-HER2 bispecific antibody using NaCl as an additive Purification by protein A affinity chromatography was performed using a 100 mM sodium acetate buffer (pH 3.7) containing 1000 mM NaCl as an additive, in the same manner as in Example 1. The purity and yield of the elution pool were analyzed in the same manner as in Example 1. As a result, the purity was relatively higher compared to purification using 100 mM sodium acetate (pH 3.7) without the additive, but the yield decreased significantly to 29.3%. 【0044】 Based on the above results, purity and yield were evaluated by reducing the concentrations of sodium acetate and the additive (NaCl). Specifically, purification by protein A affinity chromatography was performed using a 20 mM sodium acetate buffer (pH 3.7) containing 100 mM NaCl as an additive, in the same manner as in Example 1. The purity and yield of the elution pool were analyzed in the same manner as in Example 1. As a result, the purity decreased significantly to 74.2%. 【0045】 Therefore, the use of sodium acetate buffer containing NaCl did not show an improved purification effect in terms of purity and yield. 【0046】 Comparative Example 2: MgCl as an additive 2 Purification of anti-4-1BB / anti-HER2 bispecific antibody using Purification by protein A affinity chromatography was performed using a 100 mM sodium acetate buffer (pH 3.7) containing 1000 mM MgCl2 as an additive, in the same manner as in Example 1. The purity and yield of the elution pool were analyzed in the same manner as in Example 1. As a result, compared to purification using 100 mM sodium acetate (pH 3.7) without the additive, the purity was relatively higher, but the yield decreased significantly to 43.9%. 【0047】 Based on the above results, purity and yield were evaluated by reducing the concentrations of sodium acetate and the additive (MgCl2). Specifically, purification by protein A affinity chromatography was performed using a 20 mM sodium acetate buffer (pH 3.7) containing 100 mM MgCl2 as an additive, in the same manner as in Example 1. The purity and yield of the elution pool were analyzed in the same manner as in Example 1. As a result, the purity was relatively higher compared to purification using high concentrations of sodium acetate / additive, but the yield was only 66.7%. 【0048】 Therefore, the use of sodium acetate buffer containing MgCl2 did not show an improved purification effect in terms of yield.

Claims

[Claim 1] A method for purifying an anti-4-1BB / anti-HER2 bispecific antibody, (a) A step of filtering the culture supernatant of a cell line that produces anti-4-1BB / anti-HER2 bispecific antibodies to obtain a filtrate containing crude antibodies; (b) loading the filtrate onto a protein A affinity chromatography column; and (c) CaCl ２ The process includes eluting the antibody from the column in step (b) using a sodium acetate buffer containing the following: The heavy chain of the aforementioned anti-4-1BB / anti-HER2 bispecific antibody consists of the amino acid sequence of SEQ ID NO:

10. A method wherein the light chain of the anti-4-1BB / anti-HER2 bispecific antibody comprises a light chain of an anti-HER2 antibody having the amino acid sequence of SEQ ID NO:

11. [Claim 2] CaCl in the sodium acetate buffer ２ The method according to claim 1, wherein the concentration of is 30 to 500 mM. [Claim 3] CaCl in the sodium acetate buffer ２ The method according to claim 2, wherein the concentration of is 75 to 125 mM. [Claim 4] The method according to any one of claims 1 to 3, wherein the concentration of sodium acetate in the sodium acetate buffer is 20 to 100 mM. [Claim 5] The method according to any one of claims 1 to 4, wherein the pH of the sodium acetate buffer solution is pH 3.5 to pH 4.

0. [Claim 6] CaCl in the sodium acetate buffer ２ The method according to any one of claims 1 to 5, wherein the concentration of is 75 to 125 mM, and the concentration of sodium acetate in the sodium acetate buffer is 20 to 100 mM. [Claim 7] The method according to claim 6, wherein the pH of the sodium acetate buffer solution is pH 3.5 to pH 4.0.

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