JP2010070490A - Method for antibody purification - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for purifying an antibody under a pH condition not to cause an antibody denaturation or agglutination. <P>SOLUTION: The method for purifying the antibody from a mixed solution containing an antibody includes a process for adsorbing and removing impurities from the mixed solution by using a porous membrane containing an anion exchange group and a process for adsorbing the antibody by using an adsorbent carrying tryptophan and eluting and recovering the antibody after the process for removing impurities. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a protein purification technique, and particularly to a method for purifying an antibody as a biopharmaceutical.

  The antibody recognizes and binds to the target substance with high specificity. Therefore, antibodies are extremely useful as research reagents and clinical test reagents. In recent years, various therapeutic antibodies have been developed and have greatly contributed to the advancement of medical technology as epoch-making therapeutic agents in fields such as rheumatism and cancer, which have been difficult to treat conventionally. The antibody purification method roughly includes two steps of culture and purification. In the culturing step, antibody-containing blood, ascites fluid, cell culture fluid, or the like is obtained using immunized animals or cells having antibody-producing ability. Cell culture fluids and the like contain various proteins other than antibodies and impurities such as deoxyribonucleic acid (DNA). Therefore, in the purification process, it is required to reduce impurities and significantly increase the purity of the antibody.

  An important role in the purification process is protein A derived from Staphylococcus aureus, which is used as an affinity ligand, or protein G, which is a recombinant product thereof. Protein A and protein G are very specific for antibodies and have a high affinity. Therefore, an antibody purification method using protein A and protein G has been proposed (see, for example, Patent Document 1). However, protein A and protein G are usually very expensive. In the antibody purification step using protein A and protein G, only a low pH buffer solution having a pH of around 3 is used. Therefore, it is known that protein A and protein G cannot be applied to an antibody that denatures at a low pH, and cause aggregation of the antibody even if it can be applied (see, for example, Non-Patent Document 1). However, there is a concern that antibody aggregates exhibit antigenicity when administered to humans, and in the production of therapeutic antibodies, removal of aggregates and monitoring of residual amounts are required (for example, non-antibody). (See Patent Document 2).

  Furthermore, since protein A and protein G are proteins derived from microorganisms, when an antibody drug containing protein A and protein G is administered, it may cause anaphylactoid symptoms in sensitive patients (for example, Non-patent document 3). Therefore, it is indispensable to remove protein A and protein G and monitor the residual amount in the latter stage of the manufacturing process of the antibody drug.

In order to solve the above problems, a method combining ion exchange chromatography and hydrophobic chromatography is known (for example, see Patent Document 2). In addition, a method for purifying an antibody using a protein A mimetic ligand synthesized organically and not using protein A has been reported (for example, see Patent Document 3). In addition, the protein A mimetic ligand is inferior in the selective adsorptivity of the antibody as compared with the protein A itself, and thus the purity of the antibody in the eluate eluted after the adsorption is not sufficient. On the other hand, a method of desorbing an antibody from protein A using a buffer solution near weak acidity has been reported (for example, see Patent Document 3).
European Patent No. 310719 Japanese Patent Laid-Open No. 7-267997 International Publication No. 2007/064281 Pamphlet Journal of Pharmaceutical Sciences, 2007, Vol. 96, p. 1-26 Amy S. Rosenberg, “Overview Significance of Protein Aggregation to Therapeutic Protein Products”, [searched September 12, 2000], Internet <URL: http: //www.fda .gov / cder / regulatory / follow_on / 200512 / 200512_rosenberg.pdf> Eds. C. S. F. Easmon and C.I. Eds (Eds. CSF Easmon and C. Adlam), "Staphylococci and Staphylococcal infections 2" (UK), Academic Press Inc., 1983, p. 429-480

  However, the method combining ion exchange chromatography and hydrophobic chromatography in Patent Document 2 has low binding characteristics as compared with protein A and protein G, and therefore is not versatile and widely used for industrial production of antibodies. It has not reached.

  Further, in Patent Document 3, as long as an artificially synthesized compound is used, when an antibody drug containing a protein A mimetic ligand is administered, there is an unavoidable possibility that anaphylaxis-like symptoms will be caused in sensitive patients. Furthermore, in patent document 3, since it is based on the method using protein A, it has not led to the fundamental solution of the said subject.

  In view of such circumstances, the problem to be solved by the present invention is to provide a method that can be used under pH conditions that do not cause denaturation or aggregation of antibodies, and that can purify antibodies with high accuracy at low cost. It is.

  Therefore, in order to solve the above problems, the present inventors have conducted extensive research on various adsorbents and purification methods, and as a result, by filtering the liquid mixture using a porous membrane on which an anion exchange group is fixed, By combining the step of removing impurities and then the step of elution and recovery after adsorption of the antibody with an affinity column using an adsorbent with tryptophan as a low molecular ligand, it was found that the antibody can be purified with high accuracy, The method according to the present invention has been completed. Tryptophan is an essential amino acid and is not toxic unless taken in large quantities. Incidentally, the half-lethal dose (LD50) when administered intraperitoneally to mice is 4.8 g / kg.

  By using the method for purifying an antibody according to the present invention, it is possible to obtain an antibody that does not cause denaturation of the antibody and does not contain foreign proteins, artificially synthesized compounds, or antibody aggregates. In addition, the antibody can be purified with high accuracy without using a column carrier having an expensive protein A ligand.

  Hereinafter, the best mode for carrying out the present invention (hereinafter referred to as “the present embodiment”) will be described in detail. The present invention is not limited to the following embodiment, and can be implemented with various modifications within the scope of the gist.

(First embodiment)
The purification method of the present embodiment is a method for purifying the antibody from a mixed solution containing an antibody, and a step of adsorbing and removing impurities from the mixed solution using a porous membrane having an anion exchange group; The step of adsorbing and removing the impurities includes the step of adsorbing the antibody using an adsorbent carrying tryptophan and then eluting and collecting the antibody.

  First, the process of adsorbing and removing impurities in the present embodiment will be described. The porous membrane having an anion exchange group according to the present embodiment includes a porous body serving as a base material and an anion exchange group that is chemically or physically fixed to the side wall surface of the pores of the porous body. Here, it is more preferable that the porous membrane has a graft chain on the side wall surface of the pore and an anion exchange group is fixed to the graft chain because the adsorption capacity is high. The material of the graft chain is not particularly limited, but is preferably a glycidyl methacrylate or vinyl acetate polymer because it is easy to introduce into the side wall surface of the pore, and glycidyl methacrylate is easy to chemically fix the anion exchange group. The polymer of is more preferable.

  The material of the porous body is not particularly limited, but is preferably composed of a polyolefin polymer or a copolymer of olefin and halogenated olefin in order to maintain mechanical properties. Among these materials, polyethylene and polyvinylidene fluoride are preferable, and polyethylene is more preferable in that the material is particularly excellent in mechanical strength and can obtain a high adsorption capacity.

  A method of introducing a graft chain to the outermost surface of the porous body and the side wall surface of the pore and further fixing the anion exchange group to the graft chain is not limited, but for example, JP-A-2-132132 Is disclosed.

  The anion exchange group is not limited as long as it is an anion exchange group that adsorbs impurities such as DNA, host cell-derived protein (HCP), virus, and endotoxin, but diethylamino group (DEA), quaternary ammonium Examples thereof include a group (Q), a quaternary aminoethyl group (QAE), a diethylaminoethyl group (DEAE), and a diethylaminopropyl group. Among them, a diethylamino group and a quaternary ammonium group are preferable, and a diethylamino group is more preferable because chemical fixation to the graft chain introduced into the porous body is easy and a high adsorption capacity is obtained.

  The maximum pore diameter of the porous membrane is preferably 0.1 μm or more and 1.0 μm or less, more preferably 0.1 μm to 0. 0 μm, in order to cut turbid components and bacteria and obtain a high permeation flow rate. It is in the range of 6 μm, more preferably in the range of 0.22 μm to 0.5 μm.

  The form of the porous membrane is not limited as long as it is a porous body such as a flat membrane, nonwoven fabric, hollow fiber membrane, monolith, capillary, disk or cylinder. However, a hollow fiber membrane is preferable from the viewpoint of ease of production, scale-up property, and packing property of the membrane when it is molded into a module.

(Support of claim 4)
As described above, a hollow fiber porous membrane having an anion exchange group is preferably built in the module for the purpose of adsorbing and removing impurities from the mixed solution containing the antibody. The hollow fiber membrane module is a module that incorporates a hollow fiber porous membrane made of a porous hollow fiber in which an anion exchange group is chemically or physically fixed on the outermost surface of the porous body and the side wall surface of the pore. The module can be used to adsorb and remove impurities dissolved or as turbid components from the mixed solution.

  Next, a process of eluting and recovering the antibody after adsorbing the antibody using the adsorbent carrying tryptophan in the present embodiment will be described. The adsorbent is formed by supporting tryptophan on a carrier. The material and form of the carrier are not particularly limited, but particles made of natural polymers or synthetic polymers, porous membranes, porous hollow fibers, nonwoven fabrics and the like can be used. The method for immobilizing tryptophan on the carrier is not particularly limited as long as it is stable at pH 3 to 9 and the eluate concentration under pH 3 to 9 is less than the concentration that is toxic to the human body. As the adsorbent, specifically, tryptophan-immobilized polyvinyl alcohol gel (hereinafter referred to as TR-PVA carrier) or the like can be used.

  In the present embodiment, a buffer solution having a pH of 3 to 9 is used for elution of the adsorbed antibody. As the buffer solution of pH 3 to 9, for example, a phosphate buffer solution, an acetate buffer solution, a diluted hydrochloric acid solution, or the like can be used. Further, various additives such as sodium chloride and arginine may be added to the buffer solution.

(Second Embodiment)
Another purification method in the present embodiment is a method for purifying the antibody from a mixed solution containing the antibody, after the antibody is adsorbed using an adsorbent carrying tryptophan and then eluted. Then, after the purification step to be recovered, impurities are further removed by a method including at least one of the following steps.
(1) a step of purification by anion exchange chromatography;
(2) a step of purification by cation exchange chromatography,
(3) A step of purification by hydrophobic chromatography.

The step of eluting and recovering the antibody after adsorbing the antibody using an adsorbent carrying tryptophan is the same as in the first embodiment.
After eluting and recovering the antibody from the adsorbent, at least selected from the step of purification by anion exchange chromatography, the step of purification by cation exchange chromatography, and the step of purification by hydrophobic chromatography to further purify the antibody, One purification step may be performed. In these chromatography steps, a chromatography column packed with a resin on which a ligand is immobilized, or a porous membrane on which a ligand is immobilized can be used. Here, the type of ligand is not particularly limited as long as it is suitable for the purpose of antibody purification.

  The mixed solution containing the antibody applied to the present embodiment (including the first and second embodiments) is not particularly limited. For example, the present embodiment can be applied to plasma, serum, ascites, cell culture medium, and the like.

  Hereinafter, the present embodiment will be described more specifically with reference to examples and comparative examples, but the embodiment of the present invention is not limited to the following examples.

(Production Example 1) Production of anion exchange membrane module According to the method disclosed in JP-A-2-132132, a polyethylene hollow fiber having an outer diameter of 3 mm, an inner diameter of 2 mm, a pore diameter of 0.3 μm, and a porosity of 70%, Graft chains were introduced. Thereafter, a diethylamino group was fixed to the graft chain as an anion exchange group. Three hollow fibers thus obtained were bundled, and the end of the amount was fixed to a polysulfonic acid module case with an epoxy-based potting agent so as not to block the hollow portion of the hollow fiber, thereby producing a hollow fiber module having an anion exchange group. The inner diameter of the obtained module is 9 mm, the length is about 33 mm, the inner volume of the module is about 2 mL, the effective volume of the porous hollow fiber occupying in the module is 0.85 mL, and only the hollow fiber porous membrane excluding the hollow portion is used. The volume was 0.54 mL.

(Production Example 2) Preparation of antibody-containing cell culture solution Chinese hamster ovary (CHO) cell culture solution cultured in serum-free medium (salt concentration of about 0.9% by mass, protein concentration of about 1 g / L, cell density of 1. 1 × 10 ⁷ / mL) 196 mL of human γ-globulin solution (Benesys, Venoglobulin) containing 50 mg / mL of antibody was added and adjusted to pH 5.0 with hydrochloric acid, and then a 0.45 μm microfiltration membrane was added. A cell culture solution containing 1 mg / mL of antibody was prepared by permeation. The concentration of HCP, which is a typical impurity in the obtained culture broth, was measured using an ELISA method. Specifically, the cell culture solution was dropped onto a 96-well plate made by Cygnos Technologies and CHO Host Cell Protein ELISA Kit, and the HCP concentration was measured using an Ultraspec Visible Plate Reader II96 plate reader made by GE Healthcare Biosciences. . As a result, the HCP concentration in the cell culture medium was 346 μg / mL. In addition, quantification of DNA, which is another typical impurity, was performed using a Qubit (registered trademark) fluorometer after treating a cell culture solution to be evaluated using Quant-iT (registered trademark) dsDNA HS Assay Kit manufactured by Invitrogen. I went. As a result, the DNA concentration in the cell culture solution was 7340 ng / mL.

Example 1 Antibody Purification No. 1: Antibody Purification Using TR-PVA Carrier Following Anion Exchange Membrane 20 mmol / L acetic acid-0.2 mol / L NaCl buffer (pH 5) was added to the anion exchange membrane module prepared in Production Example 1. 0.0) 10 mL of the solution was allowed to equilibrate, and then 54 mL of the antibody-containing cell culture solution prepared in Production Example 2 was permeated to obtain an antibody-containing cell culture solution from which impurities were removed. Next, a column packed with 2 mL of TR-PVA support was equilibrated with 20 mL of 20 mmol / L acetic acid-0.2 mol / L NaCl buffer (pH 5.0), and then the antibody-containing cells from which impurities were removed. 20 mL of the culture solution was passed through to adsorb the antibody to the column. Thereafter, 40 mL of the buffer solution was passed through the column for washing, and then 20 mL of 0.1 mol / L sodium citrate buffer solution (pH 3.0) was passed through to elute and recover the antibody. The obtained elution and recovery solution was neutralized by adding an equal amount of 10 mmol / L sodium phosphate buffer (pH 8.2), and then the recovery solution was added with 1.5 mmol / L Tris-HCl (pH 8.0). The pH was adjusted to 8.0 to obtain a purified antibody solution.

  As a result of measuring the HCP concentration in the purified antibody solution in the same manner as in Production Example 2, it was 3.18 μg / mL. Further, the DNA concentration was measured in the same manner as in Production Example 2 and found to be 33.2 ng / mL. Further, the obtained recovered solution was diluted 10-fold, the absorbance at a wavelength of 280 nm was measured, and the antibody weight in the purified solution obtained using the antibody extinction coefficient 1.3 was 18.2 mg. If the antibody concentration in 20 mL of the antibody-containing cell culture solution after removing impurities with an anion exchange membrane is 1 mg / mL, the weight of the antibody added to the column is 20 mg. Therefore, the antibody recovery after the column treatment was 91%. Therefore, it was shown that the antibody can be recovered with a high degree of purification and a recovery rate by the purification method according to the present embodiment.

(Comparative Example 1) Antibody purification part 2: Antibody purification using TR-PVA carrier alone 30 mL of the antibody-containing cell culture solution prepared in Production Example 2 was passed through a 0.2 μm sterilization membrane (Sartorius, Minisart plus). Then, in the same manner as in Example 1, adsorption and elution recovery were performed using a TR-PVA carrier, and the pH was adjusted to 8.0 to obtain a purified antibody solution. In the same manner as in Production Example 2, the HCP concentration and DNA concentration in the purified solution of the obtained antibody were measured and found to be 15.3 μg / mL and 84.5 ng / mL, respectively. Further, the antibody recovery rate as measured in the same manner as in Example 1 was 93%. From this result, it was shown that impurities were removed even by purification using only TR-PVA support, but the degree of purification was significantly inferior compared with the case where impurities were removed in advance using an anion exchange membrane. .

(Example 2) Antibody purification part 3: Antibody purification using an anion exchange membrane after TR-PVA support 20 mL of 10 mmol / L Tris-HCl buffer (pH 8.0) was added to the anion exchange membrane module produced in Production Example 1. The solution was allowed to equilibrate. Thereafter, 22.5 mL of a pH 8.0 antibody purification solution obtained using the TR-PVA support in Comparative Example 1 was passed through the anion exchange membrane module, and 10 mL of the equilibration buffer was further passed through. Thus, a purified solution after the TR-PVA support column by an anion exchange membrane was obtained. In the same manner as in Production Example 2, the HCP concentration and DNA concentration in this antibody purified solution were measured and found to be 56 ng / mL and 1.1 ng / mL, respectively. Further, the antibody recovery rate as measured in the same manner as in Example 1 was 84%.

(Comparative Example 2) Antibody Purification No. 4: Antibody Purification Using Protein A Column After passing 25 mL of the antibody-containing cell culture solution obtained in Preparation Example 2 through a 0.2 μm sterilization membrane (Minisart plus, manufactured by Sartorius) Then, 10 mL was added to a Protein A column (HI Trap Protein A HP 1 ml, manufactured by GE Healthcare Biosciences) equilibrated with 10 mL of 20 mmol / L sodium phosphate buffer (pH 7.0) to adsorb the antibody. After washing 20 mL of the above buffer solution through the column, 10 mL of 0.1 mol / L sodium citrate buffer (pH 3.0) was passed to elute and recover the antibody. To the resulting elution and recovery solution, an equal amount of 10 mmol / L sodium phosphate buffer (pH 8.2) was added for neutralization, and then the recovery solution was added with 1.5 mol / L Tris-HCl (pH 8.0). The pH was adjusted to 8.0 to obtain a purified antibody solution.

  As a result of measuring the HCP concentration in the purified antibody solution in the same manner as in Production Example 2, it was 2.93 μg / mL. Moreover, as a result of measuring the DNA concentration in the same manner as in Production Example 2, it was 63.2 ng / mL. Further, the obtained recovered solution was diluted 10-fold, the absorbance at a wavelength of 280 nm was measured, and the recovery rate of the antibody obtained using the antibody extinction coefficient 1.3 was 90%. When this result was compared with the result of Comparative Example 1, it was shown that the degree of purification of the antibody using only the TR-PVA carrier was inferior to the degree of purification using only the protein A column. On the other hand, when compared with the results of Example 1, the degree of purification when purifying the antibody with the TR-PVA support after removing the impurities with the anion exchange membrane is equivalent to the degree of purification with the protein A column alone. It was shown that.

(Comparative Example 3) Antibody Purification No. 5: Antibody Purification Using Protein A Column followed by Anion Exchange Column 10 mmol / L Tris-HCl Buffer on Anion Exchange Chromatography Column (GE Healthcare Biosciences HiTrapQ FF 1 ml) 10 mL (pH 8.0) was passed through to equilibrate. Thereafter, 22.5 mL of a pH 8.0 antibody purification solution obtained using the protein A column in Comparative Example 2 was passed through an anion exchange chromatography column, and 10 mL of the equilibration buffer was further passed through. It collect | recovered and the refinement | purification liquid after the protein A column by an anion exchange chromatography column was obtained. When the HCP concentration and the DNA concentration in the purified solution of the obtained antibody were measured in the same manner as in Production Example 2, they were 48 ng / mL and 1.0 ng / mL, respectively. Further, the antibody recovery rate as measured in the same manner as in Example 1 was 83%. By comparing this result with Example 2, after removing impurities using the TR-PVA carrier, the degree of purification when the antibody was further purified using an anion exchange membrane was as follows. It was shown to be equivalent to the degree of purification when the antibody was purified using

  The antibody purification method according to the present invention can be used in the pharmaceutical industry, the medical industry, and the like.

Claims (5)

A method for purifying an antibody from a mixture containing the antibody,
Adsorbing and removing impurities from the mixed solution using a porous membrane having an anion exchange group;
Next to the step of adsorbing and removing the impurities, the step of adsorbing the antibody using an adsorbent carrying tryptophan and then eluting and collecting the antibody;
A method for purifying an antibody comprising   The method for purifying an antibody according to claim 1, wherein the anion exchange group is fixed to a graft chain bonded to a side wall surface of a pore of the porous membrane.   The porous membrane having the anion exchange group is stored in a module, and the impurities are adsorbed and removed from the mixed solution by allowing the mixed solution to permeate the porous membrane. Purification method. A method for purifying an antibody from a mixed solution containing the antibody, after adsorbing the antibody using an adsorbent carrying tryptophan and then eluting and recovering the antibody, the following steps A method for purifying an antibody, further removing impurities by a method comprising at least one of the methods.
(1) a step of purification by anion exchange chromatography;
(2) a step of purification by cation exchange chromatography,
(3) A step of purification by hydrophobic chromatography.   The method for purifying an antibody according to claim 4, wherein a porous membrane or resin carrying a ligand is used in the step of purifying by chromatography.