TW200927254A - Antibody production method - Google Patents

Abstract

To provide a method for producing an antibody, which uses a ligand having excellent biological safety, and which can be effected under mild conditions where the denaturation or aggregation of the antibody does not occur.Disclosed is a method for producing an antibody, which can produce an antibody that can be used as a medicinal agent from a pharmaceutical stock solution containing the antibody by utilizing the interaction with a ligand. The method is characterized by comprising the following steps in this order: a first step of contacting the pharmaceutical stock solution with an insoluble carrier having a heterocyclic aromatic amino acid ligand under the condition which is slightly or weakly acidic and contains a salt or the condition which is neutral and contains no salt, thereby causing the adsorption of the antibody to the ligand; a second step of removing a component(s) that is (are) not adsorbed to the ligand; and a third step of contacting an eluate that is slightly or weakly acidic and contains a salt at a lower concentration than that in the first step or contains no salt or an eluate that is slightly basic with the insoluble carrier, thereby causing the dissociation of the adsorbed antibody from the ligand.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing an antibody which is a pharmaceutical product, and relates to an antibody production method comprising the step of purifying an antibody of interest from a pharmaceutical raw material solution. [Prior Art] An antibody has high specificity for a target substance to which it recognizes and binds, and thus is extremely useful as a research reagent or a clinical test reagent. In particular, in recent years, we have developed a variety of therapeutic antibodies using biotechnology such as genetic recombination technology. In the field of rheumatism or cancer that was previously difficult to treat, the above-mentioned therapeutic antibodies have made significant progress in medical technology as an epoch-making therapeutic drug. offer. These are often referred to as antibody drugs. Further, the antibody can also be purified from an automatic body fluid, and an antibody purified from human plasma is called a r-globulin preparation' and is used as a pharmaceutical.

In the production of an antibody as a pharmaceutical product, a body fluid such as blood or ascites of a human or an immunized animal or a culture solution of cells having an antibody-producing ability is used as a raw material. These pharmaceutical raw material solutions contain various impurities such as proteins or DNA in addition to the target antibody, and thus have been conventionally purified by a conventional method such as fractional precipitation or ion exchange in the purification step. Further, in order to further improve the degree of purification of the antibody and also to improve productivity, a separation technique using a chromatographic method has been introduced, and a large amount of research has been conducted. For example, there are ion exchange chromatography or hydrophobic chromatography, or a method of combining the same (for example, Patent Document 1); in these methods, the pH of the solution is 136138.doc 200927254 Adsorption or dissociation is carried out by drastic changes or addition of various salts, etc., so that unnecessary components must be removed from the solution before becoming a final product. Moreover, the degree of purification is not particularly high. In any case, it cannot be matched with the following method using specificity. Further development of the above method is the affinity separation technique, and the use of a carrier having a ligand having a high affinity for a target substance in the affinity separation is a technique excellent in the recovery rate or productivity of the antibody. . Affinity separation is also actively introduced into the purification step of the antibody, and an affinity ligand has played an important role in recent years as a protein A derived from Staphyl cococus aureus. Since protein a has high specificity and affinity for the Fc region of the antibody, i.e., immunoglobulin, an antibody purification method using the ligand for the vector is known (for example, Patent Document 2). Protein A exhibits a high affinity for the Fc region of the antibody under neutral conditions, so when the antibody is purified from the pharmaceutical raw material solution, the pharmaceutical raw material solution is specifically contacted with an insoluble carrier such as protein A or the like as a ligand.吸附 Adsorb antibodies. Further, it is well understood that an antibody can be recovered at a high ratio by washing the unadsorbed component with a neutral physiological solution and removing it, and then dissociating the adsorbed antibody from the ligand by an acidic physiological solution. Thus, the antibody purification method using protein A or the like has the advantage of excellent binding specificity, but in order to recover the antibody at a high ratio, it is necessary to use a pH of around 3 (pH of 2.5 to less than 4,0). pH condition. The lower pH conditions have the advantage that they can also be used in the virus deactivation step, so that it is effective when purifying monoclonal antibodies that are stable at low pH. However, it cannot be applied to antibodies that are deactivated at low pH and lose activity. Further, 136138.doc 200927254 ' It is known that, although it is applicable at a low pH value, the high-order structure of an antibody is liable to change at a low pH value, and therefore may cause agglutination even if it does not eventually lose its activity. Generation of the body (Non-patent literature). In the production of therapeutic antibodies, it is required to use a purification step in the subsequent stage to remove aggregates and monitor residual amounts (Non-Patent Document 2). . On the other hand, in the antibody purification step using protein A or the like, there is a risk in the use of the protein A for the ligand of the insoluble carrier. First, protein A is a protein derived from microorganisms, so when protein A detached from the carrier in a purified preparation is mixed into the final product in a significant amount, if the antibody is administered to a human, it is possible to be sensitive. Symptoms causing allergies in high patients (Non-Patent Document 3) β Therefore, when protein A is used in the manufacturing step of the antibody drug, protein A should be removed at the later stage of the step or its residue should be closely monitored 4 (Non-patent literature) 4). Furthermore, with regard to the above-mentioned problems caused by low pH, a method of dissociating the adsorbed φ antibody from the protein A ligand using a buffer in the vicinity of weak acidity has also been studied (Patent Document 3), but in the end, protein A is used. Then, the risk of mixing into the final product, or the need to monitor the removal or residue caused thereby, is unsolvable. Second, in particular, when the cell culture solution is directly supplied to the coordinating body of the protein A, the protein A is sometimes cleaved due to the action of the protease present in the culture solution. As a result, not only the antibody binding ability is lowered, but also the fragment of protein A or protein A itself is mixed into the final product. Third, due to the protein A-based protein, it is less tolerant to alkaline conditions, and it is not possible to use the rinsing method commonly used in the chromatography step using other so-called synthetic ligands. Therefore, it is necessary to use (4) a special cleaning method using a surfactant such as "Τ·η (registered trademark)", trade name "TMt〇nX (registered trademark)" or SDS. The separation step of A. From the above, it has been found that in the production step of a therapeutic antibody which provides an epoch-making therapeutic opportunity, it is necessary to use a new purification technique which does not require the incorporation of a heterologous protein such as protein A and the use of a low molecular ligand. Therefore, a large number of studies have been conducted on purification methods of antibodies belonging to the affinity and separation of protein A, which can be roughly classified into a method of mimicking a ligand using protein A, and a method obtained by screening or the like. A method in which the antibody binds to a lower molecular ligand. In the former, an antibody purification method using a low molecular ligand which mimics the binding portion of the protein A and binds to the region by an antibody and is synthesized by an organic chemical method (for example, Non-Patent Document 5); In addition, a method of using an aromatic compound such as benzylated ethanolamine as a ligand (Patent Document 3), or a sulfophilic adsorption chromatography which is attracting attention as a new separation principle is used for coordination. The method of using a sulfamide compound (Patent Document 4). However, such a ligand has a possibility of applying mild solution conditions when it is bound or dissociated by an antibody, but on the other hand, it is not a constituent component of the human body but a synthetic compound, and there is a problem in this respect. The reason is that if an antibody drug mixed with such a synthetic compound is administered to a human, it may be combined with a carrier protein such as albumin in the blood to obtain antigenicity, and it is unavoidable to cause a class in a patient with high sensitivity. The possibility of allergic symptoms 136138.doc 200927254 sex. Therefore, in the post-step stage, the incorporation must be removed or the residual amount monitored closely. Therefore, it is known that if a common amino acid which is not a human ligand but a constituent of a living body including a human is used as a ligand, and the affinity oxime which uses the ruthenium film as a carrier is separated, The target protein can be efficiently purified from an aqueous solution containing protein (for example, (4) Documents 5 and 6 and Non-Patent Document 6). More specifically, Patent Document 5 discloses that a blood serum IgG solution of PH 8 is supplied. When the phenylalanine, histidine or tryptophan has been immobilized, the globulin is not detected on the permeate side, and thus the globulin is bound to the membrane. However, this is just to reveal the capture protein, and there is no record of the recovery method. On the other hand, Patent Document 6 discloses that a solution of a pH U of a bovine serum IgG solution of pH U is applied to a film which is more self-immobilized, and a solution of pH 7.4 is similarly described in Non-Patent Document 6, It is said that when a 5 % by weight ethylene glycol solution containing i ] ^ vaporized sodium is used, the bound antibody can be recovered at a high ratio. Indeed, the conditions of the material can be carried out at a milder pH than the purification conditions of the previously produced protein A, thus solving the problem caused by protein A. However, the use of a concentrated solution of ethylene glycol in the recovery of the bound antibody has become a major obstacle in the production steps for pharmaceuticals. The reason is that if ethylene glycol is metabolized in a living body, it becomes a poisonous biochemical substance, and it is difficult to use it because it is used at a very high concentration and high viscosity, so that it does not remain in the final product. In the affinity separation, as described above, even if the target substance can be adsorbed to the ligand, it is often necessary to use 136138.doc •10· 200927254 to dissociate the various undesirable additives, when used in, for example, pharmaceutical investment. When the substance is administered to the human body, not only the ligand design must be carefully considered, but also the composition of the solution used in the adsorption or dissociation must be carefully considered. As described above, various studies have been conducted on the antibody purification method using no protein A. However, it has not been known so far that the antibody production method which can fully satisfy the quality, safety or industrial productivity of a pharmaceutical product has not been known. [Patent Document 1] Japanese Patent Laid-Open No. Hei 7-267997 [Patent Document 2] European Patent No. 31071 9 (B1) No. [Patent Document 3] International Publication No. 2006/043896 (A1) [Patent Document 4] Japanese Patent Laid-Open No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. Sciences, 96(2007), 1-26 [Non-Patent Document 2] http://www.fda.gov/cder/regulatory/follow_on/ 200512/2005 12_rosenberg.pdf [Non-Patent Document 3] Staphylococci and Staphylococcal infections 2 ( Eds. CSF Easmon and C. Adlam), pp. 429-480, Academic Press Inc., London 1983 [Non-Patent Document 4] http://www.fda.gov/cber/gdlns/ptc_mab.pdf [Non-Patent Literature 5] Nature Biotechnology, 16 (1998), 190-195 [Non-Patent Document 6] Journal of Chromatography, 585 (1991), 45-51 [Summary of the Invention] [Problems to be Solved by the Invention] I36138.doc 200927254 In Industrial Manufacturing As an antibody to pharmaceuticals, it is a well-understood method of purification using protein A. Without using the various problems in the purification process of a protein, the object of the present invention is based are as follows. That is, an object of the present invention is to provide a method for producing an antibody which does not use a ligand which exhibits antigenicity or high biological activity such as protein A, but uses a ligand excellent in biological safety. And the antibody production method can be carried out under mild conditions which do not cause denaturation or aggregation depending on the stability of the antibody. [Means for Solving the Problems] In order to solve the above problems, the inventors of the present invention have conducted intensive studies on various adsorbents and purification methods. As a result, it has been found that it is useful to use a specific amino acid ligand in order to recover the antibody in the same ratio from the pharmaceutical raw material solution. Further, a purification method has been found in which not only the ligand is selected, but also the specific solution conditions previously unknown are applied when the antibody adsorbs and dissociates the ligand, thereby making the recovery rate of the target antibody from the pharmaceutical raw material solution extremely high. The present invention has thus been completed. That is, the present invention is as follows. (1) A method for producing an antibody which obtains an antibody as a pharmaceutical product from a pharmaceutical raw material solution containing an antibody by interaction with a ligand, and is characterized by comprising the following steps: a first step, which is A medicinal raw material solution having a heteropolymeric aromatic amino acid or a heterocyclic aromatic amino acid in a slightly acidic to weakly acidic and salt-containing condition, or neutral and salt-free. Contacting the insoluble carrier as a ligand, whereby the antibody is adsorbed onto the ligand; the second step 'which removes the component not adsorbed onto the ligand; and the third step, which is slightly acidic to Weakly acidic 136138.doc -12· 200927254 and containing a salt having a lower concentration than the first step or a salt-free solution or a slightly alkaline solution contacting the insoluble carrier, thereby dissociating the adsorbed antibody from the ligand . U) The method for producing an antibody according to the above (1), wherein the medicinal raw material solution is a body fluid or a cell culture solution. (3) The method for producing an antibody according to the above (1) or (2), wherein the heterocyclic aromatic amino acid is tryptophan. (4) The method for producing an antibody according to any one of the above (1) to (3), wherein the slightly acidic to weakly acidic means a pH of 6.9 or less and 3.0 or more, and the slightly alkaline means a pH of 7.1 or more. 9.0 or less. (5) The method for producing an antibody according to any one of the above (1) to (4) wherein the salt concentration in the first step is 130 mM or more. (6) The method for producing an antibody according to any one of the above (1) to (5) wherein the insoluble carrier is any one of a granular body, a fiber aggregate, and a porous film. (7) The method for producing an antibody according to any one of the above (1) to (6) wherein the anti-system is derived from an antibody of a mammal such as a bovine mouse or a mouse, or a chimera formed with human IgG, and a humanized antibody (humanized antibody) Antibody). (8) The method for producing an antibody according to the above (7), wherein the antibody is a human Ig (J). The antibody production method according to any one of the above (1) to (8) wherein the antibody is self-ligand (10) The method for producing an antibody according to any one of the above (1) to (9), wherein the non-aggregated antibody is selectively obtained from a pharmaceutical raw material solution containing the agglutinating antibody and the non-aggregated antibody. (11) A method for producing a non-agglutination 136138.doc 13 200927254 antibody by separating an agglutinating antibody from a non-agglutinating antibody, which is obtained by using an interaction with a ligand from a pharmaceutical raw material solution containing an antibody to obtain an antibody as a pharmaceutical product The method comprises the steps of: the first step, wherein the pharmaceutical raw material solution containing the agglutinated antibody and the non-agglutinated antibody is in a slightly acidic to weakly acidic state and contains a salt, or is neutral and does not contain salt. The condition τ is contacted with an insoluble carrier having a heterocyclic aromatic amine group and an H heterocyclic aromatic amino acid as a ligand, thereby adsorbing the antibody on the ligand; the second step, Suddenly, it will suck And removing the component on the ligand; and the third step of contacting the solution of the salt which is slightly acidic to weakly acidic and containing the salt lower than the first step, or the slightly alkaline solution, thereby contacting the insoluble carrier The effect of the present invention is to dissociate the adsorbed antibody from the ligand. [Effect of the Invention] According to the present invention, the amino acid which is originally retained as a cellular constituent component can be used as a ligand, and the biosafety is excellent. Body, rather than a ligand that exhibits antigenicity or higher biophysical activity in humans, such as scorpion peptone. Moreover, with a heterocyclic aromatic amino acid ligand, s, by subtle control The binding conditions of the antibody and the solution conditions at the time of dissociation can exert the following effects: purification can be carried out under mild conditions without causing denaturation or aggregation of the antibody under a wide range of conditions, and the recovery rate of the target antibody is also excellent. According to the present invention, not only the target antibody can be separated from the other components, but also the aggregates in the target antibody can be clearly separated from the non-condensed group, so that it is possible to display a fear As a result of the above, an antibody which is extremely useful as a pharmaceutical product in which a heterologous protein, a synthetic compound, or an antibody aggregate is not mixed can be efficiently obtained as a result of the above-mentioned contents. 136138.doc λ* 200927254 [Embodiment] Hereinafter, the present invention will be described in detail. In the present invention, an antibody, such as a common definition in biochemistry, refers to a glycoprotein molecule produced by a B lymphocyte which is an infection defense mechanism of a vertebrate animal ( In particular, in the present invention, it means that it can be used as a pharmaceutical for human users. That is, substantially no pathogenic microorganisms such as viruses are found in the mixture, and The antibodies present in the body of the subject are substantially the same structure. In the present invention, the chimera formed by human IgG refers to a variable region derived from a microorganism other than a human such as a mouse, but a region other than a human immunoglobulin; a humanized antibody , refers to the complementarity-determining region (CDR) in the variable region from organisms other than humans, other structural regions (such as coffee, region, FR) from humans, the immunogenicity of humanized antibodies and The chimeric antibody is further reduced compared to the antibody. The type of the antibody can be used as a pharmaceutical user, and the type (same type) or subclass thereof is not particularly limited. For example, depending on the difference in the structure of the constant region, it can be classified into five types of IgG, IgA, IgM, IgD, and IgE, and each immunoglobulin can be used. Among the human antibodies, IgG has four subclasses of IgG1 to IgG4, and IgA has two subclasses of IgAl and IgA2, and there is no particular limitation thereto. Further, if it can be used as a pharmaceutical product, an antibody that binds to an Fc region is also included in the scope of the so-called antibody of the present invention. 136138.doc •15·200927254 Related proteins. Further, the antibody may be classified according to the source or the production method, and a natural human antibody or a recombinant human antibody produced by a genetic recombination technique, or a monoclonal antibody or a plurality of antibodies may be used. Among these antibodies, the demand or importance as an antibody drug is greatest at this stage, and is particularly interesting for the application of human IgG. Further, the antibody purification conditions of the present invention are most suitable for the purification of human Ig(}.

The antibody as a pharmaceutical product is roughly produced by the following steps. Namely, the cell culture step, the cell separation step, the purification step, the virus removal step, the concentration/buffer replacement step, and the bottling step are sequentially performed. Of course, it is not limited to the process. Sometimes, the additional steps are inserted, or a part of each step is exchanged. "The above-mentioned process is a representative process when the target antibody is produced by the cell culture method, and the target is purified from the human body fluid. In the case of an antibody, the body fluid is administered to the purification step without undergoing a cell culture step and a cell separation step (IV). Widely, the so-called human body fluid 1 completely contains: a mixture of gold liquid, plasma, serum, lymph, ascites, pleural effusion, or a cage, etc., and a physiological saline solution, a buffer solution, and a sterile solution are added thereto. ^ ..., the dilution of the physiological solution of the sputum, the liquid preparation, and after the cell culture, into the purification · $ · π & the cell / knife away from the step and the liquid in the eight purification steps, refers to the ice铋 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩 铩It can also be a dilution of the physiological solution. Alternatively, the solution may be recovered by the use of ion exchange in the present invention for the recovery of the crude purified solution described in the next nucle, or by chromatography, etc., so that 136138.doc -16· 200927254 Into the purification step X, in particular the purification step using a specific amino acid ligand, the antibody-containing solution is specifically referred to as a pharmaceutical raw material solution. The purification step comprises several purification modes of one-step main purification, crude purification and main purification, or a combination of multiple purifications and multiple primary purifications, but the invention can be applied to any-purification mode 'and' can be applied to the main Purification can also be applied to crude purification. As a crude purification, purification by a different separation method such as various chromatography or membrane separation may be added after the purification of the present invention using a specific ligand. In the tenth aspect of the present invention, it is important that the purification step comprises the following three steps in sequence: the first step is to make the pharmaceutical raw material solution under conditions of slightly acidic to weakly acidic and containing a salt, or neutral and containing a salt. And contacting the insoluble carrier having a heterocyclic aromatic amino acid ligand, thereby adsorbing the antibody on the ligand; and in the second step, removing the component not adsorbed to the ligand; In the third step, the solution which is slightly acidic to weakly acidic and contains a salt having a lower concentration than the first step or the slightly alkaline solution is contacted with the insoluble φ carrier, whereby the adsorbed antibody is dissociated from the ligand. Within the scope thereof, the following modifications may be appropriately implemented: an additional step is inserted between the steps, or the first step and the second step are performed after the parent repeats the first step and the second step, or the first step of the number of cycles is repeated. Three steps and so on. First, explain the first step. The first important point in the first step is the choice of ligand. The term "ligand" as used herein refers to a binding site for a target & antibody which is chemically immobilized on the surface of an insoluble carrier. In the present invention, 'amino acid is used for the ligand', but it is well known that the amino acid system also contains the basic constitution of a human organism 136138.doc 200927254 Knife. Therefore, it is possible to avoid the antigenicity or strong physiological activity of protein A. For humans, the biological characteristics of the heterologous protein are exemplified in the case of tryptophan, and the mice are intraperitoneally administered. The LD50 at the time of the technique is 4.8 g/kg, and toxicity can be found under a large amount which is usually unimaginable. The inventors of the present invention have studied the amino acid ligands and found that if a heterocyclic aromatic amino acid is used in 2, it is possible to use a physiological solution having a mild @ value and excellent safety. Under the conditions of dissociation, the target antibody is recovered at a high ratio. A representative example of the heterocyclic aromatic amino acid is a tryptophanic acid and a histidine which are essential for human beings, and these have both the hydrophobicity of the aromatic moiety and the proton supply of the hetero atom. The detailed reason has not been determined by the fact that the adsorption rate of the antibody is low by using only the aryl (tetra) amino acid (for example, phenylalanine), and the side chain has a plurality of effects as described above, which contributes to a high ratio. The adsorption and dissociation of antibody molecules is a tryptophan ligand. The length of the ligand is not particularly limited, and may be a monoamino acid or an oligomer thereof. In the latter case, it may be an oligopeptide in which a plurality of heterocyclic aromatic groups are combined, or may be a heteroquinone in the free terminal side of other amino acid monomers or oligomers. 10,000 - detached from the carrier, it is preferred to stop at the oligo from: the length of the non-antigenicity, preferably the ligand of the monoamino acid. The so-called insoluble carrier system with the above ligand In the solid-liquid separation substrate, support, or separation element of the 'water' liquid combination function. Therefore, if the water is not substantially = there is a knife away from / / / ±, preferably J36138.doc 200927254 It is the organic solvent or acid that will not be used by the test (4). When it is selected, the material f 1 can be considered as the olefinic acid, and the polystyrene, polystyrene, A crosslinked body of poly(fluorenyl) propyl, cellulose, agarose, chitosan, a synthetic polymer, or the like, etc.: regardless of the natural polymer such as chitosan or its mountain, regardless of the material Commercially available active anaion exchange wax and resin adsorption materials can also be used.

The shape of the non-cooling carrier may be a granular material, a fiber aggregate, a porous film, or the like, and more specifically, porous f particles, non-woven fabric, woven cotton, porous flat membrane, and porous hollow fiber. membrane. In particular, many people have the ability to separate themselves, so they can also coordinate with the choice of ligands. From the viewpoint of preventing the ligand from falling off, the attachment of the ligand to the insoluble carrier is achieved by a covalent bond. The method is not particularly limited, and a known ligand immobilization technique may be applied depending on the resin component* of the carrier. For example, a method in which a carrier is irradiated with radiation to generate a radical, and then, as a base point, a glycidyl methacrylate or the like is graft-polymerized to introduce an active group, or a diglycidyl ether or a diamine is used. When the difunctional reagent introduces the active group into the surface of the carrier, the c-terminus or the N-terminus of the amino acid is reacted with the reactive groups, and the density of the ligand is set to a certain degree in terms of the amount of adsorption of the antibody. It is more important, but since the size of the antibody molecule is much larger than the size of the ligand, the higher the density, the better. If the size of the target antibody or the scale of production is considered, the ligand density can be appropriately set. 136138.doc •19· 200927254 Insoluble carrier in which the ligand is immobilized in this manner is preferably “filled in a casing of resin, glass, metal, etc.” from the viewpoint of productivity or sterility. It is used as a pipe string in which an inlet and an outlet of a liquid are provided. However, in the case of a fiber assembly or a porous film (flat film, hollow fiber membrane), the method of molding in such a manner that no short-pass is performed after being filled in the outer casing is somewhat complicated. From the viewpoint of the simplicity of the production of the column, it is preferred to use a granular body. About filling of insoluble carriers

Rate or fill capacity, if the ligand density is also considered and set according to the necessary separation size. The second important point in the first step is to bring the pharmaceutical raw material solution into contact with the above-mentioned insoluble carrier having a ligand under conditions of slightly acidic to weakly acidic and containing a salt, or neutral and salt-free. Thereby, the antibody is adsorbed to the heterocyclic aromatic amino acid ligand in a high ratio. The antibody molecule is stable to changes in pH, but may be unstable under acid or alkaline conditions depending on the type of antibody. This problem is caused when the protein A ligand is used, and this is a cause of limited use conditions. Therefore, in the affinity membrane separation of the amino acid ligand, the adsorption and dissociation of the ligand are carried out under neutral conditions while avoiding the conditions of acidity and inspectability. However, the recovery rate is increased when dissociated, not = to make: additives with significant toxicity. These points are as detailed in the prior art. ~ Research, the results are weakly acidic to moderately slightly acidic and weak. The inventors have made efforts in view of the above problems. When a heterocyclic aromatic amino acid ligand is used, the antibody between the sexual conditions is very high. The ratio is adsorbed. However, 136138.doc •20- 200927254 Must be accompanied by simultaneous summer π winter sleep under acidic conditions. “It is the opposite under neutral conditions. When it is salted under neutral conditions, the adsorption is decreased and the target antibody is lost. Change 犋 'decrease and . ^ servant can not be ignored. Under alkaline conditions, whether the helmet theory contains salt adsorption is reduced, ..., Na 疋 order contains, for example, when you want to achieve the goal and can not get a higher recovery rate. 4, the body becomes stable in the presence of salt _, if it is slightly acidic or weakly acidic and contains salt ^ _ there is no limit to the salt can be adsorbed. On the other hand, when the piece is # - ^ ^, or when If you want to avoid the lower pH value, you can avoid the lower temperature. (4) (4) Yes, the silk can be selected according to the nature of the target antibody. When the medical raw material solution is in contact with the insoluble carrier, in order to set the above solution, 'It is preferred to use a balanced solution that has been adjusted to the above solution conditions (for example, a solution in which a salt is added to a buffer) to pre-balance the carrier containing the ligand. For example, in filling = In the case of 'tubes The equivalence of the product is equal to the tens of times of the balance =: the replacement of the product can be carried out, and then, when the pharmaceutical raw material solution is loaded, when the volume of the medical raw material solution is sufficiently smaller than the column volume (for example, approximately 1) /10 or less), or when the liquidity of the pharmaceutical raw material solution (9) such as 'pH value, salt concentration, type of solute, etc.) and the solution when the antibody is adsorbed = the same can be directly loaded. However, when the volume of the pharmaceutical raw material solution When it is not negligible compared to the volume of the & column, in order to prevent the adsorption loss, it is preferable to dilute it so as to be close to the solution conditions at the time of adsorption, and then load it into the raw material solution. y' If the antibody adsorption is more strictly defined In the case of the above solution conditions, the term "slightly acidic to weakly acidic" as used in the present invention means that the pH is 65 or less, and 3 〇 is 136138.doc 21 200927254. From the P value J to 3 · 0, the adsorption rate is compared with the adsorption rate. Significantly more worried about the agglutination of antibodies' and thus poor. Therefore, from the viewpoints of the adsorption ratio of the antibody, the stability, and the action of the latter salt, the positive value of the acidic region is (3) or less, 3.0 or more, and particularly preferably the pH is 5 Å or less and 3 Å or more. On the other hand, the so-called neutral means that the PH value exceeds 6.5 and is less than 7.5. • The pH adjustment method is not particularly limited. If acid or alkali is added and the pH is adjusted to a specific pH, it is better to use a buffer for the stability of the positive change. For example, a buffer solution such as a phosphate buffer solution, an acetate buffer solution, a citrate buffer solution, or a trimethylaminomethoxine-hydrochloric acid (THs-HCl) buffer solution is used. Further, the term "salt containing salt" as used in the present invention means a solution condition in which a salt of 100 mM or more is contained in addition to the salt contained in the above buffer. If the salt concentration is lower than 100 mM, the adsorptivity decreases under weakly acidic conditions and is therefore better. The upper limit of the salt concentration is not particularly limited. However, if it is excessively added to the extent necessary, there is a possibility that the antibody is formed. Therefore, the salt concentration ❹ 1 is limited to K5 Μ or less. From the viewpoint of the adsorption rate of the antibody, it is more preferably 13 G mM or more, and particularly preferably 14 () or more. On the other hand, the term "salt-free" as used in the present invention means a solution having a salt concentration of 〇~5〇 for the purpose other than the buffering action. Under neutral conditions, if there is a salt added for the purpose other than the buffering effect, the adsorptivity will decrease significantly. Therefore, the salt concentration of the solution in contact with the ligand must be suppressed to a lower extent. . It is preferred that the edge be 'required' for the purpose of adding a salt for purposes other than buffering. In addition, when the f drug raw material solution is a body fluid or a cell culture solution, 'the salt concentration other than the 136138.doc -22·200927254 buffer must be diluted to 50 mM or less before the drug raw material solution is loaded into the column. in. The type of the salt is not particularly limited. For example, it is preferred that the electrolyte salt which is usually added to the physiological solution, that is, vaporized sodium, is not limited thereto. For example, gasification _ can also be used. Next, explain the second step. In the second step, the components not adsorbed on the ligand are removed. In the first step of the former®, the target antibody is adsorbed on the ligand, but there are unadsorbed antibodies or inclusions not adsorbed on the ligand, or inclusions weakly adsorbed on the ligand. . In the second step, the non-adsorbed antibodies or inclusions are excluded from the system by washing them. The component of the cleaning solution may be such that the pH or the salt concentration is different from the physiological solution in which the antibody is dissociated from the ligand in the subsequent third step. It is preferred to use the same solution as that used for the balance of the insoluble carrier or the solution of the pharmaceutical raw material to the insoluble carrier, and the pH or the salt concentration can be prevented from being cleaned by the solution. When the target antibody is inadvertently detached. Further, the non-specific adsorption component can be removed by appropriately selecting the ionic strength or the additive within a range in which the adsorbed antibody is not dissociated. Second, let's explain the third step. The third step is to contact a solution which is slightly acidic to weakly acidic and contains a salt having a lower concentration than the first step or a slightly alkaline solution, and is contacted with an insoluble carrier having a ligand to which the antibody is adsorbed. Thereby, the adsorbed antibody is dissociated from the ligand in a ratio. The solution is preferably a physiological solution such as physiological saline, a buffer solution, a culture medium or a sterile water. 136l3B.doc -23- 200927254 When the adsorbed antibody is dissociated from the ligand, the former is a so-called dissociation accelerator using ethylene glycol or a taste-like compound, or a high concentration of the drug can be used as a pharmaceutical. Conditions that are satisfactory in terms of productivity are not known. Therefore, the inventors of the present invention have conducted diligent research on the above aspects, and as a result, when a heterocyclic aromatic amino acid ligand is used in the field, it is not stable under neutral conditions for adsorption (four). Using a dissociation accelerator, the method predicts that a sufficient dissociation can be achieved, but a physiological solution that is slightly acidic to weakly acidic and contains a salt lower than the first step, or a slightly alkaline solution can cause the adsorbed antibody to dissociate at a high ratio. . Moreover, it has been found that if the acidity and the testability are to this extent, the denaturation of the antibody molecule is not particularly caused, and only the money contributes to the improvement of the recovery rate. Thus, for example, when the target antibody is to be stabilized in the presence of a salt, the solution is slightly acidic to weakly acidic and contains a salt having a lower concentration than the first step, or a slightly alkaline and salt-containing solution. The antibody can be dissociated from the ligand. On the other hand, when there is a limit that cannot be added to the salt, a slightly alkaline and salt-free solution is used. When it is desired to avoid acidic conditions, a slightly alkaline solution is used, and when it is desired to avoid alkaline conditions, The adsorbed antibody can be dissociated under acidic or weakly acidic conditions, and any condition can be selected depending on the nature of the target antibody. The strict definition or preferred range of "slightly acidic to weakly acidic" in the second step is the same as in the first step. When "slightly alkaline" is defined more strictly, it means that the pH is 7.5 or more and 9.0 or less. In the acidic region, if the pH value is lower than 3·〇, there is a concern that the antibody is agglutinated in the alkaline region. If the value exceeds 9.0, the activity may be decreased depending on the antibody, and thus 136I38.doc •24- 200927254 Poor. From the viewpoint of the dissociation property and stability of the adsorbed antibody, a pH value at a slightly alkaline level is preferably 8.〇 or more and 9.0 or less, and particularly preferably a pH of 8 5 or more and 9.0 or less. • If the pH is in this range, it will not cause any denaturation of the antibody. Moreover, since the dissociation property of the heterocyclic aromatic amino acid ligand and the adsorbed antibody is high, the recovery ratio of the target antibody to the purified pharmaceutical raw material solution is compared with the adsorption condition of the previous stage. Higher, almost inferior to the protein A method previously known for its high recovery. That is, the advantages of biological safety are emphasized in the present invention. The method of adjusting the pH is not limited as described in the first step. Regarding the so-called salt in the third step, when a slightly acidic to weakly acidic solution is used, it is important to contain a salt having a lower concentration than the first step. Under slightly acidic to weakly acidic conditions, if the salt concentration reaches 130 mM or more, there is a tendency for the & knife to adsorb the antibody. Thus, the salt concentration of Φ ° is better than 130 mM. Especially good is salt free. This important factor is to lower the salt concentration while maintaining a slightly acidic state, whereby the adsorbed antibody can be dissociated at a high ratio. The conditions for "salt-free" here are also as described in the first step. On the other hand, when using a microscopic elution solution, 'the effect of pH is preferred over the salt concentration, so the salt concentration is not & important, although it may or may not contain salt, but when it contains salt, Low salt concentration is preferred. The p λ & pair of salts is less than 300 mM. There is no limitation on the species of salt as described in the #th. The μ I-adsorbed antibody is dissociated from the ligand, and the dissociation accelerator may be added to the above-mentioned 136138.doc •25-200927254 dissociation dissolving solution. As the dissociation accelerator, a compound having chaotropic properties is preferred, and a water-soluble compound having a mercapto group is preferred. For example, an amino acid having a mercapto group on the side chain of arginine acid can further improve the recovery rate of the target antibody by using a slightly alkaline solution of arginine containing 0.1 to 2 Torr. Moreover, arginine is also a natural amino acid as the ligand of the present invention, so if it is used as an additive, it is not particularly difficult to remove it from the solution containing the purified antibody, and it is not necessary to pay too much attention to it in the end. It is especially good in the residue in the product. Among the three steps described above, the conditions relating to adsorption and dissociation of antibodies which are particularly important are as follows. The arrow (―) precedes the solution condition of the first step (when adsorbed), and the arrow after the third step (when dissociated) the solution strip (a) is slightly acidic to weakly acidic and contains a salt—slightly acidic to weakly acidic and contains concentration a salt lower than the first step or slightly acidic to weakly acidic and free of salt (b) slightly acidic to weakly acidic and containing a salt - slightly alkaline and free of salt φ (C) slightly acidic to weakly acidic and containing a salt ·- Slightly alkaline and containing salt (4) neutral and salt-free - slightly acidic to weakly acidic and containing a lower concentration than the salt of the first step or slightly acidic to weakly acidic and salt free (e) neutral and salt free - microtest Sex and salt-free σ) neutral and salt-free - slightly alkaline and containing salts can be selected according to the properties of the target antibody (a) ~ (f)

The virus was deactivated by acid. . Therefore, in the case of (a) to (c), it is purified in any case of the present invention. You I36138.doc • 26 - 200927254 is better for antibodies with more stable acidity. For this reason, it is particularly preferred to treat (a) under mild to weakly acidic conditions. In the present invention, as described above, adsorption or dissociation of the antibody of interest can be carried out at a high ratio by a specific ligand and solution conditions, but the mechanism thereof has not been known in detail. However, it has been inferred that it depends on the balance of electrostatic interactions and hydrophobic interactions at various pH or ionic strengths. According to the present invention, as described above, the target antibody can be obtained and other than

The fractions were separated, and the antibody of interest was obtained at a high ratio. However, the present inventors conducted a detailed study on the selectivity of the selection, and as a result, found that not only the target antibody but also other components can be separated, and surprisingly, the aggregate can be combined with the target antibody. Non-condensed groups are clearly separated. As described in the prior art, depending on the antibody, an aggregate is sometimes formed during the storage process or operation due to acidic conditions or other reasons. That is, the antibody sometimes undergoes dimerization, trimerization or multi-polymerization to a greater extent to form an aggregate, and it is feared that the aggregate exhibits antigenicity in the human body. Therefore, it is very important to produce an antibody as a pharmaceutical product in the production step of the antibody, and to reliably remove the aggregated antibody at the purification stage. In the case of clearly separating the aggregate from the non-condensed group, the conditions described in the first to third steps of the present invention can be applied, and selective separation can be carried out under such conditions. In the case of the above-mentioned (4) (neutral and without gull-weakness and no salt), or the case of (7) above (9) and not containing the weakness and the fear of eliminating the formation of aggregates under acidic conditions Containing salt) is a better condition. With the invention (IV), in: 136138.doc • 27- 200927254 size separation or size exclusion chromatography (size exciusi〇n chromatography), there is unevenness in pore size on the carrier side, so it is difficult to obtain anyway. This clear choice of separation. The reason why the agglutinating antibody and the non-aggregating antibody are clearly separated by applying specific adsorption/dissociation conditions to the heterocyclic aromatic amino acid ligand in the present invention has not been fully elucidated. The antibody monomer and the aggregate are only different in size, and it is difficult to think that it will show a large difference in the interaction with the ligand, but it is possible to infer the properties of the surface of the antibody (functionality or charge distribution, hydrophilicity/hydrophobicity). Balance, others) may change, resulting in a significant decrease in interaction with the ligand, enabling selective separation from non-agglutinated antibodies. Previously, a method of purifying an antibody containing an associated aggregate by gel filtration chromatography has been known (for example, Japanese Patent Laid-Open Publication No. Hei. No. Hei. No. Hei. No. 2-242957), In combination with the dissociation accelerator, the non-aggregate and the agglomerate can be separated by the difference in size. However, gel filtration using the size exclusion principle does not allow specific interaction with the φ vector, and can only be used for purification of the pretreatment level. Therefore, such an unexpected and useful separation using an amino acid ligand has been undetected by the inventors for the first time. In the method for producing an antibody of the present invention, the specific purification step described above is required, and as described in the detailed description, the anti-system of the pharmaceutical product is produced by the following procedure. That is, when the body fluid is used as the pharmaceutical raw material solution, it is produced in the order of the purification step, the virus removal step, the concentration/buffer replacement step, and the bottling step; when the cell culture method is employed, the cell culture step, the cell separation step, The purification step, the virus removal step I36I38.doc -28. 200927254, the concentration/buffer replacement step, and the bottling step are performed in the order. In the cell culture step, the animal cell or the antibody-producing cell into which the gene for producing the antibody of interest is introduced is cultured in a serum-free medium or the like to be propagated, and the target antibody is released or secreted outside the cell. Alternatively, the cells are mechanically stimulated or chemically disrupted to release the antibody into the culture. Then, unnecessary cell components are separated and removed by membrane filtration or centrifugation, and the antibody-containing solution is obtained in the state of a filtrate or a supernatant. Such as

The target antibody is purified in the purification step from the antibody-containing solution or body fluid, that is, the pharmaceutical raw material solution. In the purification step, for example, a separation technique having a lower specificity such as fractional precipitation, ion exchange or membrane separation is sometimes used in combination as a crude purification. In the case of medicine, it is important that the pathogenic microorganisms are not substantially mixed, and therefore, in the manufacturing step, it is necessary to prevent the incorporation of pathogenic microorganisms not only in the entire step, but also to actively set the step of removing the pathogenic microorganisms. A representative example thereof is a virus removal step, and a virus or the like is sometimes carried out by a physical method or a chemical method: a living treatment and removal. As another method, it is also preferable to use a virus containing a flat membrane or a medium::: membrane to remove the virus, and it is not necessary to use the aspect of adding ultraviolet rays or the virus removing ability. method. 2 The solution containing the purified antibody in this step is subjected to a buffer exchange for reducing the amount of the buffer used to reduce dissociation or recovery, and a pH adjuster, etc. It is filled into a sterile container of W疋 and becomes a product because of the gluten. I36138.doc •29· 200927254 Through the above-described procedure, an antibody which can be used as a pharmaceutical product without mixing heterologous proteins, synthetic compounds or antibody aggregates can be produced at a high ratio. ... & As described above, the present invention is based on the stability of the antibody to the pH value, can be used under milder conditions for an unstable antibody, and can also be used for a stable antibody T. A purification method used at lower pH values; therefore, it is extremely useful in terms of its high versatility. It is considered that the shortening of the step establishment period or the stepping of the steps can be realized, thereby making it possible to contribute to shortening the development period of the therapeutic antibody and further increasing the treatment opportunity. Furthermore, it is possible to separate not only antibodies and non-antibodies but also agglutinating antibodies and non-aggregating antibodies, or a simple alkaline cleaning method in terms of the chemical nature of the ligands, which contributes to the reduction of the number of steps or steps. The simplification can still make a big contribution to the increase in the above treatment opportunities. [Examples] φ Hereinafter, the present invention will be specifically described by way of Examples, but the present invention is not limited by the Examples. [Evaluation Method] The evaluation system of the antibody production method of the present invention is a system using a high-performance liquid phase chromatography method. That is, the liquid storage tank (balanced (cleaning) solution, dissolved liquid, column regeneration liquid), liquid feeding pump (feeding line speed of 4 em/min), sample "° loop (capacity is used) 100 PL), column (room temperature), detector (ultraviolet light, wavelength 280 nm), the system to which the discharge tube is connected, the purification target, and the concentration of antibody in each fraction recovered from the discharge tube Perform 136l38.doc 200927254 quantification. As the column column, a glass column having an inner diameter (diameter) of 5 mm and a column bed height of 1 mm was filled with 2 ml of the coordination iridium carrier. When the recovery rate of the target antibody was calculated, the absorbance of the solvent at the wavelength of 28 〇 nm from the obtained solution was subtracted from the absorbance of the solvent itself as a background value, and the actual absorbance was calculated. At this time, when the absorbance becomes a value of 0 or less, it is recorded as 〇. Regarding the actual absorbance, 丨3 was used as the light absorption coefficient of Ig(} to determine the protein content, and the ratio of the protein content in each of the dissolved fractions to the protein content in the starting material was calculated as the difference in each of the dissolved fractions.

IgG recovery rate (refer to the following formula (1)). In the following examples and comparative examples, the antibody recovery rate was evaluated by the above method unless otherwise specified. The IgG recovery rate (%) in each of the dissolved fractions = ΐ〇〇χ (protein content in the dissolved fraction / Protein content in the starting material) [Example 1] <Evaluation of ligands> The crucible will be filled with a carrier for fixing tryptophan to a porous polyvinyl alcohol particle via glycidyl methacrylate ( The column labeled as TR-PVA carrier is installed in the chromatography system. Equilibrate with a balanced solution (containing 1.5 Μ gasified sodium 20, hydrazine acetate buffer, ρΗ 5 〇) and a column volume (hereinafter referred to as π), and then contain 5 〇 mg/mL of IgG. Human igG solution (Benesis, Ven〇gl〇buHn) i〇〇吣 injected into the sample loop to balance the equilibrium / valley liquid about 1.5 CV extruded from the sample loop and added to the column 1, ', and obtained Pass the share. Next, it is used as the above balance of the cleaning liquid, and the CV is cleaned to recover the cleaning portion. Then, the eluate (containing 136138.doc •31 '200927254 with 0.5 M L-arginine as a dissociation accelerator 2 〇 squaric acid buffer, pH 8.0) 6 CV was passed through the column to recover IgG Dissolved. Further, the column regeneration liquid (1 mM hydrochloric acid solution, pH 15) was passed through the column, and the unrecovered IgG was recovered as a column regeneration portion. The evaluation results are not shown in Table 1. [Comparative Example 1] <Evaluation of Ligand> An e column packed with a carrier (hereinafter referred to as TR_pvA vector) in which amphetamine was immobilized on a porous polyvinyl alcohol particle via glycidyl methacrylate was prepared. Evaluation was carried out under the same conditions as in Example i. The evaluation results are shown in Table 1. [Table 1] Ligand Example 1 Tryptophan Comparative Example 1 Amphetamine First Step Second Step Micro Acidity (pH 5.0) / Salt (1.5 M NaCl) l^G Recovery f (%) Third Step Microalkali ί ί pH 8.0) / salt-free pass fraction 0.6 12.6 Washing fraction 2.5 35.5 Dissolving fraction 97.2 53.5 Column regenerating fraction 6.7 2.6 ------ Total 107.0 104.2 As shown in Table 1, by using as a heterocyclic aromatic The tryptophanic acid of tryptophanic acid is used as an adsorbent for adsorbing low molecular ligands of IgG, and is slightly acidic (pH 5) when adsorbed, and mildly acidic (pH 8) when dissociated. Next, IgG was obtained at a high recovery rate. On the other hand, in the case of a phenylalanine which is also an aromatic amino acid but having a side chain which is not a heterocyclic ring, the ratio of the fraction of 136138.doc •32·200927254 and the washing fraction is large, and IgG cannot be recovered at a high ratio. [Example 2] <Comparison of adsorption conditions> A column packed with a TR-PVA carrier immobilized with tryptophan was mounted in a chromatography system, and the equilibrium solution was compared under three conditions. Adsorption. That is, each equilibration solution (Condition A: 20 mM glycine buffer (1.5 3.0) containing 1.5 Μ sodium chloride, condition Β: 20 mM acetate buffer (ΡΗ 5.0) containing 1.5 Μ of sodium hydride, Condition C: 2 mM mM Phosphate® buffer (pH 7.0) without addition of salt was equilibrated at about 5 CV each. Then, the sample addition, washing, dissolving, and column regeneration were carried out in accordance with Example 以外 except that a salt-free 2 mM glycerol buffer (pH 9.0) was used as the elution solution. The evaluation results of the respective conditions A to C are shown in Table 2. [Comparative Example 2] <Comparison of adsorption conditions> A column packed with a TR_pvA carrier immobilized with tryptophan was attached to a φ chromatography system, and the adsorption was compared under the conditions of three different equilibrium solutions. . That is, to equilibrate the solution (Condition D: 20 guacamole buffer (pH 7.G) containing 1.5 Μ gasified sodium, Ε condition: 2G mM acetic acid containing no salt

The solution (pH 5.0), Condition F: salt-free 2 capsules (pH '^5) was equilibrated at about 5 Cv each. Then, the sample addition, washing, separation, and g column regeneration were carried out in accordance with the operating conditions of Example 1 except that a salt-free M mM glycine buffer (pH 9.G) was used as the elution solution. The evaluation results of the respective conditions D to F are shown in Table 2. 136138.doc •33· 200927254 [Table 2]

As shown in Table 2, when the adsorption condition was neutral and the salt was contained (condition D) was not adsorbed on the ligand immobilization (4), it also accounted for 23.7% (the sum of the parts and the cleaning group) In the case of no salt (condition C), IgG is quantitatively adsorbed. Helium, the IgG is less adsorbed in the case of microscopic or slightly acidic and salt-free, and IgG is quantitatively adsorbed in the case of slightly acidic or weak (iv) but containing a salt. From the above results, it is understood that when the ruthenium is adsorbed, it is necessary to have a slightly acidic or weakly acidic salt-containing condition or a neutral and salt-free solution condition. [Example 3] &lt;Comparison of dissociation conditions&gt; A column packed with a TR_PVA carrier immobilized with tryptophan was mounted in a chromatography system, and the dissociation of the adsorbed antibody was compared under the conditions of four different eluents. Sex. That is, according to the operating conditions of the examples, the equilibrium was carried out, 136138, d〇 (-34·200927254 attached, washed, dissolved, column regeneration, and the following five conditions were used as the elution solution (condition G: 20 mM glycine) Acid buffer (pH 3.0), condition Η: 20 mM acetate buffer (pH 5.0), Condition I: 20 mM phosphate buffer (pH 8.0), Condition J: 20 mM glycine buffer (pH 9.0), both ___________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________ The evaluation results are shown in Table 3. [Comparative Example 3] ® <Comparison of Dissociation Conditions> Except that a neutral dissolving solution (Condition K: 20 mM phosphate buffer (pH 7.0) containing no salt) was used. The operating conditions of Example 3 were carried out. The evaluation results are shown in Table 3. Further, as a result of the dissolution condition pH value of 9.0 (Condition J), the result of Condition B of Example 2 was used. [Table 3], Example 3 Comparison Example 3 G Η IJK Step ligand tryptophan first step second step slightly acidic (pH 5.0) / salt (1.5 M NaCl) third step Acidic pH 3.0 slightly acidic pH 5.0 slightly alkaline pH 8.0 slightly alkaline pH 9.0 neutral pH 7.0 IgG recovery in sowing (%) by 0.4 1.1 0.1 0.0 0.7 Washing 0.0 0.0 0.8 0.0 0.2 Dissolving 108.2 75.3 85.9 80.2 0.2 Regenerating fraction 0.4 28.1 19.3 19.9 104.0 Total 109.0 104.5 106.1 100.1 105.0 136138.doc -35- 200927254 As shown in Table 4, when the elution is carried out, the antibody cannot be obtained when the dissolution conditions are neutral and salt-free (condition K) It is recovered into the dissolving fraction, but the antibody can be efficiently recovered into the dissolving fraction when it is slightly acidic or weakly acidic and does not contain salt or when it is slightly alkaline. If compared with the result of Comparative Example 2, In the case of neutrality, if there is no salt, the adsorbed antibody hardly dissociates (condition κ), but even if the salt is dissociated at a higher concentration, it is not sufficient. If it is not under the mandatory column regeneration condition of strong acidity, the antibody is not adsorbed. It will be sufficiently dissociated (Condition D of Comparative Example 2). For the above reasons, it is considered that it is necessary to use a dissociation accelerator such as ethylene glycol under neutral conditions. From the above results, it is known that the adsorption resistance is carried out. When the dissociation (eluting time), and a slightly acidic or a weakly acidic salt containing a concentration lower than the first step, or the alkaline solution conditions necessary micro system. Further, it is considered that, in terms of a high recovery ratio of the eluted portion, it is preferably weakly acidic on the acidic side, and preferably has a pH of 7.5 or more and less than 9.0 on the slightly alkaline side. [Example 4] &lt;Effect of change in salt concentration&gt; A column packed with a TR-PV A carrier immobilized with tryptophan was attached to a chromatography system, and under two conditions of different adsorption conditions, The effect of changes in salt concentration on the dissociation of adsorbed antibodies. That is, in addition to using a balanced solution (Condition L: 20 mM glycine buffer (pH 3.0) containing 1.5 Μ of sodium hydride, condition μ: 20 mM acetate buffer (pH 5.0) containing 1.5 Μ of sodium hydride The adsorption was carried out at about 5 CV, and then the adsorption, washing, dissolving, and column regeneration were carried out in accordance with the operating conditions of Example 1 except that the solution having a salt concentration gradient was used. The salt concentration gradient is formed by continuously supplying the balanced solution with the same pH value and no salt to the balanced solution containing salt 136138.doc -36· 200927254. The salt concentration is continuously decreased from 1.5 60 in 60 minutes. To 0 Μ. The salt concentration at which each of the dissolved fractions begins to appear is calculated from the conductivity in the salt concentration gradient. The evaluation results are shown in Table 5. [Table 4] L Μ Step ligand tryptophan first step second step weakly acidic (pH 3.0) slightly acidic (pH 5.0) salt (1·5 MNaCl) third step weakly acidic (pH 3.0) slightly acidic ( pH 5.0) Salt (1.5MNaCl) concentration gradient decreased to salt-free IgG recovery (%) Passing 0.1 0.1 Washing fraction 0.8 0.3 Dissolving fraction 1 (1.5M-130mM) 1.1 (1.5 Μ-100 mM) 0.0 Dissolved fraction 2 (130 mM-&gt;0 mM) 89.4 (100 mM-0 mM) 78.6 Column Regeneration 2.5 14.5 Total 93.9 92.9 As shown in Table 5, under mild conditions of mild or weak acidity, even if the pH is not changed The salt concentration can also control the adsorption/dissociation of IgG by a line of about 100 mM. That is, if it is also compared with the results of the conditions G and Η of Example 3, in the case of slightly acidic or weakly acidic, the adsorbed antibody can be dissociated by making the salt concentration at the time of dissociation lower than the salt concentration at the time of adsorption. . [Example 5] &lt;Effect of change in pH value&gt; A column packed with a TR-PVA carrier immobilized with tryptophan was attached to a chromatography system, and the dissociation of the pH value to the adsorbed antibody was mainly studied. The effect. Namely, adsorption, washing, dissolution, and column regeneration were carried out in accordance with the operating conditions of Example 1 except that a solution having a pH gradient was used, and then a solution having a salt concentration gradient was used for 136138.doc -37-200927254. The pH gradient is formed by continuously supplying an equilibrium solution having the same salt concentration and a pH of 9.0 to an equilibrium solution containing a salt having a pH of 5.0, and continuously changing from slightly acidic to pH (pH 5.0) for 60 minutes. Alkaline (pH 9.0). Then, a balanced solution of the same pH and no salt was continuously supplied to each of the equilibration solutions containing the salt to form a salt concentration gradient, which was continuously decreased from 1.5 Torr to 0 Torr in 60 minutes. The salt concentration at which each of the dissolved fractions begins to appear is calculated from the conductivity in the salt concentration gradient. The evaluation results are shown in Table 6. [Table 5] N-step ligand tryptophan first step second step slightly acidic (pH 5.0) / salt (1·5 M NaCl) The third step is slightly acidic (pH 5.0) - the gradient changes to slightly alkaline ( 9.0) Thereafter, the salt (1.5 MNaCl)-concentration gradient drops to the salt-free IgG recovery (%) by the fraction 0.8 Washing fraction 1.5 Dissolving fraction 1 (pH change only) (pH 5.0-9.0/salt 1.5 M) 85.6 Dissolved fraction 2 (change in salt concentration only) (pH 9.0/^1.5M-&gt;0.3M) 7.3 Dissolved fraction 3 (change in salt concentration only) (pH 9.0/salt 0.3 M—0 Μ) 1.5 Column regeneration Part 6.5 Total 103.3 As shown in Table 6, when a salt is contained, only the slightly acidic condition at the time of adsorption is changed to a slightly alkaline condition, and even if the pH value is changed to the alkaline side, IgG can be recovered at a high ratio. If compared with the results of Conditions I and J of Example 3, if a slightly alkaline solution is used, the adsorbed 136138.doc -38 - 200927254 antibody can be dissociated regardless of the salt concentration. [Example 6] &lt;Purification of IgG from cell culture medium&gt; To a solution of 17.6 mL of CHO medium cultured in serum-free medium (Irvine Scientific, trade name "IS-CHO-CD"), 1 Μ acetate buffer ( The pH was 4.5) 2 mL, the pH was adjusted to about 5.0, and then 0.4 mL of a human IgG solution (Benesis, Venoglobulin) containing 50 mg/mL IgG was added to prepare a cell culture medium containing IgG. Secondly, according to the conditions of Example 3 (first and second steps: slightly acidic (pH 5.0), salt (1.5 M NaCl), the third step: slightly alkaline (pH 8 · 0), no salt) The culture solution containing the IgG cells was treated with 1 mL. Among them, the amounts of the solvents used for washing, dissolving, and column regeneration were all set to 4 CV, a pool was formed in units of 1 CV, and the first two cells were used for the following analysis. An equal volume of SDS sample buffer (TEFCO, Tris-Glycine SDS sample buffer) was added to 50 μί of the obtained solution, and heated at 100 ° C for 5 minutes, and then rapidly cooled in ice water to prepare an electrophoresis sample. According to the commonly used method, the electrophoresis samples were analyzed by SDS-PAGE. The electrophoresis apparatus uses SDS-PAGEmini (8 to 16%, 1.5 mm thick, 15 well) of TEFCO, and a molecular-weight marker is a commercially available labeling substance (GE HEALTHCARE BIO-SCIENCES, trade name) "Full-Range RainBow Molecular Markers"). After the electrophoresis was completed, the commercially available gel staining solution (Wako Pure Chemical Industries, Ltd., trade name "Quick-CBB") was used for dyeing, and the gel drying set was used to preserve the kit 136138.doc -39- 200927254 (TEFCO, GEL Dry) System) to dry. The dried colored gel is shown in Fig. 1. In the figure, the zone Μ is a molecular weight marker substance, and zones 7 and 8 are solute fractions. The arrow indicates the separation position of the igG. As is apparent from the figure, IgG was recovered in the first 1 CV (reference zone 7) of the dissolved fraction. From the above results, it was confirmed that IgG can be recovered from the cell culture solution by the antibody production method of the present invention. [Example 7] &lt;Separation of IgG agglutination&gt; A human IgG solution (Benesis, Venoglobulin) 50 mg/mL was mixed with a 0.1% aqueous solution of hydrochloric acid at a ratio of 1:4, and allowed to stand at room temperature for 1 night. An acid-denatured IgG aggregate sample (hereinafter referred to as IgG_Ag) was prepared. A column packed with a TR_PVA carrier immobilized with tryptophan was mounted in a chromatography system. Equilibrate with a balanced solution (2 mM mM acetate buffer containing 1.5 mM sodium sulphate, pH 5·0) for about 5 CV, and then inject the IgG_Ag into the sample loop three times at a time of 1 〇〇μΐ. In the above, each sample was extruded from the sample loop with the above-mentioned equilibrium solution about J, 5 cv and added to the column and a pass fraction was obtained. Next, the above-mentioned equilibration solution 5 to ό CV was used for washing, and the washing fraction was recovered. Then, the dissolving solution (20-acid buffer without salt, 卩1^9.〇) 5~6 (^ is passed through the column, and the dissolved fraction of 1 is recovered. Further, the column is further The regenerant (1 mM HCl solution, pH 15) was passed through the column, and the IgG was not recovered as a column regeneration. The uv monitor in the chromatographic system was used to identify IgG_Ag in each fraction. The measurement was measured over time.

The chromatogram of IgG-Ag is shown in Figure 2. Since the acid treatment conditions are conditions in which the igG monomer 136138.doc 200927254 is substantially quantitatively agglomerated, the peak detected in the column regeneration fraction can be regarded as an aggregate. Next, add 1.5 mL of 1 Μ glycine acid solution to 5 mL of IgG-Ag to restore the pH to about 3 to 4, and add 1 mL of undenatured human IgG solution to prepare acid-denatured IgG aggregates and not. A mixed solution of denatured IgG (hereinafter referred to as IgG-Mx). The obtained IgG-Mx was used as a sample, and the sample was added in four portions at a dose of 100 pL each time, and the same was carried out in the same manner as the above IgG-Ag.

A chromatogram of IgG-Mx is shown in Figure 3. The peak detected in the fraction of the chromatogram can be confirmed as unaggregated undenatured IgG in advance. As shown in Fig. 3, IgG-Mx was bisected into peaks corresponding to acid-denatured IgG aggregates and undenatured IgG, respectively. From the above results, it has been revealed that the undenatured IgG and the IgG aggregate can be selectively quantitatively and selectively separated by the antibody production method of the present invention. [Reference Example 1] &lt;IgG purification example using protein A immobilization carrier&gt; Each 5 CV solution (100 mM sodium citrate buffer, pH 3.0) and column regeneration solution (10 mM hydrochloric acid buffer) were used. , pH 1.5) The tube (16 mm in diameter and 25 mm in bed height) filled with the protein A immobilization carrier (Milipore, trade name "prosep-va-ultra") was sequentially washed. Next, a 1 〇CV equilibration solution (20 mM phosphate buffer containing 0.15 bismuth gas, pH 7.4) was passed through to equilibrate the carrier, and then the human jk was cleared. (CEMICON international, Normal Human Serum) Add to the column. Thereafter, the above-mentioned equilibrium 136138.doc -41 - 200927254 solution of 10 CV or more was flowed through the column for thorough cleaning. Then, 4 CV of the above-mentioned dissolving solution was passed through the column to recover IgG. Neutralization was carried out by adding a neutralizing solution (500 mM phosphate buffer, pH 9.0) equivalent to 1 CV to the recovered IgG solution. Further, the liquid passing operation up to this point was carried out at a line speed of 150 cm/hr. The neutralized IgG solution was placed in a dialysis tube with a molecular weight cut off of 15 kD and sealed, and dialyzed for 3 hours in 1 l of the above equilibrium solution, followed by a balance of 3 L. The overnight dialysis was carried out in the solution, and buffer replacement was performed. The resulting IgG solution was diluted 10-fold and the absorbance at a wavelength of 280 nm was measured. The IgG concentration was estimated to be 3 mg/mL based on the IgG absorption coefficient of 13, and the amount of IgG obtained was estimated to be 75 mg. Next, 300 pL of the above IgG solution was placed in a microcentrifuge tube and heat-treated at 100 ° C for 5 minutes. The centrifuge was operated at 3000 rpm for 5 minutes using a microcentrifuge, and the supernatant was recovered. The amount of protein A in the resulting supernatant was measured by ELISA. The ELISA system was used under the trade name "TiterZyme EIA ProteinA Enzyme Immunometric Assay Kit" from the assays company, and was carried out in accordance with the attached manual. As a result, it was estimated that the amount of protein A in the IgG solution was about 28 ng/mL, and the total amount was 28 x 25 = 700 ng. As described above, in the case of using the protein A immobilization carrier to purify IgG, about 9 ng of protein A was mixed per 1 mg of IgG. [Reference Example 2] Comparison of Human IgG Adsorption Capacity&gt; A column packed with a carrier (TR-PVA vector) immobilized on a porous polyvinyl alcohol particle was mounted in a chromatography system. Equilibrate about 10 column volumes with a balanced solution of 136138.doc • 42· 200927254 (2 mM mM acetate buffer containing 1.5 Μ sodium sulphate, pH 5.0) and then dilute to the same concentration with 5 mg/mL using the same solution The human IgG solution (Benesis, Venoglobulin) was passed through the column. The absorbance of the column was monitored and the IgG dilution was passed through until the absorbance no longer increased, and then washed with the above-mentioned equilibration solution as a cleaning solution at approximately 15 CV. Then, about 6 CV of a solution (2 mM mM disc acid buffer containing 0.5 M L-arginine as a dissociation accelerator, pH 8.0) was passed through the column, and the IgG solution was recovered as a fraction. Further, a 5 CV column regeneration liquid (1 mM hydrochloric acid solution, pH 丨5) was passed through the column, and unrecovered IgG was recovered as a column regeneration portion. The total amount of the IgG content of the obtained fraction (excluding the column regeneration fraction) was measured, and this was divided by the volume of the column carrier of 2 mL to calculate the adsorption capacity per mL of the carrier. The results are shown in Table 7. On the other hand, a column (5 mm in diameter and 〇i〇0 mm in height) filled with a protein A immobilization carrier (MiHp〇re, trade name "pr〇Sep-va-ultra") is attached to the layer. In the analysis system, Ig(} adsorption capacity is measured in the same manner as above to equilibrate the solution (2 mM phosphate buffer containing 〇·15 M vaporized sodium, pH 7.5), and then the same solution is used. Diluted into a human IgG solution at a concentration of 5 mg/mL (Benesis, Inc., (4) Flow through the column. In the same manner as above, the diluent is passed through until the absorbance of the column is no longer increased. Then, it is washed with about 1 () CV as the above-mentioned equilibrium solution of the cleaning liquid. Then, about 4 CV of the elution solution (0.1 Μ citrate buffer, pH 3 〇) is passed through the column and recovered. The IgG solution was used as a dissolving component. Further, a 5 cv tube 136138.doc -43-200927254 column regenerant (10 mM hydrochloric acid solution, pH 丨5) was passed through the column, and unrecovered IgG was recovered as a column regeneration. Adding to the lysate is 1/4 capacity relative to the IgG solution. 1 cv amount of neutralization solution (500 phosphate buffer, pH 9_0) was neutralized. The total amount of IgG content of the obtained fraction (excluding column regeneration) was measured and divided by the capacity of the column carrier 2 The adsorption capacity per mL of the carrier was calculated by mL*. The results are shown in Table 7. [Table 6] TR_-PVA# Adsorption capacity of 艚 protein A immobilized carrier (mg/mL carrier) 24.6 25.4 From the results of Table 7, it can be seen that In the antibody production method of the invention, the IgG adsorption capacity per unit carrier is about 25 mg/mL, which is inferior to the IgG adsorption capacity per unit carrier of the protein a column. The IgG was efficiently purified on the same scale as the conventional method using the protein A column. [Reference Example 3] &lt;Comparative checkability&gt; The solution used in Example 1 was used with about 1 mL of a 1 M sodium hydroxide solution. After the TR_PVA carrier was washed 4 times in about 5 mL, about 10 mL of the same solution was added, and about 3 Torr was placed at room temperature. Using the carrier thus subjected to alkali treatment, Ig was studied and calculated in the same manner as in Reference Example 2. (3 adsorption capacity. The results are shown in Table 136138.doc -44 - 200927254 [Table 7] ----- - - --- ___ None (Reference Example i) IgG adsorption amount (mg/mL carrier) 24.6 As shown in Table 8, compared with the non-alkali treated IgG adsorption amount in the reference example 2 Even if the alkali treatment adsorption capacity is carried out, almost the tryptophan ligand is resistant to alkali. '"不田钇果垅明, in the method of producing an antibody using a hydrazine ❹ Yuanfangfangxiang amino acid ligand The chiral protein &amp; ligand method is previously difficult to clean, and the ligand after use can be easily regenerated. [Industrial Applicability] According to the present invention, an antibody which is useful as a pharmaceutical and which is excellent in safety can be produced at a high ratio. Therefore, it can provide various (four) therapeutic antibodies that can be applied to rheumatism or cancer treatments that were previously difficult to treat. Of course, the therapeutic antibody also contains antibodies as biochemical reagents or clinical examination reagents, thereby making advances in medical technology. Make a big contribution. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a gel electrophoresis pattern of an antibody purified from a cell culture solution. Figure 2 is a chromatogram of an acid-denatured IgG agglutinator. Figure 3 is a chromatogram of the undenatured 1 § (mixture of 5 and condensed aggregates. 136138.doc -45-

Claims (1)

200927254 X. Patent Application: A method for producing an antibody, which obtains an antibody as a pharmaceutical product from a pharmaceutical raw material solution t containing an antibody by interaction with a ligand, and is characterized by comprising the following steps: (1) The first step is to make the pharmaceutical raw material solution under a condition of slightly acidic to weakly acidic and containing a salt, or neutral and salt-free, and having a heterocyclic aromatic amino acid or a hetero The polymer of the cyclic aromatic amino acid is contacted as an insoluble carrier of the ligand, whereby the antibody is adsorbed on the ligand; (2) the second step, which is not adsorbed on the ligand And (3) a second step of contacting a salt which is slightly acidic to weakly acidic and containing a salt having a lower concentration than the first step or a salt containing no salt, or a slightly alkaline solution, and an insoluble carrier. This dissociates the adsorbed antibody from the ligand. 〇 2. The method of producing an antibody according to the invention, wherein the pharmaceutical raw material solution is a body fluid or a cell culture solution. 3. The method of producing an antibody according to claim 1 or 2, wherein the heterocyclic aromatic amino-acid is tryptophan. 4. The method for producing an antibody according to the above, wherein the slightly acidic to weakly acidic means a pH of 6.9 or less and 3.0 or more, and the slightly alkaline means a pH of 71 or more and 9.0 or less. 5. The method of producing an antibody according to claim 1, wherein the salt concentration in the first step is 130 mM or more. The method for producing an antibody according to claim 1, wherein the insoluble carrier is any one of a granule, a fiber aggregate, and a porous membrane. 7. The method of producing an antibody according to claim 1, wherein the anti-system is derived from an antibody of a mammal such as a human, a cow, or a mouse, or a chimera formed with a human IgG, and a humanized antibody. 8. The method of producing an antibody according to claim 7, wherein the antibody is human IgG. 9. The method of producing an antibody according to claim 1, wherein a dissociation accelerator is used in dissociating the antibody from the ligand. 10. The method of producing an antibody according to claim 1, wherein the non-aggregated antibody is selectively obtained from a solution of the pharmaceutical raw material containing the agglutinating antibody and the non-aggregated antibody. π. A method for producing a non-aggregated antibody by separating an agglutination antibody from a non-agglutination antibody, which is obtained by using an interaction with a ligand to obtain an antibody as a pharmaceutical product from a pharmaceutical raw material solution containing an antibody. The steps include the following steps: (1) The first step is to make a pharmaceutical raw material solution containing agglutinated antibody and non-agglutinated antibody 'slightly acidic to weakly acidic and containing salt, or neutral and salt-free Under conditions, 'contact with an insoluble carrier having a heterocyclic aromatic amino acid or a heterocyclic aromatic amino acid as a ligand, thereby adsorbing the antibody on the ligand; (2) a second step of removing the non-adsorbed ruthenium ribs and a component of the work; and (3) a third step of making the salt slightly acidic to weakly acidic and containing a salt lower than the first step or containing no salt The eluent, or the slightly alkaline solution is contacted with the insoluble carrier, thereby dissociating the adsorbed antibody from the ligand, 136138.doc