HK1225045B - Method for purifying antibody having low isoelectric point - Google Patents

Description

Method for purifying antibodies with low isoelectric points

Technical Field

The present invention relates to a method for purifying antibodies, and in particular to a method for purifying antibodies with a low isoelectric point (pI).

Background Art

Thanks to advances in recombinant technology, various protein preparations can now be provided in stable quantities. In particular, in recent years, various antibody pharmaceuticals with higher selectivity than conventional pharmaceuticals have been developed using recombinant technology and have entered clinical trials.

In preparations containing physiologically active proteins produced by such genetic recombination techniques, it is necessary to remove proteins (host cell proteins) or DNA derived from host cells, resin ligand fragments as one of the purification raw materials, and complexes or fragments derived from the target protein. In addition, in order to ensure the viral safety of the preparation, it is necessary to show sufficient virus removal or inactivation capabilities in the purification step. Currently, according to the World Health Organization (WHO), the permissible amount of DNA in biopharmaceuticals is 100pg DNA/single dose or less. Moreover, regarding viruses, according to the World Health Organization (WHO), when retrovirus-like particles are confirmed to be present in the culture medium, the permissible amount is 1 virus particle/10 ⁶ dose or less, taking into account the removal or inactivation capabilities of the retrovirus in the purification step. In order to meet this standard, usually, the aqueous culture medium containing physiologically active proteins obtained from host cells is treated with affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydroxyapatite chromatography, hydrophobic interaction chromatography or a combination thereof to remove impurities. Furthermore, in recent years, the development of new purification ligands has been progressing, and multi-component chromatography having both ion exchange and hydrophobic interaction effects has been used for purification.

In particular, when the physiologically active protein is an antibody obtained using mammalian cells as a host, the property of protein A or protein G to bind to the Fc chain of IgG is utilized, and after treatment with affinity column chromatography using protein A or protein G, purification is performed using various chromatography methods.

For example, in Japanese Patent Application No. 5-504579 (Patent Document 1), an aqueous medium containing an antibody obtained from mammalian cell culture is applied to protein A or protein G column chromatography to adsorb the antibody to the column. Thereafter, the antibody is eluted using an acidic solution (citric acid with a concentration of approximately 0.1 M, pH 3.0-3.5), and the obtained acidic eluate is sequentially applied to ion exchange column chromatography and size exclusion column chromatography for purification.

Amino acid substitution techniques are known for controlling the isoelectric point (pI) of antibodies with the goal of improving blood retention or in vivo dynamics. Specifically, techniques for controlling the pI of antibodies by altering (modifying) amino acid residues exposed on the antibody surface are known (WO07/114319 (Patent Document 2)). The isoelectric point (pI) of natural antibodies ranges from approximately 7.5 to 9.5, indicating a relatively high pI. By lowering the pI of such antibody amino acid residues, it is expected that the plasma retention or half-life of the antibody will be prolonged, thereby reducing the dosage of the antibody as a pharmaceutical or extending the dosing interval.

However, to date, there has been no research on purification methods suitable for antibodies with low pIs that do not naturally occur, nor on problems unique to low pI antibodies during purification steps. Therefore, purification methods suitable for such low pI antibodies are completely unknown.

Prior art literature

Patent Literature

Patent Document 1: Japanese Patent No. 5-504579

Patent document 2: WO07/114319.

Summary of the Invention

Problems to be solved by the invention

An object of the present invention is to provide a method for purifying antibodies that can effectively remove impurities from a composition containing antibodies having a low pI, in particular.

Generally, the method using a Protein A column is the mainstream method for crudely purifying antibodies from culture fluids. In this case, the following is generally performed: in the step of eluting from the column, elution is performed using an acidic solution, and acid is added as needed to maintain the acidic state for a predetermined time to inactivate viruses. After neutralization, the next purification step is immediately carried out.

The present inventors have discovered that, particularly when antibodies with a low pI are purified using conventional purification procedures, new aggregates are generated after the purification steps are completed. Research into the cause has revealed a phenomenon in which, when these antibodies are left in an acidic state for a predetermined period of time and then neutralized, a portion of the antibodies gradually and irreversibly associate over the predetermined period.

Conventional antibodies have high and alkaline pI values. Therefore, after purification using protein A affinity resin, due to the alkaline nature of the resin, purification is performed using a bind/elute mode with a cation exchange resin and a pass-through mode with an anion exchange resin. Generally, it is known that aggregates are removed with a cation exchange resin, while impurities such as DNA, host cell proteins, and viruses are adsorbed and removed with an anion exchange resin. However, research on purification methods for antibodies with a low pI has revealed that, with respect to antibodies with a low pI, conventional purification methods have been insufficient in removing impurities contained in the antibody culture medium.

Means for solving problems

The present inventors conducted intensive studies to achieve the above-mentioned objectives and found that subsequent antibody aggregation can be suppressed by purifying antibodies with a low pI using a protein A column, neutralizing them, and then removing the resulting antibody aggregates after a predetermined period of time.

Furthermore, the authors discovered that purification of antibodies with low pI using an anion exchange resin in a bind/elute mode allows for more efficient removal of impurities than conventional methods. Furthermore, the authors discovered that the use of anion exchange chromatography, multi-component chromatography, or hydrophobic interaction chromatography further removes impurities.

That is, the present invention provides the following [1] to [19].

[1] A method for purifying a composition containing an antibody having a pI value of 3.0 to 8.0, the method comprising the following steps:

(a) treating a composition containing an antibody having a pI value of 3.0 to 8.0 under acidic conditions;

(b) a step of neutralizing the acidic composition obtained in step (a); and

(c) a step of removing the aggregates from the neutralized composition obtained in step (b) after at least 1 hour has passed since neutralization.

[2] The method described in [1], wherein step (a) is a virus inactivation treatment step performed after purifying the antibody with a pI value of 3.0 to 8.0 using protein A column chromatography.

[3] The method according to [1] or [2], wherein step (c) is a step of removing aggregates after holding the neutralized composition obtained in step (b) for at least 1 hour after neutralization.

[4] The method according to any one of [1] to [3], wherein the removal of the aggregates is performed by anion exchange chromatography, hydrophobic interaction chromatography, multicomponent chromatography or hydroxyapatite chromatography.

[5] The method according to any one of [1] to [4], wherein the pI value of the antibody is 5.0 to 7.5.

[6] The method according to any one of [1] to [4], wherein the pI value of the antibody is 5.0 to 6.5.

[7] A method for removing impurities from a composition containing an antibody having a pI value of 3.0 to 8.0, the method comprising the following steps:

(a) loading a composition containing an antibody having a pI value of 3.0 to 8.0 onto an anion exchange resin;

(b) A step of eluting antibodies having a pI value of 3.0 to 8.0 from an anion exchange resin using a bind/elute mode using an elution solution having a higher salt concentration than the composition of (a).

[8] The method described in [7], wherein before step (b), the method includes: a step of washing the anion exchange resin with a washing solution.

[9] The method described in [7] or [8], wherein the elution solution in step (b) is a solution containing at least one selected from sodium chloride, Tris salt, sodium sulfate salt, and sodium phosphate salt.

[10] The method according to any one of [7] to [9], further comprising the step of loading the eluted product obtained in step (b) containing antibodies having a pI value of 3.0 to 8.0 onto a chromatography column using a resin having a hydrophobic ligand and/or a multi-ligand, to obtain a flow-through fraction and/or an eluted fraction.

[11] The method according to any one of [1] to [6], wherein the aggregate is removed by the method according to any one of [7] to [10].

[12] The method according to any one of [1] to [11], wherein the antibody is a humanized antibody or a human antibody.

[13] The method according to any one of [1] to [12], wherein the antibody is an anti-IL-6 receptor antibody or an anti-IL-31 receptor antibody.

[14] A method for preparing a composition containing an antibody having a pI value of 3.0 to 8.0, wherein the content of the antibody aggregate is reduced to 3% or less by the method of any one of [1] to [13].

[15] A composition containing an antibody complex at a ratio of 3% or less, which is a composition containing an antibody having a pI value of 3.0 to 8.0.

[16] A composition containing an aggregate at a ratio of 3% or less, which is a composition containing an antibody having a pI value of 3.0 to 8.0 prepared by the method described in [14].

[17] A method for preparing a pharmaceutical composition containing an antibody having a pI value of 3.0 to 8.0, the method comprising the following steps:

1) a step of preparing an antibody having a pI value of 3.0 to 8.0 and/or a composition containing the antibody by the preparation method described in [14]; and

2) The step of mixing the antibody with a pI value of 3.0 to 8.0 and/or a composition containing the antibody prepared in step 1) with a pharmaceutically acceptable carrier and/or additive to prepare a preparation.

[18] A method for removing antibody aggregates from a composition containing antibodies having a pI value of 3.0 to 8.0, the method comprising the following steps:

(a) treating a composition containing an antibody having a pI value of 3.0 to 8.0 under acidic conditions;

(b) a step of neutralizing the acidic composition obtained in step (a); and

(c) a step of removing the aggregates from the neutralized composition obtained in step (b) after at least 1 hour has passed since neutralization.

[19] A method for purifying a composition containing an antibody having a pI value of 3.0 to 8.0, the method comprising the following steps:

(a) treating a composition containing an antibody having a pI value of 3.0 to 8.0 under acidic conditions;

(b) a step of neutralizing the acidic composition obtained in step (a); and

(c) a step of removing the aggregates from the neutralized composition obtained in step (b) after a sufficient time has passed for the formation of the aggregates.

DETAILED DESCRIPTION

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for removing antibody aggregates or impurities from a composition containing an antibody having a low isoelectric point (pI). Specifically, the present invention relates to a method for removing antibody aggregates from a composition containing an antibody having a pI value of 3.0 to 8.0, the method comprising the following steps:

(a) treating a composition containing an antibody having a pI value of 3.0 to 8.0 under acidic conditions;

(b) a step of neutralizing the acidic composition obtained in step (a); and

(c) a step of removing the aggregates from the neutralized composition obtained in step (b) after at least 1 hour has passed since neutralization.

In the present invention, the aggregates are removed from the neutralized composition obtained in step (b) after a sufficient period of time has passed for the formation of aggregates generated in the composition, thereby suppressing the formation of new aggregates after purification.

Specifically, by removing aggregates from the neutralized composition obtained in step (b) at least one hour after neutralization, the formation of new aggregates after purification can be suppressed.

That is, step (c) of the present invention can also be expressed as follows:

・After a sufficient time for the formation of the aggregate has passed, the step of removing the aggregate from the neutralized composition obtained in step (b)

・A step of removing aggregates from the neutralized composition obtained in step (b) after at least 80% of the aggregates are formed relative to the amount of aggregates that can be generated in the composition

・A step of removing aggregates from the neutralized composition obtained in step (b) after at least 80% of the aggregates formed after the neutralized composition is kept for at least 24 hours

・After the formation of the aggregates that may occur in the neutralized composition obtained in step (b) is completed by 80% or more, the step of removing the aggregates from the composition

A step of removing the aggregates from the neutralized composition obtained in step (b) at least 1 hour before the completion of the formation of the aggregates in the composition.

Furthermore, the present invention relates to a method for removing impurities from a composition containing an antibody having a pI value of 3.0 to 8.0, the method comprising the following steps:

(a) loading a composition containing an antibody having a pI value of 3.0 to 8.0 onto an anion exchange resin; and

(b) A step of eluting antibodies having a pI value of 3.0 to 8.0 from an anion exchange resin in a bind/elute mode using an elution solution having a higher salt concentration than the composition of (a).

In the above method, before step (b), the step of washing the anion exchange resin with a washing solution may be included.

In the present invention, the method for removing aggregates of antibodies having a pI value of 3.0 to 8.0 from a composition containing the antibody can also be expressed as: a method for purifying the antibody (antibody monomer) from a composition containing the antibody having a pI value of 3.0 to 8.0, a method for removing impurities from a composition containing the antibody having a pI value of 3.0 to 8.0, a method for inhibiting the association of antibodies having a pI value of 3.0 to 8.0, etc.

Furthermore, the method for removing impurities from a composition containing an antibody with a pI value of 3.0 to 8.0 can also be expressed as: a method for purifying the antibody (antibody monomer) from a composition containing an antibody with a pI value of 3.0 to 8.0, a method for removing an antibody aggregate from a composition containing an antibody with a pI value of 3.0 to 8.0, etc.

In the present invention, the composition containing an antibody can also be expressed as: a solution containing an antibody, a culture solution of an antibody, a culture medium of an antibody, etc.

The antibody used in the present invention is not particularly limited as long as it binds to the desired antigen and may be either a polyclonal antibody or a monoclonal antibody. However, monoclonal antibodies are preferred from the perspective of stable production of homogeneous antibodies.

The monoclonal antibodies used in the present invention include not only monoclonal antibodies derived from humans, mice, rats, hamsters, rabbits, goats, camels, monkeys, and the like, but also genetically modified recombinant antibodies such as chimeric antibodies, humanized antibodies, and bispecific antibodies. Furthermore, these antibodies also include genetically modified antibodies in which the constant region of the antibody is artificially modified to improve the physical properties of the antibody molecule (specifically, to modify the isoelectric point (pI) or the affinity for Fc receptors) in order to improve blood retention or in vivo kinetics.

Furthermore, the immunoglobulin class of the antibody used in the present invention is not particularly limited and may be any class such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, etc., with IgG and IgM being preferred.

Furthermore, the antibodies used in the present invention include not only antibodies having constant and variable regions (whole antibodies), but also antibody fragments such as Fv, Fab, and F(ab) ₂ , or low-molecular-weight antibodies such as monovalent or divalent or higher-valent single-chain Fv (scFv, sc(Fv) ₂ ) or scFv dimers formed by linking antibody variable regions with linkers such as peptide linkers, and whole antibodies are preferred.

The antibodies used in the present invention described above can be produced by methods well known to those skilled in the art. Hybridomas producing monoclonal antibodies can be produced using generally known techniques as follows. Specifically, a desired antigen or a cell expressing the desired antigen is used as a sensitizing antigen, and immunization is performed using the sensitizing antigen according to conventional immunization methods. The resulting immune cells are fused with known parent cells using conventional cell fusion methods, and cells (hybridomas) producing monoclonal antibodies are screened using conventional screening methods to produce hybridomas. Hybridomas can be produced, for example, according to the method of Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46). When the immunogenicity of the antigen is low, it can be combined with an immunogenic macromolecule such as albumin before immunization.

Furthermore, it is possible to use: clone antibody genes from hybridomas, insert them into appropriate vectors, import them into hosts, and use genetic recombination technology to produce recombinant antibodies (e.g., with reference to Carl, A. K. Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Specifically, reverse transcriptase is used to synthesize the cDNA of the variable region (V region) of the antibody from the mRNA of the hybridoma. When obtaining the DNA encoding the V region of the target antibody, it is connected to the DNA encoding the desired antibody constant region (C region) and then inserted into an expression vector. Alternatively, the DNA encoding the antibody V region can be inserted into an expression vector containing the DNA of the antibody C region. Inserted into the expression vector to express under the regulation of expression regulatory regions such as enhancers and promoters. Thereafter, the expression vector can be used to transform host cells to express the antibody.

In the present invention, genetically modified recombinant antibodies, such as chimeric antibodies and humanized antibodies, which have been artificially modified for the purpose of reducing heterologous antigenicity to humans, can be used. These modified antibodies can be prepared using known methods. Chimeric antibodies are antibodies composed of the variable regions of the heavy and light chains of mammals other than humans, such as mouse antibodies, and the constant regions of the heavy and light chains of human antibodies. They can be obtained by ligating DNA encoding the variable regions of mouse antibodies with DNA encoding the constant regions of human antibodies, inserting them into expression vectors, and introducing them into hosts to produce them.

Humanized antibodies are also referred to as reshaped human antibodies, and are antibodies obtained by transplanting the complementarity determining regions (CDRs; complementarity determining regions) of mammals other than humans, such as mouse antibodies, to the complementarity determining regions of human antibodies. Common genetic recombination methods are also known. Specifically, a DNA sequence is designed in which the CDRs of mouse antibodies are connected to the framework regions (framework regions; FRs) of human antibodies, and the DNA sequence is made into multiple oligonucleotides with overlapping ends, which are synthesized by the PCR method using the multiple oligonucleotides. The obtained DNA is connected to the DNA encoding the constant region of the human antibody, and thereafter, inserted into an expression vector and introduced into a host to produce, thereby obtaining (with reference to European patent application publication number EP239400, WO96/02576). For the FRs of human antibodies connected via CDRs, FRs in which the complementarity determining regions form good antigen binding sites can be selected. As needed, the amino acids in the framework regions of the antibody variable regions can be substituted so that the complementarity determining regions of the reconstructed human antibodies form appropriate antigen binding sites (Sato, K et al., Cancer Res. (1993) 53, 851-856).

As a technique for substituting amino acids in antibodies to improve antibody activity, physical properties, pharmacokinetics, safety, etc., for example, the following techniques are known. The antibodies used in the present invention also include antibodies having such amino acid substitutions (including deletions or additions).

Regarding the technology of amino acid substitution in the variable region of IgG antibodies, the following have been reported: humanization technology (Tsurushita N, Hinton PR, Kumar S., Design of humanized antibodies: from anti-Tac to Zenapax., Methods. 2005 May; 36(1): 69-83.); affinity maturation technology based on amino acid substitution in the complementarity determining region (CDR) for enhancing binding activity (Rajpal A, Beyaz N, Haber L, Cappuccilli G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea R., A general method for greatly improving the affinity of antibodies by using combinatorial libraries., Proc Natl Acad Sci U S A. 2005 Jun 14; 102(24):8466-71.); technology for improving physicochemical stability by amino acid substitution in the framework (FR) (Ewert S, Honegger A, Pluckthun A., Stability Improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering., Methods. 2004 Oct; 34(2): 184-99. Review). In addition, as a technology for performing amino acid substitution in the Fc region of an IgG antibody, a technology for enhancing antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) activity is known (Kim SJ, Park Y, Hong HJ., Antibody engineering for the development of therapeutic antibodies., Mol Cells. 2005 Aug 31; 20(1): 17-29. Review.). Furthermore, Fc amino acid substitution techniques have been reported that not only enhance such effector functions but also increase the half-life of antibodies in the blood (Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurushita N., An engineered human IgG1 antibody with longer serum half-life., J Immunol. 2006 Jan 1; 176(1): 346-56.; Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C, Ober RJ, Ward ES., Increasing the serum persistence of an IgG fragment by random mutagenesis., Nat Biotechnol. 1997 Jul; 15(7): 637-40.). Furthermore, various amino acid substitution techniques in constant regions for the purpose of improving antibody properties are also known (WO09/41613).

Moreover, methods for obtaining human antibodies are also known. For example, human lymphocytes are sensitized in vitro with a desired antigen or cells expressing a desired antigen, and the sensitized lymphocytes are fused with human myeloma cells, such as U266, to obtain desired human antibodies with antigen-binding activity (see Patent Publication No. 1-59878). Furthermore, transgenic animals with human antibody gene repertoires immunized with antigens can obtain desired human antibodies (see WO93/12227, WO92/03918, WO94/02602, WO94/25585, WO96/34096, WO96/33735). Furthermore, the technology of obtaining human antibodies by panning using human antibody libraries is also known. For example, the variable regions of human antibodies can be expressed on the surface of phages in the form of single-chain antibodies (scFv) by phage display, and phages that bind to the antigen are selected. By analyzing the selected phage genes, the DNA sequences encoding the variable regions of human antibodies that bind to the antigen can be determined. Once the DNA sequence of the antigen-binding scFv is known, an appropriate expression vector containing that sequence can be constructed to obtain human antibodies. These methods are well known and can be found in WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, and WO95/15388. The antibodies used in the present invention also include such human antibodies.

When the antibody is produced by introducing the temporarily isolated antibody gene into an appropriate host, a combination of an appropriate host and an expression vector can be used. When eukaryotic cells are used as hosts, animal cells, plant cells, and fungal cells can be used. As animal cells, known examples include: (1) mammalian cells, such as CHO, COS, myeloma, BHK (baby hamster kidney), HeLa, and Vero; (2) amphibian cells, such as Xenopus oocytes; or (3) insect cells, such as sf9, sf21, and Tn5. As plant cells, known examples include cells derived from the genus Nicotiana, such as Nicotiana tabacum, which can be cultured as callus. As fungal cells, known examples include yeast, such as the genus Saccharomyces, such as Saccharomyces serevisiae; and filamentous fungi, such as the genus Aspergillus, such as Aspergillus niger. When using prokaryotic cells, there are production systems using bacterial cells. Known bacterial cells include Escherichia coli (E. coli) and Bacillus subtilis. The target antibody gene is introduced into these cells by transformation, and the transformed cells are cultured in vitro to obtain the antibody.

Furthermore, the antibodies used in the present invention include antibody fragments or low-molecular-weight antibodies, as well as modified antibodies. For example, antibody fragments or low-molecular-weight antibodies include Fab, F(ab')2, Fv, or single-chain Fvs (scFv, sc(Fv) ₂ , etc.) obtained by linking H and L chain Fvs with appropriate linkers (Huston, JS et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883). Specifically, antibodies are treated with enzymes such as papain and pepsin to generate antibody fragments, or genes encoding these antibody fragments are constructed, introduced into expression vectors, and then expressed in appropriate host cells (for example, see Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; Bird, RE and Walker, BW, Trends Biotechnol. (1991) 9, 132-137).

As modified antibodies, antibodies bound to various molecules such as polyethylene glycol (PEG) or cytotoxic drugs can also be used (Farmaco. 1999 Aug 30; 54(8): 497-516.; Cancer J. 2008 May-Jun; 14(3): 154-69.). The antibodies used in the present invention also include these modified antibodies. Such modified antibodies can be obtained by chemically modifying antibodies. These methods are already established in this field.

Examples of antibodies used in the present invention include, but are not limited to, anti-tissue factor antibodies, anti-IL-6 receptor antibodies, anti-IL-6 antibodies, HM1.24 antigen monoclonal antibodies, anti-parathyroid hormone-related peptide antibodies (anti-PTHrP antibodies), anti-glypican-3 antibodies, anti-ganglioside GM3 antibodies, anti-TPO receptor agonist antibodies, coagulation factor VIII replacement antibodies, anti-IL31 receptor antibodies, anti-HLA antibodies, anti-AXL antibodies, anti-CXCR4 antibodies, anti-NR10 antibodies, and bispecific antibodies of factor IX and factor X.

Preferred reshaped humanized antibodies used in the present invention include, but are not limited to, humanized anti-interleukin-6 (IL-6) receptor antibodies (tocilizumab, hPM-1, or MRA) (see WO92/19759), humanized anti-HM1.24 antigen monoclonal antibodies (see WO98/14580), humanized anti-parathyroid hormone-related peptide antibodies (anti-PTHrP antibodies) (see WO98/13388), humanized anti-tissue factor antibodies (see WO99/51743), anti-glypican-3 humanized IgG1κ antibodies (see PCT/JP05/013103), anti-NR10 humanized antibodies (see WO2009/072604), and bispecific humanized antibodies against Factor IX and Factor X. Particularly preferred humanized antibodies used in the present invention include humanized anti-IL-6 receptor antibodies, humanized anti-NR10 antibodies, and bispecific humanized antibodies of Factor IX and Factor X.

Preferred human IgM antibodies include anti-ganglioside GM3 recombinant human IgM antibodies (see WO05/05636 ).

As low-molecular-weight antibodies, anti-TPO receptor agonist diabody (Diabody) (see WO02/33072), anti-CD47 agonist diabody (Diabody) (see WO01/66737), and the like are preferred.

In the present invention, antibodies with low isoelectric points (low pI antibodies) refer to antibodies with low isoelectric points, which are particularly rare in nature. Examples of the isoelectric points of such antibodies include, but are not limited to, 3.0 to 8.0, preferably 5.0 to 7.5, more preferably 5.0 to 7.0, more preferably 5.0 to 6.8, further preferably 5.0 to 6.5, and particularly preferably 5.0 to 6.0. It should be noted that natural (or normal) antibodies are generally considered to have an isoelectric point in the range of 7.5 to 9.5.

Furthermore, the antibodies used in the present invention are preferably pI-modified antibodies whose pI is lowered by altering the amino acid residues exposed on the antibody surface. Such pI-modified antibodies are antibodies whose pI is lowered by at least 1, preferably at least 2, and more preferably at least 3, compared to the pI of the antibody before the alteration. As described in the Examples below, the isoelectric point of Mab1, which was controlled by altering the amino acid sequence of Mab3 (isoelectric point: 9.4), was 5.8. Furthermore, the isoelectric point of Mab2, which was controlled by altering the amino acid sequence of the fully humanized NS22 antibody (isoelectric point: 7.8) produced using the method described in Example 12 of WO2009/072604, was 5.6.

Examples of antibodies with improved isoelectric points include, but are not limited to, the anti-IL-6 receptor antibody Mab1 (H chain/SEQ ID NO: 1, L chain/SEQ ID NO: 2) described in WO2009/041621, and the fully humanized NS22 antibody (H chain/SEQ ID NO: 3, L chain/SEQ ID NO: 4), which is an anti-NR10 humanized antibody prepared by the method described in Example 12 of WO2009/072604.

In the case of the H chain variable region, amino acid residues exposed on the antibody surface include, but are not limited to, amino acid residues selected from the group consisting of the following amino acid residues based on Kabat numbering: H1, H3, H5, H8, H10, H12, H13, H15, H16, H19, H23, H25, H26, H31, H39, H42, H43, H44, H46, H61, H62, H64, H65, H68, H71, H72, H73, H75, H76, H81, H82b, H83, H85, H86, H105, H108, H110, and H112. In the case of the L chain variable region, amino acid residues selected from the group consisting of the following amino acid residues based on Kabat numbering: L1, L3, L7, L8, L9, L11, L12, L16, L17, L18, L20, L22, L24, L27, L38, L39, L41, L42, L43, L45, L46, L49, L53, L54, L55, L57, L60, L63, L65, L66, L68, L69, L70, L74, L76, L77, L79, L80, L81, L85, L100, L103, L105, L106, and L107 can be mentioned, but are not limited thereto.

In the present invention, "modification" refers to substitution of an original amino acid residue with another amino acid residue, deletion of an original amino acid residue, addition of a new amino acid residue, etc., and preferably refers to substitution of an original amino acid residue with another amino acid residue.

Among amino acids, there are known amino acids with a charge. Generally, lysine (K), arginine (R), and histidine (H) are known as amino acids with a positive charge (positively charged amino acids). Aspartic acid (D), glutamic acid (E), etc. are known as amino acids with a negative charge (negatively charged amino acids). Amino acids other than these are known to be considered as uncharged amino acids.

The amino acid residue to be modified in the present invention can be appropriately selected from the amino acid residues included in either group (a) or (b) below, but is not particularly limited to these amino acids.

(a) Glutamic acid (E), aspartic acid (D);

(b) Lysine (K), arginine (R), and histidine (H).

Furthermore, when the amino acid residue before modification already has a charge, modification to an uncharged amino acid residue is also a preferred approach.

That is, as modifications in the present invention, there are:

(1) Substitution from a charged amino acid to an uncharged amino acid;

(2) Substitution of a charged amino acid into an amino acid with an opposite charge;

(3) Substitution from an uncharged amino acid to a charged amino acid.

The isoelectric point can be measured by isoelectric electrophoresis known to those skilled in the art. Furthermore, the theoretical isoelectric point can be calculated using gene and amino acid sequence analysis software (Genetyx, etc.).

Antibodies in which the charge of amino acid residues is altered can be obtained as follows: altering the nucleic acid encoding the antibody, culturing the nucleic acid in a host cell, and purifying the antibody from the host cell culture. In the present invention, "altering the nucleic acid" refers to altering the nucleic acid sequence so that codons corresponding to the amino acid residues introduced by the alteration are formed. More specifically, it refers to altering the nucleotide sequence of the nucleic acid so that the codons for the amino acid residues before the alteration become codons for the amino acid residues introduced by the alteration. That is, the codons encoding the amino acid residues to be altered are replaced by codons encoding the amino acid residues introduced by the alteration. Regarding such alterations in nucleic acids, those skilled in the art can appropriately perform such alterations using known techniques, such as site-specific mutagenesis, PCR mutation introduction methods, and the like.

In a preferred embodiment of the present invention, the generated aggregates can be effectively removed from a composition containing an antibody having a pI value of 3.0 to 8.0 by a method comprising the following steps:

(a) treating a composition containing an antibody having a pI value of 3.0 to 8.0 under acidic conditions;

(b) a step of neutralizing the acidic composition obtained in step (a); and

(c) a step of removing the aggregates from the neutralized composition obtained in step (b) after at least 1 hour has passed since neutralization.

Examples of the antibody complex having a pI of 3.0 to 8.0 in the present invention include, but are not limited to, 1.5-mers, dimers, trimers, tetramers, and pentamers.

The method of the present invention effectively removes low-pI antibody aggregates produced in solution and suppresses the risk of subsequent formation of new aggregates. In the present invention, examples of acidic conditions for treating the antibody-containing composition include, but are not limited to, pH 2.0 to 4.0, preferably pH 3.0 to 3.9, and more preferably pH 3.1 to 3.8.

Examples of methods for treating an antibody-containing composition under acidic conditions include, but are not limited to, methods of adding a known acid such as hydrochloric acid, citric acid, phosphoric acid, or acetic acid to the antibody-containing composition.

In the present invention, the antibody-containing composition treated under acidic conditions is preferably maintained for a predetermined time. Examples of the maintenance time include, but are not limited to, 15 minutes to 4 hours, preferably 30 minutes to 2 hours, and more preferably 1 to 1.5 hours.

In the present invention, the step of neutralizing the acidic composition refers to the step of neutralizing the composition containing the antibody having a pI value of 3.0 to 8.0 that has been treated with acid. The pH value after neutralization is generally 4.5 to 8.5, preferably 6.5 to 8.5, and more preferably 7.0 to 8.5, but is not limited thereto.

The results of the Examples of this application have demonstrated that, in the antibody purification methods of the present invention, when the step of increasing the pH of the acidic composition (e.g., neutralization step) is performed intermittently, aggregates continue to form each time. Therefore, in the antibody purification methods or aggregate removal methods of the present invention, it is preferred that the step of removing generated antibody aggregates be performed after the step of increasing the pH (e.g., neutralization step) is completed. Furthermore, it is preferred that the step of increasing the pH again not be performed after aggregate removal.

Furthermore, the present invention may further include the following step: neutralizing the acidic composition containing the antibody, maintaining the composition at a pH lower than the pI of the antibody for a predetermined time, adjusting the pH of the composition to a value higher than the pI of the antibody, and maintaining the composition for a predetermined time.

More specifically, the present invention relates to a method for purifying a composition containing antibodies having a pI value of 3.0 to 8.0, the method comprising the following steps:

(a) treating a composition containing an antibody having a pI value of 3.0 to 8.0 under acidic conditions;

(b) neutralizing the acidic composition obtained in step (a) and maintaining the composition at a pH lower than the pI of the antibody;

(c) a step of adjusting the pH of the neutralization composition obtained in step (b) to a value higher than the pI of the antibody; and

(d) a step of removing the aggregates from the neutralized composition obtained in step (b) after at least 1 hour has passed since neutralization.

Examples of pH values lower than the pI of the antibody include, but are not limited to, pH values that are, for example, 0.3 or more, preferably 0.5 or more, and more preferably 1.0 or more lower than the pI of the antibody. On the other hand, examples of pH values higher than the pI of the antibody include, but are not limited to, pH values that are, for example, 0.3 or more, preferably 0.5 or more, and more preferably 1.0 or more higher than the pI of the antibody. Furthermore, examples of the time for which the neutralized composition is maintained include, but are not limited to, 1 hour or more (e.g., 1 hour to 7 days, preferably 1 to 3 days), preferably 2 hours or more (e.g., 2 hours to 7 days, preferably 2 to 72 hours), more preferably 6 hours or more (e.g., 6 hours to 7 days, preferably 6 to 72 hours), and even more preferably 12 hours or more (e.g., 12 hours to 7 days, preferably 12 to 72 hours).

Neutralization can be performed using a buffer. The buffer used for neutralization is generally not particularly limited as long as it is a buffer for adjusting pH, and examples thereof include Tris and disodium hydrogen phosphate, with Tris being preferred, but not limited thereto.

The present invention is characterized by the discovery, for the first time, that irreversible aggregates continuously form over a certain period of time after neutralization of an acidic solution containing an antibody. This phenomenon is not observed for conventional antibodies (antibodies with a pI of approximately 9) whose pI remains unchanged. While the cause of the formation of irreversible aggregates remains unclear, it is believed that these may be due to stress on low-pI antibodies in acidic solutions, changes in the charge of antibody molecules caused by adjusting the pH of the composition near the antibody pI, or by adjusting the pH of the composition across the antibody pI. In any case, in the method of the present invention, after neutralization of an antibody with a pI of 3.0 to 8.0 that has been treated with acid, the generated antibody aggregates are removed after a certain period of time, thereby preventing the formation of new aggregates after the removal of the aggregates.

More specifically, the time from neutralization to removal of antibody aggregates is typically 1 hour or more (e.g., 1 hour to 7 days, preferably 1 to 3 days), preferably 2 hours or more (e.g., 2 hours to 7 days, preferably 2 to 72 hours), more preferably 6 hours or more (e.g., 6 hours to 7 days, preferably 6 to 72 hours), further preferably 12 hours or more (e.g., 12 hours to 7 days, preferably 12 to 72 hours), particularly preferably 20 hours, 23 hours, 24 hours, or 66 hours or more, but is not limited thereto.

Alternatively, in the present invention, after a sufficient time for aggregate formation has elapsed, the aggregate is removed from the neutralized composition. In the present invention, a sufficient time for aggregate formation refers to the time required for the antibodies in the neutralized composition to form aggregates to form aggregates. The sufficient time for aggregate formation in the present invention encompasses not only the time required for all antibodies to form aggregates to form aggregates, but also the time required for at least 50%, preferably at least 70%, more preferably at least 80%, and particularly preferably at least 90% of the antibodies to form aggregates to form aggregates. Those skilled in the art can determine the time from neutralization to removal of antibody aggregates, taking into account the aggregate formation time shown in Example 1.

Alternatively, in the present invention, after at least 50% or more, preferably 70% or more, more preferably 80% or more, and particularly preferably 90% or more of the aggregates that can be produced in the neutralization composition are formed, the aggregates can be removed from the composition. In the present invention, the amount of aggregates that can be produced in the neutralization composition refers to the amount of aggregates formed by the antibodies in the neutralization composition that are expected to form aggregates. Examples of the amount of aggregates that can be produced in the neutralization composition include, but are not limited to, the amount of aggregates formed after neutralization, typically for at least 1 hour (e.g., 1 hour to 7 days, preferably 1 to 3 days), preferably for at least 2 hours (e.g., 2 hours to 7 days, preferably 2 to 72 hours), more preferably for at least 6 hours (e.g., 6 hours to 7 days, preferably 6 to 72 hours), and particularly preferably 24 hours.

Alternatively, in the present invention, aggregates can be removed from the composition after the formation of aggregates that can be induced in the neutralization composition has been completed by at least 50%, preferably at least 70%, more preferably at least 80%, and particularly preferably at least 90%. In the present invention, the formation of aggregates that can be induced in the neutralization composition means that the antibodies expected to form aggregates form aggregates.

Alternatively, in the present invention, the aggregate can be removed from the neutralization composition at least one hour before aggregate formation is complete. In the present invention, the completion of aggregate formation means that aggregate formation of the antibodies expected to form an aggregate is complete. The completion of aggregate formation in the present invention refers not only to the completion of aggregate formation of all antibodies expected to form an aggregate, but also to the completion of aggregate formation of at least 50%, preferably at least 70%, more preferably at least 80%, and particularly preferably at least 90% of the antibodies expected to form an aggregate.

Whether the formation of antibody complexes is complete can be confirmed by, for example, analyzing the neutralized composition of the present invention by size exclusion chromatography (SEC) over time after neutralization treatment as described in the Examples below, and plotting the amount of complexes produced.

In the present invention, the generated aggregates can be removed by known methods such as anion exchange chromatography, multicomponent chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography. Anion exchange chromatography or hydroxyapatite chromatography is particularly preferred.

Furthermore, the aggregate of the present invention can be effectively removed by using a purification step based on a bind/elute mode of anion exchange chromatography as described later.

In the present invention, the anion exchange resin is not limited as long as it exhibits anion exchange function. Examples of the anion exchange resin include:

YMC-BioPro (YMC company),

Q Sepharose High Performance (GE Healthcare),

Q Sepharose Fast Flow (GE Healthcare),

Q Sepharose XL (GE Healthcare),

Capto Q ImpRes (GE Healthcare),

Capto Q (GE Healthcare),

Capto DEAE (GE Healthcare company),

SOURCE 30Q (GE Healthcare),

SOURCE 15Q (GE Healthcare),

POROS HQ (Life technologies company),

POROS D (Life technologies),

POROS PI (Life Technologies),

Eshumuno Q (Merck Millipore),

Fractogel TMAE (Merck Millipore),

Fractogel DEAE (Merck Millipore),

Macro-Prep Q (Bio-Rad Laboratories),

Macro-Prep DEAE (Bio-Rad Laboratories),

Giga Cap Q-650M (TOSOH),

Giga Cap DEAE-650M (TOSOH),

Q HyperCel (PALL), etc., but not limited to these.

Furthermore, as resins used for hydroxyapatite column chromatography, there can be exemplified:

Ceramic Hydroxyapatite (Bio-Rad Laboratories),

Ceramic Fluoloapatite (Bio-Rad Laboratories),

MPC Ceramic Hydroxyfluoloapatite (Bio-Rad Laboratories),

HA Ultragel (PALL) and the like, but not limited thereto.

Furthermore, as resins used for multicomponent chromatography, there can be exemplified:

Capto Adhere (GE Healthcare),

Capto MMC (GE Healthcare),

Eshumuno HCX (Merck Millipore), but not limited to these.

Furthermore, as resins used for hydrophobic interaction chromatography, there can be exemplified:

Phenyl Sepharose High Performance (GE Healthcare),

Butyl Sepharose High Performance (GE Healthcare),

Phenyl Sepharose 6 Fast Flow (GE Healthcare),

Butyl-S Sepharose 6 Fast Flow (GE Healthcare),

Butyl Sepharose 4 Fast Flow (GE Healthcare),

Octyl Sepharose 4 Fast Flow (GE Healthcare),

Capto Phenyl ImpRes (GE Healthcare),

Capto Phenyl (GE Healthcare),

Capto Butyl (GE Healthcare),

Capto Octyl (GE Healthcare),

Fractogel Phenyl (Merck Millipore),

Fractogel Propyl (Merck Millipore),

TOYOPEARL Butyl (TOSOH Corporation),

TOYOPEARL Ether (TOSOH Corporation),

TOYOPEARL Hexyl (TOSOH Corporation),

TOYOPEARL Phenyl (TOSOH Corporation),

TOYOPEARL PPG (TOSOH Corporation),

TOYOPEARL SuperButyl (TOSOH Corporation),

TOYOPEARL Butyl-600 (TOSOH Corporation),

Macro-Prep HIC (Bio-Rad Laboratories) and the like, but not limited thereto.

Whether the aggregates have been removed can be determined by methods known to those skilled in the art, such as size exclusion chromatography (SEC), but is not limited thereto.

In the present invention, the antibody-containing composition treated under acidic conditions can be purified by known purification methods such as protein A column chromatography. Specifically, the method for removing aggregates of the present invention can include a step of "purifying the composition containing an antibody having a pI value of 3.0 to 8.0 by protein A column chromatography" before the step of "treating (a) the composition containing an antibody having a pI value of 3.0 to 8.0 under acidic conditions."

Furthermore, in the present invention, after the aforementioned steps (a) to (c) for removing antibody aggregates having a pI value of 3.0 to 8.0, a purification step utilizing anion exchange chromatography in a bind/elute mode (bind/elute fraction) as described below, and/or a purification step utilizing multicomponent chromatography in a flow-through mode (flow-through fraction) or hydrophobic interaction chromatography in a flow-through mode may be included. Combining these steps also removes impurities other than aggregates, allowing for efficient purification of antibodies having a pI value of 3.0 to 8.0.

Specifically, the present invention relates to a method for effectively removing impurities from a composition containing an antibody having a pI value of 3.0 to 8.0, the method comprising the following steps:

(a) a step of loading a composition containing an antibody having a pI value of 3.0 to 8.0 onto an anion exchange resin; and

(b) A step of eluting antibodies having a pI value of 3.0 to 8.0 from an anion exchange resin in a bind/elute mode using an elution solution having a higher salt concentration than the composition of (a).

In the above method, before step (b), the method further comprises the step of washing the anion exchange resin with a washing solution.

As described later, in the present invention, the elution fraction from the anion exchange resin may be further subjected to multi-layer chromatography or hydrophobic interaction chromatography to further remove impurities.

The impurities to be removed can be any substance other than the target protein. Examples of impurities include, but are not limited to, host cell proteins (host cell proteins) or DNA, protein A (eluate from the column), complexes or fragments derived from the target protein, viruses, endotoxins, culture medium components such as Hy-Fish (FL), IGF, insulin, antibiotics, and defoaming agents. In the present invention, it is preferred to remove host cell proteins or DNA, protein A, complexes derived from the target protein (e.g., antibody complexes), and viruses, but is not limited to these.

The viruses removed by the method of the present invention are not particularly limited. The viruses of the present invention include DNA viruses and RNA viruses. Examples of DNA viruses include parvoviruses such as MVM, and examples of RNA viruses include retroviruses such as MuLV and reoviruses such as Reo3, but are not limited to these. Specific examples of viruses removed by the method of the present invention include, for example, MuLV, PRV, Reo3, MVM, SV40, VSV, herpes simplex virus, CHV, Sindbis virus, mumps virus, vaccinia virus, measles virus, rubella virus, influenza virus, herpes zoster virus, cytomegalovirus, parainfluenza virus, Epstein-Barr virus, HIV, HA, HB, NANB, ATL, ECHO, parvovirus, and preferably include, but are not limited to, viruses such as MuLV, Reo3, MVM, PRV, and SV40.

The present invention, leveraging the characteristics of low-pi antibodies, has established for the first time a purification method using an anion column bind/elute mode, which has been unattainable for antibodies with higher pIs. Furthermore, the present invention establishes for the first time a purification method that further removes impurities by combining this method with multi-layer chromatography or hydrophobic interaction chromatography.

In the method of the present invention, purification of a composition containing an antibody with a pI of 3.0 to 8.0 using an anion exchange resin is typically performed using a column equilibrated with a buffer containing Tris, BIS-TRIS, or histidine at a concentration of 1 to 100 mmol/L, chloride ions or acetate ions as counterions, and a pH of 6 to 9. Preferably, the column is equilibrated with a buffer containing Tris at a concentration of 10 to 50 mmol/L, chloride ions or acetate ions as counterions, and a pH of 7 to 8, but the method is not limited thereto.

Secondly, the method of the present invention may include the step of washing the anion exchange resin adsorbed with an antibody having a pI value of 3.0 to 8.0. The washing is performed under the same conditions as the usual equilibration conditions; alternatively, the washing is performed using a buffer having a lower concentration, or an equal or higher pH value than the elution conditions. Specifically, the washing is performed using a column equilibrated with the following buffer: a buffer having a concentration of 1 to 100 mmol/L of Tris, BIS-TRIS, or histidine, with chloride ions or acetate ions as counterions, and a pH value of pH 6 to pH 9. Preferably, the washing is performed using the following buffer: a buffer having a concentration of 10 to 50 mmol/L of Tris, with chloride ions or acetate ions as counterions, and a pH value of pH 7 to pH 8, but is not limited thereto.

Next, in the method of the present invention, the antibody is eluted from the anion exchange resin in a bind/elute mode using an elution solution having a higher salt concentration than that of the composition containing an antibody with a pI value of 3.0 to 8.0. Elution conditions are typically performed using a buffer solution containing Tris, BIS-TRIS, and histidine at a concentration of 1 to 500 mmol/L, chloride ions or acetate ions as counterions, and, if necessary, sodium chloride, potassium chloride, sodium sulfate, or sodium phosphate, at a pH of 6 to 9. Preferably, the buffer solution is used, but is not limited to, a buffer solution containing Tris at a concentration of 10 to 500 mmol/L, chloride ions or acetate ions as counterions, and, if necessary, 50 to 500 mmol/L of sodium chloride, sodium phosphate, or sodium sulfate, at a pH of 7 to 8. Examples of salt concentrations higher than that of the composition containing an antibody with a pI value of 3.0 to 8.0 include, but are not limited to, 5 mmol/L or higher, preferably 10 mmol/L or higher. Examples of the elution solution include, but are not limited to, solutions containing at least one selected from the group consisting of sodium chloride, Tris salts, sodium sulfate salts, and sodium phosphate salts.

In the present invention, the eluted fraction (eluate) containing the antibody having a pI value of 3.0 to 8.0 obtained from the anion exchange resin can be further purified using multiplex chromatography (for example, a resin having both hydrophobic interaction and anion exchange).

Purification of an antibody-containing composition using a multi-component chromatography resin is typically performed using a buffer solution containing Tris, BIS-TRIS, and histidine at a concentration of 1 to 500 mmol/L, chloride ions or acetate ions as counterions, and, if necessary, sodium chloride, potassium chloride, sodium sulfate, ammonium sulfate, sodium citrate, or arginine, at a pH of 4 to 9. Preferably, the purification is performed using a column equilibrated with a buffer solution containing Tris at a concentration of 10 to 500 mmol/L, chloride ions or acetate ions as counterions, and, if necessary, 50 to 500 mmol/L of sodium chloride and/or sodium sulfate, at a pH of 6 to 7, but the purification is not limited thereto.

The eluted fraction from anion exchange chromatography obtained by the method of the present invention is loaded onto a multicomponent chromatography column to obtain a fraction containing the target antibody with a pI of 3.0 to 8.0 as a flow-through fraction and/or an eluted fraction. Typically, the loaded fraction is pre-adjusted to the same pH as that used for equilibration, and, if necessary, the same salt as that used in the equilibration buffer may be added.

In the present invention, the flow-through fraction refers to the fraction recovered from the fraction loaded on the column that is not adsorbed to the column (impurities are adsorbed to the column, but the target substance is not). On the other hand, the eluted fraction refers to the fraction recovered from the fraction loaded on the column by passing through a buffer solution having a higher salt concentration than that of the loaded fraction (the target substance (and, if necessary, impurities) are adsorbed to the column).

Furthermore, in the present invention, the eluted fraction (eluate) containing the antibody having a pI value of 3.0 to 8.0 obtained from the anion exchange resin can be further purified using hydrophobic interaction chromatography.

Purification of an antibody-containing composition using a hydrophobic interaction chromatography resin is generally performed using a buffer solution containing Tris, BIS-TRIS, or histidine at a concentration of 1 to 500 mmol/L, chloride ions or acetate ions as counterions, and, if necessary, sodium chloride, potassium chloride, sodium sulfate, ammonium sulfate, sodium citrate, or arginine, at a pH of 4 to 9. Preferably, the purification is performed using a column equilibrated with a buffer solution containing Tris at a concentration of 10 to 500 mmol/L, chloride ions or acetate ions as counterions, and, if necessary, 50 to 500 mmol/L of sodium chloride and/or sodium sulfate, at a pH of 7 to 8, but the purification is not limited thereto.

The eluted fraction from anion exchange chromatography obtained by the method of the present invention is loaded onto hydrophobic interaction chromatography to obtain a fraction containing the target antibody as a flow-through fraction and/or an eluted fraction. Typically, the loaded fraction is pre-adjusted to the same pH as that used for equilibration, and, if necessary, the same salt as that used in the equilibration buffer may be added.

In the method of the present invention, by preparing the buffer solution and the loaded component to have a composition that does not contain chloride ions, a rust-preventing effect on the buffer solution tank and the component tank can be expected.

In the method of removing impurities from a composition containing an antibody having a pI value of 3.0 to 8.0 of the present invention, the composition loaded on an anion exchange resin can be purified by a known purification method such as protein A column chromatography before loading on the anion exchange resin. Furthermore, the composition loaded on the anion exchange resin can be subjected to the following steps (a) to (c):

(a) maintaining a composition containing an antibody having a pI value of 3.0 to 8.0 under acidic conditions;

(b) a step of neutralizing the acidic composition obtained in step (a); and

(c) a step of removing the aggregates from the neutralized composition obtained in step (b) after at least 1 hour has passed since neutralization.

Whether or not the impurities have been removed can be determined by size exclusion chromatography (SEC) when the impurities are proteins, but the method is not limited thereto.

Furthermore, when the impurity is DNA, qPCR method, threshold method, etc. can be used, but it is not limited to these.

When the impurities are cell-derived proteins (Host cell protein/HCP), ELISA using anti-HCP antibodies can be used, but the method is not limited to this.

When the impurity is protein A, ELISA using an anti-protein A antibody can be used, but the method is not limited to this.

When the impurities are viruses, qPCR, tissue infection, plaque assay, etc. can be used, but are not limited to these.

When the impurity is IGF, the ELISA method using an anti-IGF antibody can be used, but the method is not limited to this.

When the impurity is insulin, the ELISA method using anti-insulin antibodies can be used, but the method is not limited to this.

When the impurity is FL, ELISA using an anti-FL antibody can be used, but the method is not limited to this.

When the impurity is a defoaming agent, NMR can be used, but it is not limited to this.

When the impurity is endotoxin, it can be measured by a colorimetric method or a turbidimetric method based on the reaction of activating LAL (Limulus Amebocyte Lysate), a component of a horseshoe crab blood cell extract, but the method is not limited to these.

When the impurities are antibiotics, their concentrations are measured by ELISA using antibodies that specifically recognize antibiotics such as gentamicin, but the method is not limited to these.

Furthermore, the present invention relates to a method for producing an antibody having a pI value of 3.0 to 8.0, comprising the step of removing aggregates and/or impurities of the antibody from a composition containing the antibody having a pI value of 3.0 to 8.0. Furthermore, the present invention relates to a method for producing a composition containing the antibody having a pI value of 3.0 to 8.0, comprising the step of:

(a) obtaining a composition containing an antibody having a pI value of 3.0 to 8.0; and

(b) A step of purifying antibodies having a pI value of 3.0 to 8.0 from the composition of step (a) using the purification method described in this specification.

Purification of antibodies includes removing antibody aggregates and/or impurities from a composition containing the antibodies. In the composition containing antibodies having a pI value of 3.0 to 8.0 obtained in the present invention, the aggregate content can be, for example, 5% or less, preferably 4% or less, and particularly preferably 3% or less, but is not limited to these. In the present invention, the aggregate content refers to the ratio of antibodies forming aggregates relative to the antibodies contained in the composition.

Furthermore, the present invention relates to antibodies having a pI value of 3.0 to 8.0 obtained by the purification methods or production methods described herein, or compositions containing such antibodies. Furthermore, the present invention relates to antibodies having a pI value of 3.0 to 8.0 obtained by the purification methods or production methods described herein, or pharmaceutical compositions containing such antibodies. The pharmaceutical compositions of the present invention may contain pharmaceutically acceptable carriers and/or additives.

Furthermore, the present invention relates to a method for preparing a pharmaceutical composition containing an antibody having a pI value of 3.0 to 8.0, the method comprising the following steps:

1) a step of obtaining an antibody having a pI value of 3.0 to 8.0 by the method described in this specification; and

2) The step of mixing the antibody with a pI value of 3.0 to 8.0 prepared in step 1) with a pharmaceutically acceptable carrier and/or additive to prepare a preparation.

The pharmaceutical composition of the present invention can be a solution preparation (a solution preparation containing an antibody) or a lyophilized agent. The solution preparation of the present invention further comprises a solution before the lyophilization process or a solution after redissolution in the preparation step of the lyophilized agent. The solution preparation of the present invention is preferably a solution preparation prepared without lyophilizing the solution preparation during the preparation process. Moreover, the lyophilized agent of the present invention can be obtained by lyophilizing the solution preparation of the present invention by methods well known to those skilled in the art.

Furthermore, the preparation of the present invention may contain additives or carriers such as cryoprotectants, suspending agents, solubilizers, isotonic agents, preservatives, adsorption inhibitors, diluents, excipients, pH adjusters, analgesics, sulfur-containing reducing agents, and antioxidants as needed.

Examples of cryoprotectants include sugars such as trehalose, sucrose, and sorbitol, but are not limited thereto.

Examples of the solubilizing agent include, but are not limited to, polyoxyethylene hydrogenated castor oil, polysorbate 80, niacinamide, polyoxyethylene sorbitan monolaurate, polyethylene glycol, and castor oil fatty acid ethyl ester.

Examples of isotonic agents include, but are not limited to, sodium chloride, potassium chloride, and calcium chloride.

Examples of the preservative include, but are not limited to, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, sorbic acid, phenol, cresol, and chlorocresol.

Examples of the adsorption inhibitor include, but are not limited to, human serum albumin, lecithin, dextran, ethylene oxide/propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hydrogenated castor oil, and polyethylene glycol.

Examples of the sulfur-containing reducing agent include, but are not limited to, compounds having a mercapto group such as N-acetylcysteine, N-acetylhomocysteine, lipoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and thioalkanoic acids having 1 to 7 carbon atoms.

Examples of antioxidants include, but are not limited to, erythorbic acid, butylated hydroxytoluene, butylated hydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbyl palmitate, L-ascorbyl stearate, sodium bisulfite, sodium sulfite, tripentyl gallate, propyl gallate, or chelating agents such as disodium ethylenediaminetetraacetic acid (EDTA), sodium pyrophosphate, and sodium metaphosphate.

The preparation of the present invention can be administered in any form, either orally or parenterally, and is generally administered parenterally. Specifically, it can be administered by injection, transdermal administration, transmucosal administration, nasal administration, or transpulmonary administration. Examples of injectable dosage forms include systemic or local administration by subcutaneous injection, intravenous injection, and intramuscular injection. Although subcutaneous injection is limited in the amount of the injection solution, a large amount of antibody can be administered per administration (approximately 100 to 200 mg). Therefore, the preparation of the present invention is particularly suitable for subcutaneous administration (injection).

It should be noted that all prior art documents cited in this specification are incorporated into this specification as reference.

Example

Hereinafter, the present invention will be described in further detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1. Inhibition of complex formation in subsequent steps by viral inactivation and post-neutralization retention

In this example, the following antibodies are provided.

Mab1: The anti-IL-6 receptor antibody described in WO2009/041621, with a pI of 5.8 achieved by modifying the amino acids of Mab3. The amino acid sequence of the Mab1 antibody is represented by H chain/SEQ ID NO: 1, and L chain/SEQ ID NO: 2.

Mab2: A fully humanized NS22 antibody, an anti-NR10 (IL-31 receptor) antibody, produced using the method described in Example 12 of WO2009/072604. This antibody is classified as IgG2 and has a pI value of 5.6 reduced by altering the amino acid sequence. The amino acid sequence of Mab2 is represented by SEQ ID NO: 3 for the H chain and SEQ ID NO: 4 for the L chain.

Mab3: Tocilizumab (H chain/SEQ ID NO: 5, L chain/SEQ ID NO: 6), pI value: 9.4.

The antibodies were expressed using a CHO cell stably expressing strain by methods known to those skilled in the art, purified by methods known to those skilled in the art including protein A column chromatography, and used for evaluation of removal of complexes in the following examples.

Mab1 and Mab2 were respectively simulated in the viral inactivation steps in the actual production and purification steps, 1 mol/L hydrochloric acid was added to the purified antibody solution, and then maintained at a pH below 3.8 for more than 30 minutes. The retained components were neutralized with 1-2 mol/LTris at a pH above 6.5. Using the components with different retention times after neutralization, the aggregates were removed using a method known to those skilled in the art including hydroxyapatite column chromatography or a method including anion exchange chromatography, and purified to a high purity. The components from which the aggregates were removed were further retained, and the amount of aggregates corresponding to the retention time after purification was calculated using size exclusion chromatography (SEC) using the area percentage method.

In hydroxyapatite column chromatography, for Mab1, after the column was equilibrated with 10 mmol/L phosphate buffer (pH 6.5), a neutralized component was loaded. After the column was washed with 10 mmol/L phosphate buffer (pH 6.5), the salt concentration was increased by 500 mmol/L NaCl and 10 mmol/L phosphate buffer (pH 6.5), thereby eluting Mab1. For Mab2, after the column was equilibrated with 10 mmol/L phosphate buffer (pH 6.5), a neutralized component was loaded. After the column was washed with 100 mmol/L MES and 5 mmol/L phosphate buffer (pH 6.0), the salt concentration was increased by 200 mmol/L NaCl and 17.5 mmol/L phosphate buffer (pH 6.6), thereby eluting Mab2.

In anion exchange chromatography, for Mab1, the column was equilibrated with 20 mmol/L Tris-acetate buffer (pH 8.0), and the neutralized fraction was loaded. After washing the column with 20 mmol/L Tris-acetate buffer (pH 8.0), the salt concentration was increased with 267 mmol/L Tris-acetate buffer (pH 8.0), allowing Mab1 to be eluted. For Mab2, the column was equilibrated with 20 mmol/L Tris-HCl buffer (pH 7.0), and the neutralized fraction was loaded. After washing the column with 20 mmol/L Tris-HCl buffer (pH 7.0), the salt concentration was increased with 350-360 mmol/L NaCl and 20 mmol/L Tris-HCl buffer (pH 7.0-7.2), allowing Mab2 to be eluted.

Mab3 was similarly maintained with hydrochloric acid and then neutralized, and the amount of the aggregate during the maintenance time after neutralization was calculated.

Size exclusion chromatography (SEC) was performed to analyze the amount of antibody aggregates at each retention time. Each sample was diluted to approximately 1.0 g/L using the following mobile phase and analyzed using a G3000SWXL column (Tosoh). The mobile phase used was 50 mmol/L phosphate buffer (pH 7.5) containing 300 mmol/L NaCl, and the analysis was performed at a flow rate of 0.5 ml/min. Peaks eluting earlier than the monomer were analyzed as aggregates, and the monomer and aggregate content (%) was calculated using the area percentage method.

The retention time after neutralization and the amount of aggregates (ratio of aggregates) after hydroxyapatite column chromatography for Mab1 and Mab2 are shown in Table 1. The amount of aggregates after anion exchange chromatography is shown in Table 2. The amount of aggregates corresponding to the retention time after neutralization for Mab1, Mab2, and Mab3 is shown in Table 3. These results clearly show that for both Mab1 and Mab2, even if aggregates are removed by chromatography immediately after neutralization, aggregates will re-form. In contrast, by allowing the aggregates to stand for a predetermined time after neutralization and then purifying, almost no increase in the amount of aggregates after purification is observed. On the other hand, for Mab3, no increase in aggregates after neutralization is observed, indicating that a retention time until the next step is unnecessary.

[Table 1]

[Table 2]

[Table 3]

※1 After storage at low pH 3.1 for 1 hour, neutralize at pH 7.0 (acetic acid concentration in the antibody solution after purification using a Protein A column: 50 mM)

※2 After storage at low pH 3.1 for 1 hour, neutralize at pH 7.0 (acetic acid concentration in the antibody solution after purification using a Protein A column: 20 mM)

※3 After storage at low pH 3.6 for 1 hour, neutralize at pH 7.0 (acetic acid concentration in the antibody solution after purification using a Protein A column: 50 mM)

※4 After storage at low pH 3.4 for 1 hour, neutralize at pH 7.0 (acetic acid concentration in the antibody solution after purification using a Protein A column: 20 mM)

*5 After storage at a low pH of 3.4 for 1 hour, neutralize at pH 7.0 (HCl concentration in the antibody solution after purification using a Protein A column: 2.5 mM).

Example 2. Purification of low pI antibodies

Example 2-1. Removal of Aggregates of Mab1 in the Sodium Chloride-Containing Eluent of Anion Exchange Chromatography

The anti-IL-6 receptor antibody Mab1 is a modified antibody with a low pI (pI <8; pI 5.8). It was expressed using a CHO cell stably expressing strain using methods known to those skilled in the art and purified to a high purity using methods known to those skilled in the art, including protein A column chromatography, and used for purification in the following examples.

Mab1 was prepared to simulate the viral inactivation step in actual production and purification procedures. 1 mol/L hydrochloric acid was added to the purified antibody solution and maintained at a pH below 3.6 for at least 30 minutes. The retained fraction was neutralized with 1 mol/L Tris at a pH above 7. The solution was then maintained for at least 24 hours for purification.

Purification was performed using the commercially available resins listed in Table 4 for anion exchange chromatography. The column was equilibrated with 20 mmol/L Tris-HCl buffer (pH 8.0) and then loaded with the neutralized fraction. After washing with 20 mmol/L Tris-HCl buffer (pH 8.0), the salt concentration was increased using 20 mmol/L Tris-HCl and 100-150 mmol/L NaCl buffer (pH 8.0) to elute Mab1. Size exclusion chromatography (SEC) was used to calculate the amount of purified aggregates using the area percentage method.

[Table 4]

Size exclusion chromatography (SEC) was performed to analyze the amount of antibody aggregates at each retention time. Each sample was diluted to approximately 1.0 g/L using the following mobile phase and analyzed using a G3000SWXL column (Tosoh). The mobile phase used was 50 mmol/L phosphate buffer (pH 7.5) containing 300 mmol/L NaCl, and the analysis was performed at a flow rate of 0.5 ml/min. Peaks eluting earlier than the monomer were analyzed as aggregates, and the monomer and aggregate content (%) was calculated using the area percentage method.

The amount of the aggregates of Mab1 after purification by anion exchange chromatography is shown in Table 5. From these results, it was clarified that the monomers and aggregates of Mab1 were separated by anion exchange chromatography.

[Table 5]

.

Example 2-2. Removal of aggregates of Mab2 in the sodium chloride-containing eluent of anion exchange chromatography

The anti-IL-31 receptor antibody Mab2 is a modified antibody with a low pI (pI <8; pI 5.6). It was expressed using a stable expression strain of CHO cells using methods known to those skilled in the art and purified to a high purity using methods known to those skilled in the art including protein A. It was used for purification in the following examples.

Mab2 simulated the viral inactivation step in actual production and purification procedures. 1 mol/L hydrochloric acid was added to the purified antibody solution and maintained at a pH below 3.6 for at least 30 minutes. The retained fraction was neutralized with 1 mol/L Tris at a pH above 7 and then maintained for at least 20 hours before purification.

Purification was performed using the commercially available resins listed in Table 6 for anion exchange chromatography. The column was equilibrated with 20 mmol/L Tris-HCl buffer (pH 7.0 or 8.0), and the neutralized fraction was loaded. After washing the column with 20 mmol/L Tris-HCl buffer (pH 7.0), the salt concentration was increased using 20 mmol/L Tris-HCl, 200-300 mmol/L NaCl buffer (pH 7.0), allowing Mab2 to be eluted. Size exclusion chromatography (SEC) was used to calculate the amount of purified complexes using the area percentage method.

[Table 6]

The amount of aggregates of Mab2 after purification by anion exchange chromatography is shown in Table 7. From these results, it was clarified that monomers and aggregates of Mab2 were separated by anion exchange chromatography.

[Table 7]

.

Example 2-3. Removal of Aggregates of Mab1 in Various Eluates of Anion Exchange Chromatography

Mab1 was prepared to simulate the viral inactivation step in actual production and purification procedures. 1 mol/L HCl was added to the purified antibody solution and maintained at a pH below 3.6 for at least 30 minutes. The retained fraction was neutralized with 1 mol/L Tris at a pH above 7.0 and then maintained for at least 24 hours before purification.

Purification was performed using the commercially available resins listed in Table 8 for anion exchange chromatography. The column was equilibrated with 20 mmol/L Tris-HCl buffer (pH 8.0) or 20 mmol/L Tris-acetate buffer (pH 8.0), and then loaded with the neutralized fraction. After washing the column with 20 mmol/L Tris-HCl buffer (pH 8.0) or 20 mmol/L Tris-acetate buffer (pH 8.0), Mab1 was eluted by increasing the salt concentration with 240-270 mmol/L Tris-acetate buffer (pH 8.0), 40-60 mmol/L sodium phosphate buffer (pH 8.0), or a buffer containing 20-50 mmol/L sodium sulfate in 20 mmol/L Tris-HCl (pH 8.0). Size exclusion chromatography (SEC) was used to calculate the amount of purified complexes using the area percentage method.

[Table 8]

The amount of the aggregates of Mab1 after purification by anion exchange chromatography is shown in Table 9. From these results, it was clarified that the monomers and aggregates of Mab1 were separated by anion exchange chromatography.

[Table 9]

.

Example 2-4. Virus Clearance Ability of Mab1 in Tris-Containing Eluate of Anion Exchange Chromatography

Mab1 was prepared to simulate the viral inactivation step in actual production and purification procedures. 1 mol/L hydrochloric acid was added to the purified antibody solution and maintained at a pH below 3.6 for at least 30 minutes. The retained fraction was neutralized with 1 mol/L Tris at a pH above 7 and then maintained for at least 20 hours for purification.

Purification was performed using commercially available POROS HQ resin (manufactured by Life Technologies) as anion exchange chromatography. After equilibration with 20 mmol/L Tris-acetate buffer (pH 7.8), the column was loaded with a substance obtained by adding the retroviral model virus MuLV to the neutralized fraction. After washing the column with 20 mmol/L Tris-acetate buffer (pH 7.8), the salt concentration was increased using 225-275 mmol/L Tris-acetate buffer (pH 7.8) to elute Mab1. After purification, the viral titers of the loaded and eluted fractions were measured to calculate the viral load.

The virus-clearing ability of Mab1 during purification by anion exchange chromatography is shown in Table 10. These results indicate that purification by anion exchange chromatography using Tris-acetate can effectively remove viruses.

[Table 10]

.

Example 2-5. Removal of Aggregates of Mab2 in Tris-Containing Eluents from Anion Exchange Chromatography

Mab2 simulated the viral inactivation step in actual production and purification procedures. 1 mol/L hydrochloric acid was added to the purified antibody solution and maintained at a pH below 3.6 for at least 30 minutes. The retained fraction was neutralized with 1 mol/L Tris at a pH above 7 and then maintained for at least 20 hours before purification.

Purification was performed using the commercially available resins listed in Table 11 for anion exchange chromatography. The column was equilibrated with 20 mmol/L Tris-HCl buffer (pH 7.0 or 8.0), and the neutralized fraction was loaded. After washing the column with 20 mmol/L Tris-acetate buffer (pH 7.0 or 8.0), the salt concentration was increased with 300-500 mmol/L Tris-acetate (pH 7.0 or 8.0) to elute Mab2. Size exclusion chromatography (SEC) was used to calculate the amount of purified complexes using the area percentage method.

[Table 11]

The amount of the aggregates of Mab2 after purification by anion exchange chromatography is shown in Table 12. From these results, it was clarified that the monomers and aggregates of Mab2 were separated by anion exchange chromatography.

[Table 12]

.

Example 2-6. Virus Clearance Ability of Mab2 in Tris-Containing Eluate of Anion Exchange Chromatography

Mab2 simulated the viral inactivation step in actual production and purification procedures. 1 mol/L hydrochloric acid was added to the purified antibody solution and maintained at a pH below 3.6 for at least 30 minutes. The retained fraction was neutralized with 1 mol/L Tris at a pH above 7 and then maintained for at least 20 hours before purification.

Purification was performed using commercially available POROS PI resin (manufactured by Life Technologies) as anion exchange chromatography. After equilibrating the column with 20 mmol/L Tris-HCl buffer (pH 8.0), the neutralized fraction was loaded with a retroviral model virus, MuLV, added to the column. After washing the column with 20 mmol/L Tris-HCl buffer (pH 8.0), the salt concentration was increased using 470 mmol/L Tris-HCl buffer (pH 8.0), thereby eluting Mab2. After purification, the viral titers of the loaded and eluted fractions were measured to calculate the viral load.

The virus-clearing ability of Mab2 during purification by anion exchange chromatography is shown in Table 13. From these results, it was clarified that viruses can be removed by purification using Tris-HCl by anion exchange chromatography.

[Table 13]

.

Example 2-7. Purification of Mab1 using anion exchange chromatography and multicomponent chromatography

Mab1 was expressed using a stable CHO cell line using methods known to those skilled in the art, and was purified to a high purity using methods known to those skilled in the art including protein A column chromatography. It was then purified using anion exchange chromatography as shown in Examples 2-3 and 2-4 and used for purification in the following examples.

The pH of the eluted fraction from the anion exchange chromatography was adjusted to 6.5 ± 0.3 using acetic acid. Purification was performed using the commercially available Capto Adhere (manufactured by GE Healthcare) as the resin for multi-layer chromatography (hydrophobic + anion exchange). The column was equilibrated with 250 ± 25 mmol/L Tris-acetate buffer (pH 6.5) and loaded with the pH-adjusted fraction. Mab1 was recovered from the flow-through fraction.

Through this series of purification steps, impurity DNA derived from the purification step was removed to <1.0 pg/mg-Mab1 (below the limit of quantification), host cell proteins were removed to <17 ng/mg-Mab1 (below the limit of quantification), and leached protein A was removed to <0.4 ng/mg-Mab1 (below the limit of quantification). Furthermore, as demonstrated in Example 2-3, Mab1 aggregates were removed. Regarding viral clearance, in addition to the sufficient viral clearance demonstrated in Example 2-4, a high viral clearance of 5.42 Log ₁₀ was also achieved against the retroviral model virus MuLV in the multi-step chromatography step.

DNA was measured by quantitative PCR, host cell protein was measured by ELISA using anti-host cell protein antibodies, and leached protein A was measured by ELISA using anti-protein A antibodies.

Example 2-8. Purification of Mab2 using anion exchange chromatography and hydrophobic interaction chromatography

Mab2 was expressed using a stable CHO cell line using methods known to those skilled in the art. After purification to a high purity using methods known to those skilled in the art including protein A, it was purified using anion exchange chromatography as shown in Examples 2-2 and 2-6 and used for purification in the following examples.

Salt (e.g., sodium sulfate) is added to the eluted fraction from anion exchange chromatography to a final concentration of 250 mmol/L or higher, the pH is adjusted to 6.8-8.0, and purification is performed using a hydrophobic interaction chromatography resin (e.g., a resin having phenyl groups). After equilibration with a buffer (pH 6.8-8.0) having the same concentration as the added salt, the fraction is loaded and Mab2 is recovered in the flow-through fraction.

Through this series of purification steps, impurity DNA derived from the steps was removed to 1.9 pg/mg-Mab2, host cell proteins were removed to <8.0 ng/mg-Mab2 (below the limit of quantification), and leached protein A was removed to <0.4 ng/mg-Mab2 (below the limit of quantification). Furthermore, as demonstrated in Example 2-2, Mab2 aggregates were removed. Regarding viral clearance, in addition to the sufficient viral clearance demonstrated in Example 2-6, high viral clearance of 5.94 Log ₁₀ was achieved against the retroviral model virus MuLV and 1.55 Log ₁₀ against the parvovirus model virus MVM in the hydrophobic interaction chromatography step.

DNA was measured by quantitative PCR, host cell protein was measured by ELISA using anti-host cell protein antibodies, and leached protein A was measured by ELISA using anti-protein A antibodies.

Example 3. Different association formation depending on the pH value after neutralization after virus inactivation

In this example, the following antibodies are provided.

Mab1: The anti-IL-6 receptor antibody described in WO2009/041621, with a pI of 5.8 achieved by modifying the amino acids of Mab3. The amino acid sequence of the Mab1 antibody is represented by H chain/SEQ ID NO: 1, and L chain/SEQ ID NO: 2.

The above antibodies were expressed using a CHO cell stable expression strain by methods known to those skilled in the art, and purified by methods known to those skilled in the art, including protein A column chromatography, to evaluate the amount of complex formation in the following examples.

Mab1 simulated the viral inactivation step in the actual production and purification steps, and 1 mol/L hydrochloric acid was added to the purified antibody solution, and the pH was maintained below 3.8 for more than 30 minutes. The retained components were neutralized with 2 mol/L Tris at pH 5.0, which is lower than the pI of the antibody, and pH 7.0, which is higher than the pI. After neutralization, samples were collected over time and the amount of aggregates was measured. In addition, for the samples neutralized with pH 5.0, 2 mol/L Tris was further added after 22 hours of neutralization and maintenance, and the pH was maintained at pH 7.0, which is higher than the pI. Samples were collected over time and the amount of aggregates was measured. The amount of aggregates was calculated using size exclusion chromatography (SEC) using the area percentage method.

Size exclusion chromatography (SEC) was performed to analyze the amount of antibody aggregates at each retention time. Each sample was diluted to approximately 1.0 g/L using the following mobile phase and analyzed using a G3000SWXL column (Tosoh). The mobile phase used was 5 mmol/L phosphate buffer (pH 7.5) containing 300 mmol/L NaCl, and the analysis was performed at a flow rate of 0.5 ml/min. Peaks eluting earlier than the monomer were analyzed as aggregates, and the monomer and aggregate content (%) was calculated using the area percentage method.

The increase in the amount of aggregates over time when maintained at pH 7.0 and the increase in the amount of aggregates over time when maintained at pH 5.0 for 22 hours are shown in Table 14 (when maintained at pH 7.0) and Table 15 (when the pH was increased to 7.0 after 22 hours of maintenance at pH 5.0). These results clearly show that even if the increase in aggregates over time is stable when Mab1 is neutralized at pH 5.0 (lower than the pI of Mab1), the increase in aggregates over time is observed again when the pH is raised to pH 7.0 (higher than the pI of Mab1) and maintained, and then stabilizes after the prescribed period of maintenance.

[Table 14]

[Table 15]

.

Industrial Applicability

The present invention provides a purification method that can effectively remove antibody aggregates or impurities contained in compositions containing antibodies, particularly those with low pIs. The present invention also provides a formulation containing antibodies in which aggregate formation is suppressed. The purification method of the present invention is useful in the preparation of biopharmaceuticals requiring high purity.

Claims (18)

1. A method for purifying a composition containing an antibody with a pI value of 5.0 to 6.5, the method comprising the following steps: (a) The step of treating a composition containing an antibody with a pI value of 5.0 to 6.5 under acidic conditions at pH 2.0 to 4.0; (b) Neutralizing the acidic composition obtained in step (a) to a pH of 6.5 or higher; (c) After neutralization, maintain the neutralized composition obtained in step (b) for at least 6 hours, and (d) The step of removing the associative compound from the neutralized composition obtained in step (c). 2. The method of claim 1, wherein step (a) is a virus inactivation treatment step performed after purifying the antibody with a pI value of 5.0 to 6.5 by protein A column chromatography. 3. The method according to any one of claims 1 to 2, wherein the associative aggregates are removed by anion exchange chromatography, hydrophobic interaction chromatography, multi-component chromatography or hydroxyapatite chromatography. 4. The method of any one of claims 1 to 2, wherein the removal of the associative organism is carried out by a method comprising the steps of: (i) Loading a composition containing an antibody with a pI value of 5.0 to 6.5 onto a chromatography plate using an anion exchange resin; (ii) the step of eluting antibodies with a pI value of 5.0–6.5 from chromatography using anion exchange resin in a binding/elution mode using an elution solution with a higher salt concentration than that of the composition in (i); and (iii) The step of loading the elution product containing the antibody with a pI value of 5.0 to 6.5 obtained in step (ii) onto a resin having hydrophobic ligands and/or multiple ligands for chromatography to obtain the flow-through component and/or the elution component. 5. The method of claim 4, wherein, prior to step (ii), the method comprises: cleaning the anion exchange resin with a cleaning solution. 6. The method of claim 4, wherein the elution solution in step (ii) is a solution containing at least one selected from sodium chloride, Tris salt, sodium sulfate, and sodium phosphate. 7. The method according to any one of claims 1 to 2, wherein the antibody is a humanized antibody or a human antibody. 8. The method according to any one of claims 1 to 2, wherein the antibody is an anti-IL-6 receptor antibody or an anti-IL-31 receptor antibody. 9. A method for preparing a composition containing an antibody with a pI value of 5.0 to 6.5, wherein the proportion of antibody conjugates is 3% or less by the method according to any one of claims 1 to 8. 10. A method for preparing a pharmaceutical composition containing an antibody with a pI value of 5.0 to 6.5, the method comprising the following steps: 1) The step of preparing an antibody with a pI value of 5.0 to 6.5 and/or a composition containing the antibody by the preparation method according to claim 9; and 2) The step of mixing the antibody with a pI value of 5.0 to 6.5 prepared in step 1) and/or a composition containing the antibody with a pharmaceutically acceptable carrier and/or additives to prepare a formulation. 11. A method for removing antibody conjugates from a composition containing an antibody with a pI value of 5.0 to 6.5, the method comprising the following steps: (a) The step of treating a composition containing an antibody with a pI value of 5.0 to 6.5 under acidic conditions at pH 2.0 to 4.0; (b) Neutralizing the acidic composition obtained in step (a) to a pH of 6.5 or higher; (c) After neutralization, maintain the neutralized composition obtained in step (b) for at least 6 hours; and (d) The step of removing the associative compound from the neutralized composition obtained in step (c). 12. The method of claim 11, wherein step (a) is a virus inactivation step performed after purifying the antibody with a pI value of 5.0 to 6.5 by protein A column chromatography. 13. The method of any one of claims 11 to 12, wherein the removal of the associative aggregates is carried out by anion exchange chromatography, hydrophobic interaction chromatography, multi-component chromatography or hydroxyapatite chromatography. 14. The method of any one of claims 11-12, wherein the removal of the associative organism is carried out by a method comprising the steps of: (i) Loading a composition containing an antibody with a pI value of 5.0 to 6.5 onto a chromatography plate using an anion exchange resin; (ii) the step of eluting antibodies with a pI value of 5.0–6.5 from chromatography using anion exchange resin in a binding/elution mode using an elution solution with a higher salt concentration than that of the composition in (i); and (iii) The step of loading the elution product containing the antibody with a pI value of 5.0 to 6.5 obtained in step (ii) onto a resin having hydrophobic ligands and/or multiple ligands for chromatography to obtain the flow-through component and/or the elution component. 15. The method of claim 14, wherein, prior to step (ii), the method comprises: cleaning the anion exchange resin with a cleaning solution. 16. The method of claim 14, wherein the elution solution in step (ii) is a solution comprising at least one selected from sodium chloride, Tris salt, sodium sulfate, and sodium phosphate. 17. The method of any one of claims 11 to 12, wherein the antibody is a humanized antibody or a human antibody. 18. The method of any one of claims 11 to 12, wherein the antibody is an anti-IL-6 receptor antibody or an anti-IL-31 receptor antibody.