JP2010241761A - Method for purifying antibody monomer using anion exchange group-immobilized porous membrane - Google Patents
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Landscapes
- Treatment Of Liquids With Adsorbents In General (AREA)
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Abstract
Description
本発明は、アニオン交換基が固定された多孔質吸着膜を用いた、抗体モノマーの精製方法に関する。 The present invention relates to a method for purifying an antibody monomer using a porous adsorption membrane having an anion exchange group immobilized thereon.
近年、バイオテクノロジー産業において、タンパク質の大量精製が重要な課題となっている。特に医薬の分野において、抗体医薬の需要が急速に拡大しており、効率的に大量のタンパク質を産生可能及び精製可能な技術の確立が強く望まれている。 In recent years, mass purification of proteins has become an important issue in the biotechnology industry. Particularly in the field of medicine, the demand for antibody drugs is rapidly expanding, and it is strongly desired to establish a technique capable of efficiently producing and purifying a large amount of protein.
一般的に、タンパク質は、動物由来の細胞株を用いる細胞培養によって産生される。この細胞培養液からタンパク質を精製する通常の操作においては、最初に、細胞培養液を遠心分離し、濁質成分を沈降除去する。次いで、遠心分離で除去しきれない約１μｍ以下の細胞デブリを、精密ろ過膜を用いるサイズろ過により除去する。さらに無菌化するために、最大細孔径が０．２２μｍ以下のろ過膜を用いて無菌化ろ過を施して、目的タンパク質を含む無菌溶液を得る（ハーベスト工程）。続いて、プロテインＡに代表されるアフィニティクロマトグラフィーを初めとする、複数のクロマトグラフィー技術の組み合わせによる精製プロセスを用いて、Ｈｏｓｔ Ｃｅｌｌ Ｐｒｏｔｅｉｎ（ＨＣＰ）、ＤＮＡ、目的タンパク質の凝集体、エンドトキシン、ウィルス、カラムから脱離したプロテインＡ及びプロテインＡと抗体との凝集体などの夾雑物を、この無菌溶液から除去し、目的タンパク質を分離・精製する（ダウンストリーム工程）。 In general, proteins are produced by cell culture using animal-derived cell lines. In a normal operation for purifying protein from the cell culture solution, first, the cell culture solution is centrifuged to remove sediment components from the turbid components. Next, cell debris of about 1 μm or less that cannot be removed by centrifugation is removed by size filtration using a microfiltration membrane. For further sterilization, sterilization filtration is performed using a filtration membrane having a maximum pore size of 0.22 μm or less to obtain a sterilized solution containing the target protein (harvest process). Subsequently, using a purification process including a combination of a plurality of chromatographic techniques including affinity chromatography typified by protein A, Host Cell Protein (HCP), DNA, target protein aggregate, endotoxin, virus, Contaminants such as protein A desorbed from the column and aggregates of protein A and antibody are removed from this sterile solution, and the target protein is separated and purified (downstream process).
以上説明した従来のタンパク質の精製方法の対象となる細胞培養液中の目的タンパク質の濃度は、現状では通常１ｇ／Ｌ程度である。また、夾雑物の濃度も、目的タンパク質の濃度とほぼ同程度又はそれ以下であると考えられ、かかる濃度においては、上記のハーベスト工程及びダウンストリーム工程を含む従来のタンパク質の精製方法は充分有効である。 The concentration of the target protein in the cell culture medium to be subjected to the conventional protein purification method described above is usually about 1 g / L at present. In addition, the concentration of impurities is considered to be approximately the same as or lower than the concentration of the target protein. At such a concentration, the conventional protein purification method including the harvesting step and the downstream step is sufficiently effective. is there.
しかしながら、抗体医薬の需要が急速に拡大し、抗体医薬に用いられるタンパク質の大量生産が指向されたため、近年では細胞培養液中のタンパク質濃度を高める細胞培養技術が急速に発達し、細胞培養液中の目的タンパク質の濃度が１０ｇ／Ｌ又はそれ以上に達することもある。同時に、細胞培養液中の夾雑物の濃度も同様に増加し、従来のタンパク質の精製方法では、目的タンパク質の精製が困難になりつつある。 However, since the demand for antibody drugs has expanded rapidly and mass production of proteins used in antibody drugs has been directed, cell culture technology for increasing protein concentration in cell culture media has been rapidly developed in recent years. The concentration of the target protein may reach 10 g / L or more. At the same time, the concentration of contaminants in the cell culture medium increases as well, and the purification of the target protein is becoming difficult with the conventional protein purification methods.
特に、細胞培養液中の目的とする抗体タンパク質の濃度の増大に伴い、そのモノマーの凝集体、例えば２量体、３量体などの多量体の濃度も顕著に増加する傾向にある。凝集体は、生体内への投与時に、補体の活性又はアナフィラキシーをもたらすことによって、抗体医薬の安全性に有害な影響を及ぼす可能性が指摘されており、その有効な除去方法が近年希求されている。このため、凝集体を含む各種の夾雑物を有効に除去し、抗体医薬品として使われる抗体タンパク質、すなわちモノクローナル抗体、ポリクローナル抗体、ヒト化抗体、ヒト抗体及び免疫グロブリンなどを精製することを目的としたクロマトグラフィー工程が多数報告されている。 In particular, as the concentration of the target antibody protein in the cell culture medium increases, the concentration of monomer aggregates such as dimers and trimers tends to increase significantly. It has been pointed out that aggregates may adversely affect the safety of antibody drugs by bringing about complement activity or anaphylaxis when administered in vivo, and effective removal methods have recently been desired. ing. Therefore, it was intended to effectively remove various contaminants including aggregates and purify antibody proteins used as antibody drugs, ie, monoclonal antibodies, polyclonal antibodies, humanized antibodies, human antibodies and immunoglobulins. Many chromatographic processes have been reported.
イオン交換クロマトグラフィーは、抗体と夾雑物との等電点の相違を利用して、これらを分離する方法であり、特にアニオン交換クロマトグラフィーは、一般に等電点の値が抗体タンパク質より小さいＨＣＰ、ＤＮＡ、ウィルスなどの夾雑物の除去に多用されている。しかしながら、等電点がモノマーとほぼ等しい凝集体を、アニオン交換クロマトグラフィーを用いて除去するためには、他の夾雑物の除去よりも精密な条件の制御が要求される。 Ion exchange chromatography is a method of separating these by utilizing the difference in isoelectric point between the antibody and the contaminant, and in particular, anion exchange chromatography is generally HCP having an isoelectric point smaller than that of the antibody protein, It is frequently used to remove contaminants such as DNA and viruses. However, in order to remove an aggregate having an isoelectric point almost equal to that of the monomer using anion exchange chromatography, it is necessary to control the conditions more precisely than the removal of other impurities.
例えば、特許文献１には、抗体の等電点の近傍のｐＨに抗体モノマーと凝集体の混合液を調整して、アニオン交換クロマトグラフィーカラムに通液して透過液を回収し、さらに同じｐＨの緩衝液を通液して洗浄液を回収し、これらの回収液を抗体モノマーの精製液とする抗体モノマーの精製方法が開示されている。この精製方法は、凝集体はモノマーに比べて多くの電荷点を有するため、僅かではあるがアニオン交換基により固定されやすいという原理に基づいている。特許文献２には、抗体モノマーと凝集体をともにアニオン交換クロマトグラフィーカラムに吸着させた後、溶出液の塩濃度を徐々に上昇させるグラジエント溶出により、先に溶出される抗体モノマーの溶出ピークを回収する、抗体モノマーの精製方法が開示されている。 For example, in Patent Document 1, a mixture of antibody monomers and aggregates is adjusted to a pH in the vicinity of the isoelectric point of the antibody, and the mixture is passed through an anion exchange chromatography column to collect the permeate. A method for purifying an antibody monomer is disclosed in which a washing solution is collected by passing through the buffer solution, and the collected solution is used as a purified solution of the antibody monomer. This purification method is based on the principle that aggregates have a small number of charge points as compared to monomers, and thus are easily fixed by anion exchange groups. In Patent Document 2, the antibody monomer and the aggregate are adsorbed on an anion exchange chromatography column, and then the elution peak of the antibody monomer eluted first is collected by gradient elution in which the salt concentration of the eluate is gradually increased. A method for purifying antibody monomers is disclosed.
また、近年、効率的に抗体を夾雑物から分離精製するために、複数のリガンドを有する混合モードの樹脂を充填したクロマトグラフィーカラムを用いることが、広く検討されている。このようなクロマトグラフィーカラムに用いられるリガンドとしては、ミックスモードリガンド又はマルチモーダルリガンドと呼ばれるリガンドが用いられている。このようなリガンドを用いるクロマトグラフィーにおいては、電荷と疎水性の複数の相互作用の差を利用するため、より高精度な分離が可能になるばかりでなく、複数の夾雑物を同時にかつ有効に除去可能になることが期待される。 In recent years, in order to efficiently separate and purify antibodies from impurities, it has been widely studied to use a chromatography column packed with a mixed mode resin having a plurality of ligands. As a ligand used in such a chromatography column, a ligand called a mixed mode ligand or a multimodal ligand is used. Chromatography using such ligands uses the difference between multiple interactions between charge and hydrophobicity, allowing not only more accurate separation but also more effective removal of multiple contaminants simultaneously. It is expected to be possible.
例えば、特許文献３には、アニオン交換基と疎水性基よりなるマルチモーダルリガンドのクロマトグラフィーを用いたフロースルーモードにより、夾雑物を吸着して抗体モノマーを回収する方法、特に遊離したプロテインＡリガンドと抗体モノマーよりなる凝集体を吸着除去する方法が記載されている。特許文献４には、四級アンモニウム基、水素結合基及び疎水性基を有するマルチモーダルのクロマトグラフィーを用いて、ＤＮＡ、ウィルス、エンドトキシン、凝集体、ＨＣＰなどの殆どの夾雑物を吸着し、抗体モノマーをフロースルーにより回収する方法が記載されており、特にＨＣＰはほぼ完全に除去されることが記載されている。特許文献５には、メルカプト基及び芳香族ピリジン環を有するミックスモードのクロマトグラフィーを用いて、抗体モノマーのみを吸着し、凝集体を非吸着画分として除去することにより、結合モードで抗体モノマーを精製する方法が記載されている。 For example, Patent Document 3 discloses a method for recovering antibody monomers by adsorbing impurities by a flow-through mode using chromatography of a multimodal ligand comprising an anion exchange group and a hydrophobic group, particularly a free protein A ligand. And a method for adsorbing and removing aggregates composed of antibody monomers. Patent Document 4 adsorbs most contaminants such as DNA, viruses, endotoxins, aggregates, and HCPs by using multimodal chromatography having a quaternary ammonium group, a hydrogen bonding group, and a hydrophobic group. A method for recovering the monomer by flow-through is described, and in particular, it is described that HCP is almost completely removed. In Patent Document 5, by using mixed mode chromatography having a mercapto group and an aromatic pyridine ring, only the antibody monomer is adsorbed, and the aggregate is removed as a non-adsorbed fraction. A method of purification is described.
凝集体を選択的にカラムに吸着させ、より有効に凝集体を除去することを目的とした、フロースルーモードで抗体モノマーを精製回収する方法として、特許文献６には、ヒドロキシアパタイトのクロマトグラフィーを適用した例が記載されている。 As a method for purifying and recovering antibody monomers in the flow-through mode for the purpose of selectively adsorbing the aggregates to the column and removing the aggregates more effectively, Patent Document 6 discloses a chromatography of hydroxyapatite. An applied example is described.
上述のとおり、細胞培養液中の多種多様な夾雑物から抗体を高精度で分離する方法は、数多く提案され、報告されている。しかしながら、全ての夾雑物を有効かつ迅速に除去して抗体を精製することは、現状でも困難であり、特にアフィニティクロマトグラフィー後の中間精製において、微量に残存するＨＣＰ、凝集体などを医薬品として要求されるレベルにまで除去し、高精度な抗体モノマーを回収することは、細胞培養液中の目的タンパク質濃度が大幅に向上しつつある状況下、さらに困難になることが懸念される。 As described above, many methods have been proposed and reported for separating antibodies with high accuracy from a wide variety of contaminants in cell culture media. However, it is still difficult to purify antibodies by removing all contaminants effectively and quickly. Particularly, in the intermediate purification after affinity chromatography, trace amounts of HCP, aggregates, etc. are required as pharmaceutical products. It is feared that it is more difficult to recover the highly accurate antibody monomer in a situation where the concentration of the target protein in the cell culture medium is greatly improved.
特許文献１及び２に開示された、アニオン交換クロマトグラフィーカラムを用いた方法では、凝集体の除去のために極僅かな電荷相互作用の相違を用いるため、非常に精密な条件の制御と分解能が要求され、高流速で通液することによる迅速な処理は困難である。 In the method using an anion exchange chromatography column disclosed in Patent Documents 1 and 2, a very slight difference in charge interaction is used for the removal of aggregates. It is required and rapid processing by passing liquid at a high flow rate is difficult.
特許文献３及び４に開示されたマルチモーダルのリガンドについても、同様にクロマトグラフィーカラムに用いるビーズのみをリガンドの固定対象としており、高流速で通液することが困難である。また、特許文献５及び６に開示されたミックスドモードのリガンドについても、クロマトグラフィーカラムに用いるビーズのみをリガンドの固定対象としており、高流速で通液することが困難である。これは、クロマトグラフィーに用いるビーズは多孔体粒子であり、本質的に溶液が拡散によって多孔体の細孔内に侵入することによってのみ、タンパク質とリガンドとの接触が行われるためである。そのため、通常ビーズによるカラムクロマトグラフィーの場合、有効に吸着がなされるための通液流速は１００Ｖ／ｈ（１時間あたりカラム体積の１００倍の溶液を通液）程度である。 Similarly, for the multimodal ligands disclosed in Patent Documents 3 and 4, only beads used in the chromatography column are targeted for fixation of the ligand, and it is difficult to pass the solution at a high flow rate. In addition, with the mixed mode ligands disclosed in Patent Documents 5 and 6, only the beads used in the chromatography column are intended for ligand fixation, and it is difficult to pass the solution at a high flow rate. This is because the beads used in the chromatography are porous particles, and the protein and the ligand are contacted essentially only by the solution entering the pores of the porous body by diffusion. For this reason, in the case of column chromatography using ordinary beads, the flow rate for effective adsorption is about 100 V / h (100 times the volume of the column per hour).
全ての夾雑物を有効かつ迅速に除去して抗体を精製することの困難さは、フロースルーモード並びに結合モードの両方において、有効に抗体を分離精製するための有効なｐＨ、塩濃度、溶液組成などの条件、すなわちプロセスウィンドウが狭く、安定で汎用的な精製条件を決定することが容易ではないことにも起因する。この状況はより効率的に夾雑物を除去することを目的とした混合モードリガンドのクロマトグラフィーにおいても同様である。 The difficulty of purifying antibodies by effectively and quickly removing all contaminants is effective pH, salt concentration, solution composition to effectively separate and purify antibodies in both flow-through mode and binding mode. This is also due to the fact that the process window is narrow and it is not easy to determine stable and versatile purification conditions. This situation is similar in mixed-mode ligand chromatography aimed at more efficiently removing contaminants.
加えて、中間精製において夾雑物を除去する工程は、高精度が要求されるために、高流速処理が可能な吸着膜ではなく、分解能が高いクロマトグラフィーカラムによるプロセスが指向される。特にＨＣＰのような、アフィニティグロマトグラフィーにより低濃度にまで除去された状態から、さらに除去することが必要とされる夾雑物の除去においては、なおさらプロセスウィンドウが狭いために条件の設定がより困難となり、さらに凝集体の除去については抗体モノマーと近似した相互作用の性質のために特に条件の設定が困難であり、これまで吸着膜などを用いた迅速な精製は不可能であった。このため、これまでに多孔質膜を用いて抗体モノマーから夾雑物を除去する方法については報告されていない。 In addition, since the process of removing impurities in the intermediate purification requires high accuracy, a process using a chromatographic column with high resolution is directed to an adsorption membrane capable of high flow rate processing. Especially in the removal of impurities that need to be further removed from a state that has been removed to a low concentration by affinity chromatography, such as HCP, it is more difficult to set conditions due to the narrow process window. Furthermore, for the removal of aggregates, it is difficult to set conditions especially due to the nature of interaction similar to antibody monomers, and rapid purification using an adsorption membrane or the like has been impossible so far. For this reason, no method has been reported so far to remove impurities from antibody monomers using a porous membrane.
かかる状況に鑑み、本発明の解決しようとする課題は、高濃度の抗体モノマー及び高濃度の夾雑物を含む溶液から、簡便で高速処理が可能でプロセスウィンドウが広い方法で、ＨＣＰ、凝集体などの夾雑物を有効にかつ迅速に除去することのできる、抗体モノマーの精製方法を提供することである。 In view of such a situation, the problem to be solved by the present invention is that a solution containing a high concentration of antibody monomer and a high concentration of contaminants can be easily and rapidly processed and has a wide process window, such as HCP and aggregates. It is an object of the present invention to provide a method for purifying antibody monomers, which can effectively and rapidly remove impurities.
本発明者らは、上記の課題を解決するために鋭意検討した結果、アニオン交換基が表面に固定されている多孔膜を用いることが、凝集体をはじめとするこれまで困難であった夾雑物の除去に有効であることを見出し、本発明を完成した。 As a result of intensive studies to solve the above problems, the present inventors have found that it has been difficult until now to use a porous membrane having an anion exchange group fixed on the surface, including aggregates. As a result, the present invention was completed.
すなわち、本発明は、抗体モノマー及び夾雑物を含む混合液からの抗体モノマーの精製方法であって、アニオン交換基が固定された多孔膜に、混合液を通液し、夾雑物及び／又は抗体モノマーを多孔膜に吸着させる工程を含む、抗体モノマーの精製方法に関する。
本発明は、また、前記多孔膜が、アニオン交換基がグラフト鎖を介して固定された多孔膜であって、各グラフト鎖は1以上の側鎖を有し、前記側鎖がアニオン交換基を有する多孔膜である、前記の抗体モノマーの精製方法に関する。
本発明は、また、前記グラフト鎖が、グリシジルメタクリレートの重合体を含む、前記の抗体モノマーの精製方法に関する。
本発明は、また、前記アニオン交換基がジエチルアミノ基である、前記の抗体モノマーの精製方法に関する。
本発明は、また、前記混合液の、ｐＨが６〜９及び塩濃度が０Ｍ〜０．２Ｍであるか、又は、ｐＨが４〜７及び塩濃度が０．０５Ｍ〜２Ｍであり、前記多孔膜に吸着させる工程において、夾雑物を多孔膜に吸着させる、前記の抗体モノマーの精製方法に関する。
本発明は、また、前記抗体モノマーの等電点が７．５以上である、前記の抗体モノマーの精製方法に関する。
本発明は、また、前記抗体モノマーの等電点が７．５未満であり、前記混合液のｐＨが６〜９及び塩濃度が０Ｍ〜０．１Ｍであり、前記多孔膜に吸着させる工程において、抗体モノマーを多孔膜に吸着させ、さらにその後、ｐＨが４〜９及び塩濃度が０．１Ｍ〜２Ｍの溶出液を前記多孔膜に通液し、前記多孔膜に吸着した抗体モノマーを溶出回収する工程を含む、前記の抗体モノマーの精製方法に関する。
本発明は、また、前記夾雑物が、抗体モノマーの凝集体である、前記の抗体モノマーの精製方法に関する。
That is, the present invention is a method for purifying an antibody monomer from a mixed solution containing an antibody monomer and contaminants, wherein the mixed solution is passed through a porous membrane on which an anion exchange group is fixed, and the contaminants and / or antibodies. The present invention relates to a method for purifying antibody monomers, comprising a step of adsorbing monomers on a porous membrane.
In the present invention, the porous membrane is a porous membrane in which an anion exchange group is fixed via a graft chain, each graft chain has one or more side chains, and the side chain has an anion exchange group. It is related with the purification method of the said antibody monomer which is a porous membrane which has.
The present invention also relates to the method for purifying the antibody monomer, wherein the graft chain contains a polymer of glycidyl methacrylate.
The present invention also relates to the method for purifying the antibody monomer, wherein the anion exchange group is a diethylamino group.
In the present invention, the mixed solution may have a pH of 6 to 9 and a salt concentration of 0 M to 0.2 M, or a pH of 4 to 7 and a salt concentration of 0.05 M to 2 M. The present invention relates to the method for purifying antibody monomers, wherein in the step of adsorbing to a membrane, impurities are adsorbed to a porous membrane.
The present invention also relates to a method for purifying the antibody monomer, wherein the antibody monomer has an isoelectric point of 7.5 or more.
In the process of the present invention, the isoelectric point of the antibody monomer is less than 7.5, the pH of the mixed solution is 6-9 and the salt concentration is 0M-0.1M, and the porous membrane is adsorbed. The antibody monomer is adsorbed on the porous membrane, and then the eluate having a pH of 4-9 and a salt concentration of 0.1M-2M is passed through the porous membrane, and the antibody monomer adsorbed on the porous membrane is eluted and recovered. And a method for purifying the antibody monomer.
The present invention also relates to the method for purifying the antibody monomer, wherein the contaminant is an aggregate of the antibody monomer.
本発明のアニオン交換基が固定された多孔膜を用いた抗体モノマーの精製方法によれば、高濃度の抗体モノマー及び高濃度の夾雑物を含む溶液についても、凝集体を含む夾雑物の除去を簡便で高速処理が可能でプロセスウィンドウが広い方法で行うことができ、抗体モノマーの精製を有効かつ迅速に実施することができる。 According to the method for purifying an antibody monomer using a porous membrane having an anion exchange group immobilized thereon according to the present invention, impurities including aggregates can be removed from a solution containing a high concentration of antibody monomer and a high concentration of contaminants. It can be carried out by a method that is simple and capable of high-speed processing and has a wide process window, and can purify antibody monomers effectively and rapidly.
以下、本発明を実施するための形態（以下、「本実施の形態」という。）について詳細に説明する。なお、本発明は、以下の本実施の形態に限定されるものではなく、その要旨の範囲内で種々変形して実施することができる。 Hereinafter, a 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.
本実施の形態は、アニオン交換基が固定された多孔膜を用いて、抗体モノマーと夾雑物を含む混合液のろ過を行って、混合液中の夾雑物及び／又は抗体モノマーを該多孔膜に吸着させる工程を含む、抗体モノマーの精製方法に関する。 In the present embodiment, a mixed liquid containing an antibody monomer and impurities is filtered using a porous film on which an anion exchange group is fixed, and impurities and / or antibody monomers in the mixed liquid are applied to the porous film. The present invention relates to a method for purifying antibody monomers, comprising a step of adsorbing.
本実施の形態における「抗体」とは、生体内に侵入した抗原と抗原抗体反応を起こし、抗原に対して免疫性を生体に与えるタンパク質の総称であり、具体的には、モノクローナル抗体、ポリクローナル抗体、ヒト化抗体、ヒト抗体及び免疫グロブリンなどを指す。特に本実施の形態における精製方法が、抗体医薬の製造工程で用い得る方法であるという観点からは、上記の抗体のうち、抗体医薬となり得る抗体が好ましい場合もある。本実施の形態における「抗体モノマー」とは、上記の抗体のうち、単量体で存在するものを指す。 “Antibody” in the present embodiment is a general term for proteins that cause an antigen-antibody reaction with an antigen that has entered the living body and immunize the living body with respect to the antigen. Specifically, monoclonal antibodies, polyclonal antibodies , Humanized antibody, human antibody, immunoglobulin and the like. In particular, from the viewpoint that the purification method in the present embodiment is a method that can be used in the production process of an antibody drug, among the above antibodies, an antibody that can be an antibody drug may be preferable. “Antibody monomer” in the present embodiment refers to the above-mentioned antibody that exists as a monomer.
本実施の形態における「夾雑物」とは、抗体モノマーを精製しようとする対象溶液（混合液）中に含まれる、抗体モノマー以外の不純物を指し、例えば、抗体を細胞培養により産生させる際に培養槽内で産生される目的抗体モノマー以外の不純物が挙げられ、より具体的には、抗体モノマーの凝集体、誤って折りたたまれた抗体タンパクの種、ＨＣＰ、エンドトキシン、ＤＮＡ、プロテアーゼ、遊離プロテインＡ、ウィルス及びバクテリアなどが挙げられる。 The “contaminants” in the present embodiment refers to impurities other than antibody monomers contained in a target solution (mixed solution) for purifying antibody monomers. For example, culture is performed when antibodies are produced by cell culture. Impurities other than the target antibody monomer produced in the tank are mentioned. More specifically, antibody monomer aggregates, misfolded antibody protein species, HCP, endotoxin, DNA, protease, free protein A, Examples include viruses and bacteria.
本実施の形態における「凝集体」（「抗体モノマーの凝集体」ということもある）とは、１種又は複数種の抗体モノマー同士の複合体、又は抗体モノマーと他のタンパク質等の化合物との複合体を指し、例えば、抗体モノマーの２量体又は３量体などの多量体、遊離したプロテインＡと抗体との複合体などを含む。 The “aggregate” in this embodiment (sometimes referred to as “aggregate of antibody monomers”) is a complex of one or more types of antibody monomers, or a combination of an antibody monomer and another compound such as a protein. It refers to a complex, and includes, for example, a multimer such as a dimer or trimer of antibody monomers, a complex of free protein A and an antibody, and the like.
本実施の形態における「抗体モノマー及び夾雑物を含む混合液」は、上記の抗体モノマー及び夾雑物を含有する溶液（又は含有する可能性のある溶液）であれば特に制限されず、本実施の形態の抗体モノマーの精製方法により抗体モノマーを精製しようとする対象溶液である。例えば、抗体の産生において用いられる細胞培養液又はその除濁液、あるいはそれらのダウンストリーム工程でのクロマトグラフィー工程中又は工程後の部分精製液等が挙げられる。これらの中でも、ダウンストリーム工程における、プロテインＡに代表されるアフィニティクロマトグラフィー工程前の除濁液、又はアフィニティクロマトグラフィー工程後の部分的に精製された溶液が、溶液が澄明であること、また夾雑物が部分的に除去されていることで後段の精製工程における負荷がより低減されることから、特に好適である。抗体モノマー及び夾雑物を含む混合液中の、抗体モノマー及び夾雑物の濃度は、特に限定されないが、本実施の形態における抗体モノマーの精製方法が、特に高濃度の抗体モノマー及び夾雑物を含有する混合液に対しても効率よく実施することができるという観点からは、例えば、混合液は、抗体モノマー及び夾雑物を合わせて２ｇ／L含んでいてもよく、抗体モノマーを５ｇ／Ｌ以上、夾雑物を１ｇ／Ｌ以上それぞれ含んでいてもよい。 The “mixed solution containing antibody monomers and contaminants” in the present embodiment is not particularly limited as long as it is a solution (or a solution that may be contained) containing the above antibody monomers and contaminants. This is a target solution to be purified by a method for purifying antibody monomers in the form. Examples thereof include a cell culture solution used for antibody production or a turbid solution thereof, or a partially purified solution during or after the chromatography step in the downstream step thereof. Among these, the turbid solution before the affinity chromatography step represented by protein A in the downstream step, or the partially purified solution after the affinity chromatography step is clear and contaminated. It is particularly suitable because the load in the subsequent purification step is further reduced by removing the substances partially. The concentration of the antibody monomer and the contaminant in the mixed solution containing the antibody monomer and the contaminant is not particularly limited, but the method for purifying the antibody monomer in the present embodiment particularly includes a high concentration of the antibody monomer and the contaminant. From the viewpoint that the mixture can be efficiently carried out with respect to the mixed solution, for example, the mixed solution may contain 2 g / L of the antibody monomer and impurities together, and the antibody monomer may contain 5 g / L or more. Each product may contain 1 g / L or more.
本実施の形態において用いられる「アニオン交換基が固定された多孔膜」とは、基材となる多孔膜の多孔質体及びその細孔の表面にアニオン交換基が固定されている多孔膜であり、特に多孔膜への抗体モノマー及び／又は夾雑物の吸着性を高めるという観点からは、アニオン交換基が表面に固定された多孔膜であることが好ましい。 The “porous membrane with anion exchange groups fixed” used in the present embodiment is a porous body of a porous membrane as a base material and a porous membrane with anion exchange groups fixed on the surface of the pores. In particular, from the viewpoint of enhancing the adsorptivity of antibody monomers and / or contaminants to the porous membrane, a porous membrane having an anion exchange group immobilized on the surface is preferable.
多孔膜の基材は、特に限定はされないが、基材が疎水性であることで、タンパク質と基材の間に疎水性相互作用が生じ得ること、また機械的性質を保持するという観点から、ポリオレフィン系重合体から構成されていることが好ましい。ポリオレフィン系重合体の例としては、例えば、エチレン、プロピレン、ブチレン及びフッ化ビニリデンなどのオレフィンの単独重合体、前記オレフィンの２種以上の共重合体、又は１種もしくは２種以上の前記オレフィンとパーハロゲン化オレフィンとの共重合体などが挙げられる。これらの重合体の２種以上の混合物であってもよい。パーハロゲン化オレフィンの例としては、テトラフルオロエチレン及びクロロトリフルオロエチレンなどが挙げられる。 The base material of the porous membrane is not particularly limited, but from the viewpoint that the base material is hydrophobic, a hydrophobic interaction can occur between the protein and the base material, and mechanical properties are maintained. It is preferably composed of a polyolefin polymer. Examples of polyolefin polymers include, for example, homopolymers of olefins such as ethylene, propylene, butylene and vinylidene fluoride, two or more types of copolymers of the olefins, or one or more types of the olefins. Examples thereof include copolymers with perhalogenated olefins. A mixture of two or more of these polymers may be used. Examples of perhalogenated olefins include tetrafluoroethylene and chlorotrifluoroethylene.
これらの中でも、疎水性と機械的強度に特に優れ、かつ高い吸着容量が得られる素材であるという観点から、多孔膜の基材として、ポリエチレン又はポリフッ化ビニリデンが好ましく、ポリエチレンがより好ましい。 Among these, polyethylene or polyvinylidene fluoride is preferable as the base material of the porous film, and polyethylene is more preferable from the viewpoint that it is a material that is particularly excellent in hydrophobicity and mechanical strength and that can obtain a high adsorption capacity.
本実施の形態において、アニオン交換基は、液中で負に帯電したタンパク質等を吸着することができればよく、例えば、特に限定されないが、ジエチルアミノ基（ＤＥＡ、Ｅｔ2Ｎ−）、四級アンモニウム基（Ｑ、Ｒ3Ｎ+−）、四級アミノエチル基（ＱＡＥ、Ｒ3Ｎ+−（ＣＨ2）2−）、ジエチルアミノエチル基（ＤＥＡＥ、Ｅｔ2Ｎ−（ＣＨ2）2−）、ジエチルアミノプロピル基（ＤＥＡＰ、Ｅｔ2Ｎ−（ＣＨ2）3−）などが挙げられる。ここで、Ｒは、特に限定されず、同一のＮに結合するＲが同一又は異なっていてもよく、好適には、アルキル基、フェニル基、アラルキル基などの炭化水素基を表す。四級アンモニウム基としては、例えば、トリメチルアミノ基（トリメチルアンモニウム基、Ｍｅ3Ｎ+−）などが挙げられる。多孔膜への化学的な固定が容易であり、高い吸着容量が得られるという観点からは、アニオン交換基としてはＤＥＡ及びＱが好ましく、ＤＥＡがより好ましい。 In the present embodiment, the anion exchange group is not particularly limited as long as it can adsorb a negatively charged protein or the like in the liquid. For example, a diethylamino group (DEA, Et 2 N—), a quaternary ammonium group (Q, R 3 N + - ), quaternary aminoethyl group (QAE, R 3 N + - (CH 2) 2 -), diethylaminoethyl group (DEAE, Et 2 N- (CH 2) 2 -), diethylamino And a propyl group (DEAP, Et 2 N— (CH 2 ) 3 —). Here, R is not particularly limited, and R bonded to the same N may be the same or different, and preferably represents a hydrocarbon group such as an alkyl group, a phenyl group, or an aralkyl group. Examples of the quaternary ammonium group include a trimethylamino group (trimethylammonium group, Me 3 N + -). From the viewpoint of easy chemical fixation to the porous membrane and high adsorption capacity, DEA and Q are preferred as the anion exchange group, and DEA is more preferred.
アニオン交換基の多孔膜への固定の方法は、特に制限されないが、一般に、エポキシ又はアミンのような反応性の高い官能基を多孔膜の基材表面に導入し、その後、該官能基にアニオン交換基を有する化合物を結合させる方法によって行うことができる。 The method for immobilizing the anion exchange group on the porous membrane is not particularly limited, but generally, a highly reactive functional group such as epoxy or amine is introduced on the substrate surface of the porous membrane, and then the anion is added to the functional group. It can be performed by a method of bonding a compound having an exchange group.
上記のアニオン交換基が表面に固定された多孔膜のうち、例えば特開平２−１３２１３２号公報又はJournal of Chromatography A, 689(1995) 211-218に記載されている多孔膜は、多孔質体及びその細孔の表面にグラフト鎖が固定され、さらに該グラフト鎖が有する側鎖にアニオン交換基が固定されていることを特徴とする。このような多孔膜は、基材に固定された各グラフト鎖が１以上の側鎖を有し、その側鎖にアニオン交換基が１以上固定された構造を有するため、アニオン交換基は細孔空間内に立体的に分布する。そのため、電荷点を有するタンパク質などに対して吸着点の数が多く、そのため吸着量が増加するとともに、相互作用の小さなタンパク質などの吸着性が高いことを本発明者は見出した。 Among the porous membranes in which the anion exchange groups are fixed on the surface, for example, the porous membrane described in JP-A-2-132132 or Journal of Chromatography A, 689 (1995) 211-218 is a porous body and A graft chain is fixed to the surface of the pore, and an anion exchange group is fixed to a side chain of the graft chain. Such a porous membrane has a structure in which each graft chain fixed to the substrate has one or more side chains, and one or more anion exchange groups are fixed to the side chains. It is distributed three-dimensionally in space. Therefore, the present inventor has found that the number of adsorption points is large with respect to a protein having a charge point and the like, so that the adsorption amount is increased and the adsorption property of a protein having a small interaction is high.
本実施の形態において、「グラフト鎖」とは、上記の多孔質の基材の内部又は表面に結合した、基材と同種又は異種の分子鎖である。多孔膜の表面及び細孔に、グラフト鎖を導入し、さらに、該グラフト鎖にアニオン交換基を固定する方法としては、限定されるものではないが、例えば、特開平２−１３２１３２号公報に開示される方法が挙げられる。 In the present embodiment, the “graft chain” is a molecular chain of the same kind or different kind from the base material bonded to the inside or the surface of the porous base material. A method for introducing a graft chain into the surface and pores of the porous membrane and further immobilizing an anion exchange group on the graft chain is not limited. For example, it is disclosed in JP-A-2-132132. The method to be mentioned is mentioned.
グラフト鎖としては、例えば、グリシジルメタクリレート、酢酸ビニル、ヒドロキシプロピルアセテート又はこれらのいずれか２種以上の重合体を含む分子鎖が挙げられるが、アニオン交換基を導入しやすいことから、グリシジルメタクリレート又は酢酸ビニルの重合体が好ましく、グリシジルメタクリレートの重合体がより好ましい。多孔膜に対するグラフト鎖の結合率（グラフト率）は、例えば後述の実施例等に記載の手法を用いて測定することができ、より高い吸着容量及び力学的に安定な強度をともに確保するという観点から、好ましくは２０％〜２００％、より好ましくは２０％〜１５０％、更に好ましくは３０％〜７０％である。 Examples of the graft chain include glycidyl methacrylate, vinyl acetate, hydroxypropyl acetate, or a molecular chain containing any two or more of these polymers, but glycidyl methacrylate or acetic acid is easy to introduce an anion exchange group. Vinyl polymers are preferred, and glycidyl methacrylate polymers are more preferred. The rate of graft chain binding to the porous membrane (graft rate) can be measured, for example, using the method described in the examples described later, and the viewpoint of ensuring both higher adsorption capacity and mechanically stable strength. Therefore, it is preferably 20% to 200%, more preferably 20% to 150%, still more preferably 30% to 70%.
アニオン交換基のグラフト鎖への固定の例として、グラフト鎖がグリシジルメタクリレートの重合体である場合、この重合体が有するエポキシ基を開環し、ジエチルアミンなどのアミン及びジエチルアンモニウム又はトリメチルアンモニウムなどのアンモニウム塩を付加することにより、アニオン交換基をグラフト鎖に固定することができる。グラフト鎖に対するアニオン交換基の置換率は、後述の実施例等に記載の手法を用いて測定することができ、より高い吸着容量を得るという観点から、好ましくは２０％〜１００％、より好ましくは５０％〜１００％、更に好ましくは７０％〜１００％である。 As an example of fixing an anion exchange group to a graft chain, when the graft chain is a polymer of glycidyl methacrylate, the epoxy group of this polymer is opened, and an amine such as diethylamine and ammonium such as diethylammonium or trimethylammonium By adding a salt, the anion exchange group can be fixed to the graft chain. The substitution rate of the anion exchange group with respect to the graft chain can be measured using the method described in Examples and the like described below, and from the viewpoint of obtaining a higher adsorption capacity, preferably 20% to 100%, more preferably It is 50% to 100%, more preferably 70% to 100%.
多孔膜の最大細孔径は、溶液中の抗体モノマー及び／又は夾雑物を有効に吸着し、かつ高い透過流速を得るために、上記のアニオン交換基の固定及びグラフト鎖の導入前の状態で、好ましくは０．１μｍ〜１．０μｍであり、より好ましくは０．１μｍ〜０．８μｍであり、さらに好ましくは０．２μｍ〜０．６μｍである。 In order to effectively adsorb antibody monomers and / or contaminants in the solution and obtain a high permeation flow rate, the maximum pore diameter of the porous membrane is as described above before the fixation of the anion exchange group and the introduction of the graft chain. Preferably it is 0.1 micrometer-1.0 micrometer, More preferably, it is 0.1 micrometer-0.8 micrometer, More preferably, it is 0.2 micrometer-0.6 micrometer.
多孔膜中の細孔の占める体積である空孔率は、多孔膜の形状を保持しかつ通液時の圧損が実用上問題のない程度であれば、特に限定されないが、好ましくは５％〜９９％であり、より好ましくは１０％〜９５％であり、さらに好ましくは３０％〜９０％である。 The porosity, which is the volume occupied by the pores in the porous membrane, is not particularly limited as long as the shape of the porous membrane is maintained and the pressure loss during liquid passage is practically acceptable, but preferably 5% to 99%, more preferably 10% to 95%, still more preferably 30% to 90%.
上記細孔径及び空孔率の測定は、Ｍａｒｃｅｌ Ｍｕｌｄｅｒ著「膜技術」（株式会社アイピーシー）などに記載されているような、当業者にとって公知の方法により行うことができる。例えば、電子顕微鏡による観察、バブルポイント法、水銀圧入法、透過率法などの測定方法が挙げられる。例えば最大細孔径の測定は、後述の実施例等に記載のバブルポイント法による評価を適切に用いることができる。 The pore diameter and porosity can be measured by methods known to those skilled in the art, such as described in “Membrane Technology” by Marcel Mulder (IPC Co., Ltd.). Examples thereof include measurement methods such as observation with an electron microscope, bubble point method, mercury intrusion method, and transmittance method. For example, the measurement by the bubble point method described in the below-mentioned Examples etc. can be used appropriately for the measurement of the maximum pore diameter.
多孔膜の形態は、溶液を通液することが可能な形態であれば特に限定されず、例えば、平膜、不織布、中空糸膜、モノリス、キャピラリー、円板又は円筒状などが挙げられる。これらの形態の中でも、製造のし易さ、スケールアップ性、モジュール成型した際の膜のパッキング性などの観点からは、中空糸膜が好ましい。 The form of the porous membrane is not particularly limited as long as the solution can be passed therethrough, and examples thereof include a flat membrane, a nonwoven fabric, a hollow fiber membrane, a monolith, a capillary, a disc, and a cylindrical shape. Among these forms, a hollow fiber membrane is preferable from the viewpoints of ease of production, scale-up property, and packing property of the membrane when it is molded into a module.
本実施の形態において、中空糸多孔膜とは、中空部分を有する円筒状又は繊維状の多孔膜であり、中空糸の内層と外層が貫通孔である細孔によって連続しており、その細孔によって内層から外層、あるいは外層から内層に、液体又は気体が透過する性質を有する多孔体を意味する。中空糸の外径及び内径は、物理的に多孔膜が形状を保持することができ、かつモジュール成型可能であれば、特に限定されない。 In the present embodiment, the hollow fiber porous membrane is a cylindrical or fibrous porous membrane having a hollow portion, and the inner layer and outer layer of the hollow fiber are continuous by pores that are through holes, and the pores Means a porous body having a property of allowing liquid or gas to permeate from the inner layer to the outer layer or from the outer layer to the inner layer. The outer diameter and inner diameter of the hollow fiber are not particularly limited as long as the porous membrane can physically hold the shape and can be molded into a module.
上記の本実施の形態の多孔膜に、抗体モノマー及び夾雑物を含む混合液を通液し、夾雑物及び／又は抗体モノマーを多孔膜に吸着させる工程を、以下に説明する。 A process of passing the mixed solution containing the antibody monomer and impurities through the porous film of the present embodiment and adsorbing the impurities and / or antibody monomer to the porous film will be described below.
本実施の形態による典型的な抗体モノマーの精製方法の例としては：
混合液中の夾雑物を多孔膜に吸着させる場合には、吸着により夾雑物を混合液から除去し、透過液を抗体モノマーの精製液として回収し；
混合液中の抗体モノマーを多孔膜に吸着させる場合には、まず非吸着の夾雑物を透過液として回収して除去し、その後溶出液を通液して多孔膜に吸着した抗体モノマーを溶出し、抗体モノマーの精製液として回収し；
混合液中の夾雑物及び抗体モノマーの両方を多孔膜に吸着させる場合には、例えば、まず大部分の非吸着の夾雑物を透過液に回収して除去し、その後吸着した残りの夾雑物を保持したまま抗体モノマーのみを溶出する条件の溶出液を用いて抗体モノマーを溶出して回収する、あるいは抗体モノマーのみを保持したまま吸着した夾雑物を先に溶出して除去し、次いで抗体モノマーを溶出して回収する、ことにより抗体モノマーの精製液として回収する方法が挙げられる。
Examples of typical antibody monomer purification methods according to this embodiment include:
When the impurities in the mixed solution are adsorbed on the porous membrane, the impurities are removed from the mixed solution by adsorption, and the permeate is collected as a purified antibody monomer solution;
When adsorbing the antibody monomer in the mixed solution to the porous membrane, first remove the non-adsorbed impurities as permeate and remove it, and then pass the eluate through to elute the antibody monomer adsorbed on the porous membrane. Recovered as a purified antibody monomer solution;
When adsorbing both contaminants and antibody monomer in the mixed solution to the porous membrane, for example, first, most of the non-adsorbed impurities are collected and removed in the permeate, and then the remaining adsorbed impurities are removed. The antibody monomer is eluted and collected using an eluate that elutes only the antibody monomer while being retained, or the adsorbed contaminants are first eluted and removed while retaining only the antibody monomer. The method of recovering as a purified antibody monomer solution by elution and recovery.
抗体モノマーの等電点（ｐＩ）は通常、６．５〜８．５の範囲であり、一方ＨＣＰ、ＤＮＡ、ウィルスなどの夾雑物の多くのｐＩは６以下にあるため、上記の多孔膜に通液する溶液のｐＨ範囲及び塩濃度(電気伝導度)を好適に制御することによって、夾雑物の多くが多孔膜のアニオン交換基に吸着し、透過液を抗体モノマーの精製液として回収することができる。夾雑物のうち、凝集体のｐＩは抗体モノマーに近いかほぼ等しいが、抗体モノマーと凝集体の相違点は、凝集体は抗体モノマーの複合体であるため、ｐＩが抗体モノマーと同程度であっても、凝集体の方が一つの分子が有する電荷点の数が多い点である。このため、凝集体は僅かながら抗体モノマーよりも、アニオン交換基を有する担体に吸着しやすい性質を有し、溶液のｐＨ及び塩濃度を適切に調整することにより、凝集体を多孔膜のアニオン交換基に吸着させ、透過液を抗体モノマーの精製液として回収することができる。 The isoelectric point (pI) of antibody monomers is usually in the range of 6.5 to 8.5, while many pIs of contaminants such as HCP, DNA, and virus are 6 or less. By suitably controlling the pH range and salt concentration (electrical conductivity) of the solution to be passed, most of the impurities are adsorbed on the anion exchange group of the porous membrane, and the permeate is recovered as a purified antibody monomer solution. Can do. Among the contaminants, the pI of the aggregate is close to or nearly equal to that of the antibody monomer. However, the difference between the antibody monomer and the aggregate is that the aggregate is a complex of antibody monomers, so that the pI is about the same as that of the antibody monomer. However, the aggregate has a larger number of charge points in one molecule. For this reason, the aggregate is slightly more easily adsorbed to a carrier having an anion exchange group than the antibody monomer, and the aggregate is anion-exchanged in the porous membrane by appropriately adjusting the pH and salt concentration of the solution. The permeate can be recovered as a purified antibody monomer solution.
特に、アニオン交換基がグラフト鎖を介して多孔膜に固定された多孔膜（好ましくは各グラフト鎖が１以上の側鎖を有し、各側鎖がアニオン交換基を有する多孔膜）を用いる場合、多孔膜においてアニオン交換基が立体的に固定されていることになる。その結果、抗体モノマーや凝集体などのタンパク質は、電荷点において複数のアニオン交換基によって立体的に固定される。そのため、凝集体は抗体モノマーよりも、より強固に多孔膜に吸着され、抗体モノマーの精製を容易に行うことができる。 In particular, a porous membrane in which an anion exchange group is fixed to the porous membrane via a graft chain (preferably a porous membrane in which each graft chain has one or more side chains and each side chain has an anion exchange group) is used. In the porous membrane, the anion exchange group is sterically fixed. As a result, proteins such as antibody monomers and aggregates are sterically fixed by a plurality of anion exchange groups at the charge point. Therefore, the aggregate is more firmly adsorbed to the porous membrane than the antibody monomer, and the antibody monomer can be easily purified.
さらに、タンパク質は本質的に疎水性相互作用の性質も有するため、特に基材が疎水性の性質を有する多孔膜を用いることで、抗体モノマーの精製をより容易に行うことができると考えられる。 Furthermore, since proteins inherently have hydrophobic interaction properties, it is considered that antibody monomers can be purified more easily by using a porous membrane having a hydrophobic property as a base material.
混合液中の夾雑物を多孔膜に吸着させ、吸着により夾雑物を混合液から除去し、透過液を抗体モノマーの精製液として回収する場合、夾雑物（特に凝集体）の多孔膜への吸着性が抗体モノマーよりも顕著になる条件に混合液のｐＨ及び塩濃度を調整する。具体的には、ｐＨ６〜９及び塩濃度が０Ｍ〜０．２Ｍの範囲に混合液を調整する。 When adsorbing impurities in the mixed solution to the porous membrane, removing the impurities from the mixed solution by adsorption, and collecting the permeate as a purified antibody monomer solution, adsorption of impurities (especially aggregates) to the porous membrane The pH and salt concentration of the mixed solution are adjusted to conditions where the property becomes more remarkable than that of the antibody monomer. Specifically, the mixed solution is adjusted to a pH of 6 to 9 and a salt concentration of 0 M to 0.2 M.
塩濃度の調整に用いる塩としては、例えば、塩化ナトリウム、硫酸ナトリウム、酢酸ナトリウム、硫酸アンモニウムなどの他、クエン酸、リン酸又はグリシンの金属塩が挙げられるが、これらに限定されるものではない。また、ｐＨの調整は通常、簡便には塩酸又は水酸化ナトリウムの添加によって行うことができるが、これに限定されず、当業者に公知のｐＨ調整方法を適宜用いることができる。溶液のｐＨ及び塩濃度の測定は、例えば市販の測定機器を用いて、当業者に公知の手法を用いて行うことができる。 Examples of the salt used for adjusting the salt concentration include, but are not limited to, sodium chloride, sodium sulfate, sodium acetate, ammonium sulfate, and metal salts of citric acid, phosphoric acid, or glycine. Moreover, although pH adjustment can usually be performed simply by adding hydrochloric acid or sodium hydroxide, it is not limited to this, The pH adjustment method well-known to those skilled in the art can be used suitably. The pH and salt concentration of the solution can be measured by using a method known to those skilled in the art, for example, using a commercially available measuring instrument.
上記のｐＨ及び塩濃度の混合液を、本実施の形態における多孔膜に通液することにより、凝集体、ＨＣＰ、ＤＮＡ、エンドトキシン、ウィルスなどのｐＩが６以下の夾雑物が多孔膜に吸着される。混合液のｐＨ及び塩濃度を、精製対象とする抗体モノマーのｐＩに応じて調整することにより、夾雑物（特に凝集体）がより選択的に多孔膜に吸着され、抗体モノマーの精製を、より有効に行うことができる。一般的な抗体モノマーのｐＩは８近辺にあるという観点からは、混合液のｐＨ及び塩濃度は、好ましくはｐＨ６〜９及び塩濃度が０Ｍ〜０．２Ｍ、より好ましくはｐＨ７〜８．５及び塩濃度が０Ｍ〜０．１Ｍ、さらに好ましくはｐＨ７．５〜８．５及び塩濃度が０Ｍ〜０．０５Ｍである。具体的には、精製対象とする抗体モノマーによって異なるが、抗体モノマーの等電点が７．５以上、例えば７．５〜８．５の範囲にある場合、混合液のｐＨ及び塩濃度を上記の範囲とすれば、抗体モノマーの精製を有効に行い得る。 By passing the mixed solution having the above pH and salt concentration through the porous membrane in the present embodiment, contaminants such as aggregates, HCP, DNA, endotoxin, and viruses having a pI of 6 or less are adsorbed on the porous membrane. The By adjusting the pH and salt concentration of the mixed solution according to the pi of the antibody monomer to be purified, impurities (especially aggregates) are more selectively adsorbed to the porous membrane, and the purification of the antibody monomer is further improved. It can be done effectively. From the viewpoint that the pI of a general antibody monomer is around 8, the pH and salt concentration of the mixed solution are preferably pH 6-9 and the salt concentration 0 M to 0.2 M, more preferably pH 7 to 8.5 and The salt concentration is 0M to 0.1M, more preferably pH 7.5 to 8.5 and the salt concentration is 0M to 0.05M. Specifically, although depending on the antibody monomer to be purified, when the isoelectric point of the antibody monomer is 7.5 or more, for example, in the range of 7.5 to 8.5, the pH and salt concentration of the mixed solution are set as above. If it is in the range, it is possible to effectively purify the antibody monomer.
上記のｐＨ及び塩濃度の混合液のほか、ｐＨ４〜７及び塩濃度が０．０５Ｍ〜２Ｍの混合液においても、凝集体を含む夾雑物が選択的に多孔膜に吸着され、精製された抗体モノマーが透過液として回収されることを本発明者らは見出した。この場合、混合液のｐＨ及び塩濃度の好ましい範囲は、抗体モノマーの種類によって異なるが、好ましくはｐＨ４〜７及び塩濃度が０．０５Ｍ〜２Ｍ、より好ましくはｐＨ５〜７及び塩濃度が０．１Ｍ〜１Ｍ、さらに好ましくはｐＨ５．５〜６．５及び塩濃度が０．２Ｍ〜１Ｍである。なお、上記の混合液と同じｐＨ及び塩濃度を有する洗浄液で、混合液通液後の多孔膜を洗浄することで、抗体モノマーをより高収率で精製することができる。 In addition to the above-mentioned mixed solution of pH and salt concentration, in the mixed solution of pH 4 to 7 and salt concentration of 0.05M to 2M, a purified antibody in which contaminants including aggregates are selectively adsorbed on the porous membrane The inventors have found that the monomer is recovered as a permeate. In this case, the preferred range of pH and salt concentration of the mixed solution varies depending on the type of antibody monomer, but preferably pH 4 to 7 and salt concentration 0.05 M to 2 M, more preferably pH 5 to 7 and salt concentration 0. 1M to 1M, more preferably pH 5.5 to 6.5 and salt concentration is 0.2M to 1M. The antibody monomer can be purified at a higher yield by washing the porous membrane after passing through the mixture with a washing solution having the same pH and salt concentration as the above mixture.
混合液中の抗体モノマーを多孔膜に吸着させ、まず非吸着の夾雑物を透過液として回収して除去し、その後溶出液を通液して多孔膜に吸着した抗体モノマーを溶出し、抗体モノマーの精製液として回収する場合、抗体モノマーの多孔膜への吸着性が夾雑物（特に凝集体）よりも顕著になる条件に混合液のｐＨ及び塩濃度を調整する。特に抗体モノマーの等電点が７．５未満、例えば６．０〜７．５の範囲にある場合、この方法を用いて抗体モノマーの精製を有効に行い得る。具体的には、精製対象とする抗体モノマーによって異なるが、抗体モノマーの等電点が７．５未満、例えば６．０〜７．５の範囲にある場合、混合液のｐＨ及び塩濃度は、好ましくはｐＨ６〜９及び塩濃度が０Ｍ〜０．１Ｍであり、より好ましくはｐＨ７〜８．５及び塩濃度０Ｍ〜０．０５Ｍである。その後、多孔膜に吸着した抗体モノマーを溶出するための溶出液のｐＨ及び塩濃度は、ｐＨ４〜９及び塩濃度０．１Ｍ〜２Ｍが好ましく、ｐＨ４〜８及び塩濃度０．３Ｍ〜２Ｍがより好ましい。抗体モノマーを溶出する前に、抗体モノマー及び夾雑物を含む混合液と同じｐＨ及び塩濃度を有する洗浄液で、多孔膜を予め洗浄しておくと、溶出した際の抗体モノマーの精製度が向上するため好ましい。 The antibody monomer in the mixed solution is adsorbed on the porous membrane, and first, non-adsorbed impurities are collected and removed as a permeate, and then the eluate is passed through to elute the antibody monomer adsorbed on the porous membrane. In the case of recovering as a purified liquid, the pH and salt concentration of the mixed liquid are adjusted to conditions such that the adsorptivity of the antibody monomer to the porous membrane becomes more prominent than impurities (particularly aggregates). In particular, when the isoelectric point of the antibody monomer is less than 7.5, for example, in the range of 6.0 to 7.5, the antibody monomer can be effectively purified using this method. Specifically, although depending on the antibody monomer to be purified, when the isoelectric point of the antibody monomer is less than 7.5, for example, in the range of 6.0 to 7.5, the pH and salt concentration of the mixed solution are: Preferably, the pH is 6-9 and the salt concentration is 0M-0.1M, more preferably the pH is 7-8.5 and the salt concentration is 0M-0.05M. Thereafter, the pH and salt concentration of the eluate for eluting the antibody monomer adsorbed on the porous membrane are preferably pH 4-9 and salt concentration 0.1M-2M, more preferably pH 4-8 and salt concentration 0.3M-2M. preferable. Before the antibody monomer is eluted, if the porous membrane is washed in advance with a washing solution having the same pH and salt concentration as the mixture containing the antibody monomer and impurities, the purity of the antibody monomer at the time of elution is improved. Therefore, it is preferable.
このように、抗体モノマー及び夾雑物を含む混合液のｐＨ及び塩濃度を調整し、本実施の形態における多孔膜に通液することによって、凝集体を含む夾雑物を容易に除去し、抗体モノマーの精製を簡便に行うことができる。この際、通液する溶液と、多孔膜に固定されたアニオン交換基との接触は強制対流によってなされるため、カラムクロマトグラフィーの場合と比べて極めて早い通液流速、例えば、１０００Ｖ／ｈ又はそれ以上の流速でも抗体モノマーの精製を行うことができる。 In this way, by adjusting the pH and salt concentration of the mixed solution containing the antibody monomer and contaminants and passing through the porous membrane in the present embodiment, the contaminants containing aggregates can be easily removed, and the antibody monomer Can be easily purified. At this time, contact between the solution to be passed through and the anion exchange group fixed to the porous membrane is made by forced convection, so that the flow rate is much faster than that in the case of column chromatography, for example, 1000 V / h or more. The antibody monomer can be purified even at the above flow rate.
本実施の形態の方法及び多孔膜を用い、本明細書の記載を参照することにより、高濃度の抗体モノマー及び高濃度の夾雑物を含む溶液から、凝集体を含む夾雑物の除去を簡便で高速処理が可能でプロセスウィンドウが広い方法で行うことができ、有効かつ迅速に抗体モノマーを精製することができる。従って、精製された抗体モノマーを工業的に効率的に得ることが可能となる。 By using the method of the present embodiment and the porous membrane, and referring to the description of the present specification, it is possible to easily remove contaminants containing aggregates from a solution containing a high concentration of antibody monomer and a high concentration of contaminants. High-speed processing can be performed by a method having a wide process window, and antibody monomers can be purified effectively and rapidly. Therefore, it becomes possible to obtain the purified antibody monomer industrially efficiently.
以下、参考例、実施例及び比較例（本明細書中において、単に「実施例等」ともいう。）に基づいて本実施の形態をさらに具体的に説明するが、本発明の範囲は以下の実施例のみに限定されない。 Hereinafter, the present embodiment will be described more specifically based on reference examples, examples, and comparative examples (also simply referred to as “examples” in the present specification), but the scope of the present invention is as follows. It is not limited only to the examples.
［参考例１］ アニオン交換基が表面に固定された多孔膜モジュールの作成
（ｉ）中空糸多孔膜へのグラフト鎖の導入
外径３．０ｍｍ、内径２．０ｍｍ、空孔率７０％、下記（ｉｖ）に記載のバブルポイント法で測定した最大細孔径が０．３μｍのポリエチレン製中空糸多孔膜を密閉容器に入れて、容器内の空気を窒素で置換した。その後、容器の外側からドライアイスで冷却しながら、γ線２００ｋＧｙを照射し、ラジカルを発生させた。得られたラジカルを有するポリエチレン製中空糸多孔膜をガラス反応管に入れて、２００Ｐａ以下に減圧することにより、反応管内の酸素を除いた。ここに４０℃に調整したグリシジルメタクリレート（ＧＭＡ）３体積部、メタノール９７体積部よりなる反応液を、中空糸多孔膜の２０質量部に注入した後、１２分間密閉状態で静置してグラフト重合反応を施し、中空糸多孔膜にグラフト鎖を導入した。なお、ＧＭＡ及びメタノールよりなる反応液は予め窒素でバブリングして、反応液内の酸素を窒素置換した。
[Reference Example 1] Preparation of porous membrane module having anion exchange groups fixed on the surface (i) Introduction of graft chain into hollow fiber porous membrane Outer diameter 3.0 mm, inner diameter 2.0 mm, porosity 70%, A polyethylene hollow fiber porous membrane having a maximum pore diameter of 0.3 μm measured by the bubble point method described in (iv) was placed in a sealed container, and the air in the container was replaced with nitrogen. Thereafter, while cooling with dry ice from the outside of the container, γ rays 200 kGy were irradiated to generate radicals. The obtained polyethylene hollow fiber porous membrane having radicals was put in a glass reaction tube and the pressure in the reaction tube was reduced to 200 Pa or less to remove oxygen in the reaction tube. A reaction solution consisting of 3 parts by volume of glycidyl methacrylate (GMA) adjusted to 40 ° C. and 97 parts by volume of methanol was injected into 20 parts by mass of the hollow fiber porous membrane, and then left to stand for 12 minutes in a sealed state to perform graft polymerization. The reaction was performed to introduce graft chains into the hollow fiber porous membrane. The reaction solution composed of GMA and methanol was previously bubbled with nitrogen to replace oxygen in the reaction solution with nitrogen.
グラフト重合反応後、反応管内の反応液を捨てた。次いで、反応管内にジメチルスルホキシドを入れて中空糸多孔膜を洗浄することにより、残存したグリシジルメタクリレート、そのオリゴマー及び中空糸多孔膜に固定されなかったグラフト鎖を除去した。洗浄液を捨てた後、さらにジメチルスルホキシドを入れて２回洗浄を行った。次いでメタノールを用いて同様にして洗浄を３回行った。洗浄後の中空糸多孔膜を乾燥し、重量を測定したところ、中空糸多孔膜の重量はグラフト鎖導入前の１３８％であり、基材重量に対するグラフト鎖の重量の比として定義されるグラフト率は３８％であった。導入したグラフト鎖は、ＧＭＡが重合した分子鎖であり、その主鎖は（−ＣＨ2ＣＲＣＨ3−）の繰り返し構造である。ここでＲは側鎖であり、−ＣＯＯＣＨ2ＣＨＯＣＨ2の構造を有する。 After the graft polymerization reaction, the reaction solution in the reaction tube was discarded. Next, dimethyl sulfoxide was placed in the reaction tube to wash the hollow fiber porous membrane, thereby removing the remaining glycidyl methacrylate, its oligomer, and the graft chain not fixed to the hollow fiber porous membrane. After discarding the washing solution, dimethyl sulfoxide was further added and washing was performed twice. Subsequently, washing was performed three times in the same manner using methanol. The hollow fiber porous membrane after washing was dried and weighed. The weight of the hollow fiber porous membrane was 138% before graft chain introduction, and the graft ratio defined as the ratio of the weight of the graft chain to the weight of the base material Was 38%. The introduced graft chain is a molecular chain obtained by polymerizing GMA, and its main chain has a repeating structure of (—CH 2 CRCH 3 —). Here, R is a side chain and has a structure of —COOCH 2 CHOCH 2 .
このグラフト率から、下記式（Ｉ）を用いて、基材ポリエチレンの骨格単位であるＣＨ2基（分子量１４）のモル数に対する導入されたＧＭＡ（分子量１４２）のモル数は３．７５％であると算出された。
導入ＧＭＡのモル数％＝（グラフト率／１４２）／（１００／１４）×１００
・・・（Ｉ）
From this graft ratio, using the following formula (I), the number of moles of introduced GMA (molecular weight 142) with respect to the number of moles of CH 2 group (molecular weight 14) which is the skeleton unit of the base polyethylene is 3.75%. It was calculated that there was.
Number of moles of introduced GMA% = (graft ratio / 142) / (100/14) × 100
... (I)
固体ＮＭＲ法により、グラフト反応後の中空糸多孔膜中のポリエチレン骨格単位ＣＨ2基のモル数と、グラフト鎖を構成するＧＭＡに特有なエステル基（ＣＯＯ基）のモル数の比を測定した。測定は、グラフト反応後の中空糸多孔膜を凍結粉砕した粉末サンプル０．５ｇを用いて、Ｂｒｕｋｅｒ Ｂｉｏｓｐｉｎ社製ＤＳＸ４００を使用し、核種を13Ｃとして、Ｈｉｇｈ Ｐｏｗｅｒ Ｄｅｃｏｕｐｌｉｎｇ法（ＨＰＤＥＣ法）の定量モードにより、待ち時間１００ｓ、積算１０００回の条件で、室温下で行った。 The ratio of the number of moles of the polyethylene skeleton unit CH 2 group in the hollow fiber porous membrane after the graft reaction and the number of moles of ester groups (COO groups) unique to GMA constituting the graft chain was measured by solid-state NMR. For measurement, 0.5 g of a powder sample obtained by freeze-pulverizing the hollow fiber porous membrane after the graft reaction was used, DSX400 manufactured by Bruker Biospin was used, the nuclide was 13 C, and the quantitative mode of the High Power Decoupling method (HPDEC method) Thus, the measurement was performed at room temperature under a condition of a waiting time of 100 s and an accumulation of 1000 times.
得られたＮＭＲスペクトルのエステル基に対応するピーク面積と、ＣＨ2基に対応するピーク面積との比が、ＧＭＡとＣＨ2基のモル数の比に対応することから、測定結果よりＣＨ2基のモル数に対する導入されたＧＭＡのモル数を算出したところ、３．８％の値が得られた。これは上記グラフト率３８．５％に相当し、グラフト反応後のサンプルを固体ＮＭＲ法で測定することにより、グラフト率が得られることが示された。 A peak area corresponding to the ester group of the resulting NMR spectrum, the ratio of the peak area corresponding to the CH 2 group, since it corresponds to the ratio of the number of moles of GMA and CH 2 groups, measurement results from the CH 2 group When the number of moles of introduced GMA relative to the number of moles was calculated, a value of 3.8% was obtained. This corresponds to the grafting rate of 38.5%, and it was shown that the grafting rate can be obtained by measuring the sample after the grafting reaction by the solid state NMR method.
（ｉｉ）アニオン交換基（３級アミノ基）のグラフト鎖への固定
乾燥したグラフト鎖を導入した中空糸多孔膜をメタノールに１０分以上浸漬して膨潤させた後、純水に浸漬して水置換した。ジエチルアミン５０体積部、純水５０体積部の混合溶液よりなる反応液を、上記（ｉ）で得られたグラフト反応後の中空糸多孔膜の乾燥重量に対して２０質量部、ガラス反応管に入れ、３０℃に調整した。ここに純水浸漬後のグラフト鎖を導入した中空糸多孔膜を挿入し、２１０分間静置して、グラフト鎖のエポキシ基をジエチルアミノ基に置換することにより、アニオン交換基としてジエチルアミノ基がグラフト鎖を介して固定された中空糸多孔膜を得た。得られた中空糸多孔膜は外径３．３ｍｍ、内径２．１ｍｍであり、中空糸多孔膜においてグラフト鎖の有するエポキシ基の８０％がジエチルアミノ基によって置換されていた。
(Ii) Fixation of anion exchange group (tertiary amino group) to graft chain After swelled by immersing a hollow fiber porous membrane into which a dried graft chain has been introduced for 10 minutes or more, it is immersed in pure water to obtain water. Replaced. A reaction solution comprising a mixed solution of 50 parts by volume of diethylamine and 50 parts by volume of pure water is placed in a glass reaction tube at 20 parts by mass with respect to the dry weight of the hollow fiber porous membrane after the graft reaction obtained in (i) above. , Adjusted to 30 ° C. A hollow fiber porous membrane into which a graft chain after immersion in pure water was introduced was inserted here and allowed to stand for 210 minutes to replace the epoxy group of the graft chain with a diethylamino group, whereby the diethylamino group was grafted as an anion exchange group. A hollow fiber porous membrane fixed via the was obtained. The obtained hollow fiber porous membrane had an outer diameter of 3.3 mm and an inner diameter of 2.1 mm. In the hollow fiber porous membrane, 80% of the epoxy groups of the graft chain were substituted with diethylamino groups.
置換率Ｔはエポキシ基のモル数Ｎ0のうち、ジエチルアミノ基に置換されたモル数Ｎ1として下記式（ＩＩ）を用いて算出した。
Ｔ＝１００×Ｎ1／Ｎ0
＝１００×｛（ｗ1−ｗ0）／Ｍ1｝／｛ｗ0（ｄｇ／（ｄｇ＋１００））／Ｍ2｝
・・・（ＩＩ）
式（ＩＩ）中、Ｍ1はジエチルアンモニウムの分子量（７３．１４）、ｗ0はグラフト重合反応後の中空糸多孔膜の重量、ｗ1はジエチルアミノ基置換反応後の中空糸多孔膜の重量、ｄｇはグラフト率、Ｍ2はＧＭＡの分子量（１４２）である。
The substitution rate T was calculated using the following formula (II) as the number of moles N 1 substituted with a diethylamino group out of the number of moles N 0 of the epoxy group.
T = 100 × N 1 / N 0
= 100 × {(w 1 −w 0 ) / M 1 } / {w 0 (dg / (dg + 100)) / M 2 }
... (II)
In formula (II), M 1 is the molecular weight of diethylammonium (73.14), w 0 is the weight of the hollow fiber porous membrane after the graft polymerization reaction, w 1 is the weight of the hollow fiber porous membrane after the diethylamino group substitution reaction, dg is the graft ratio and M 2 is the molecular weight of GMA (142).
固体ＮＭＲ法により、上記と同様の方法で、ジエチルアミノ基を導入した中空糸多孔膜中の、ポリエチレン骨格単位ＣＨ2基のモル数に対する、ＧＭＡに特有なエステル基のモル数の比を測定したところ、３．７５％という値が得られた。これはグラフト率３８．５％に対応し、この結果よりジエチルアミノ基の導入によるグラフト率の変化はないことが確認された。 When the ratio of the number of moles of ester groups peculiar to GMA to the number of moles of polyethylene skeleton unit CH 2 groups in the hollow fiber porous membrane into which diethylamino groups were introduced was measured by the solid NMR method in the same manner as described above. A value of 3.75% was obtained. This corresponds to a grafting rate of 38.5%. From this result, it was confirmed that there was no change in the grafting rate due to the introduction of diethylamino groups.
（ｉｉｉ）アニオン交換基が固定された中空糸多孔膜モジュールの作製
（ｉｉ）で得られた、アニオン交換基としてジエチルアミノ基がグラフト鎖を介して固定された中空糸多孔膜３本を束ね、中空糸多孔膜の中空部を閉塞しないようにエポキシ系ポッティング剤で両末端をポリスルホン酸製モジュールケースに固定して、アニオン交換が固定された中空糸多孔膜モジュールを作製した。得られたモジュールの内径は０．９ｃｍ、長さは約３．３ｃｍ、モジュールの内容積は約２ｍＬ、モジュール内に占める中空糸多孔膜の有効体積は０．８５ｍＬ、中空部分を除いた中空糸多孔膜のみの体積は０．５３ｍＬであった。これを、以下の実施例等において、評価モジュールとして用いた。
(Iii) Production of hollow fiber porous membrane module in which anion exchange groups are fixed Bundled three hollow fiber porous membranes obtained in (ii) in which diethylamino groups are fixed via graft chains as anion exchange groups Both ends of the yarn porous membrane were fixed to a polysulfonic acid module case with an epoxy potting agent so as not to block the hollow portion of the yarn porous membrane, thereby producing a hollow fiber porous membrane module in which anion exchange was fixed. The obtained module has an inner diameter of 0.9 cm, a length of about 3.3 cm, an inner volume of the module of about 2 mL, an effective volume of the hollow fiber porous membrane in the module of 0.85 mL, and a hollow fiber excluding the hollow portion. The volume of the porous membrane alone was 0.53 mL. This was used as an evaluation module in the following examples.
（ｉｖ）バブルポイント法
基材としての中空糸多孔膜の最大細孔径を、バブルポイント法を用いて測定した。長さ８ｃｍの中空糸多孔膜の一方の末端を閉塞し、他方の末端に圧力計を介して窒素ガス供給ラインを接続した。この状態で窒素ガスを供給してライン内部を窒素に置換した後、中空糸多孔膜をエタノールに浸漬した。この時、エタノールがライン内に逆流しないように極僅かに窒素で圧力をかけた状態で、中空糸多孔膜を浸漬した。中空糸多孔膜を浸漬した状態で、窒素ガスの圧力をゆっくりと増加させ、中空糸多孔膜から窒素ガスの泡が安定して出始めた圧力Ｐを記録した。これより、最大細孔径をｄ、表面張力をγとして、下記式（ＩＩＩ）に従って、中空糸多孔膜の最大細孔径を算出した。
ｄ＝Ｃ1γ／Ｐ・・・（ＩＩＩ）
式（ＩＩＩ）中、Ｃ1は定数である。エタノールを浸漬液としたときのＣ1γ＝０．６３２（ｋｇ／ｃｍ）であり、上式にＰ（ｋｇ／ｃｍ2）を代入することにより、最大細孔径ｄ（μｍ）を求めた。
(Iv) Bubble Point Method The maximum pore diameter of the hollow fiber porous membrane as the substrate was measured using the bubble point method. One end of a hollow fiber porous membrane having a length of 8 cm was closed, and a nitrogen gas supply line was connected to the other end via a pressure gauge. In this state, nitrogen gas was supplied to replace the inside of the line with nitrogen, and then the hollow fiber porous membrane was immersed in ethanol. At this time, the hollow fiber porous membrane was immersed in a state where a slight pressure of nitrogen was applied so that ethanol did not flow back into the line. With the hollow fiber porous membrane immersed, the pressure of nitrogen gas was slowly increased, and the pressure P at which nitrogen gas bubbles started to stably emerge from the hollow fiber porous membrane was recorded. From this, the maximum pore diameter of the hollow fiber porous membrane was calculated according to the following formula (III), where d is the maximum pore diameter and γ is the surface tension.
d = C 1 γ / P (III)
Wherein (III), C 1 is a constant. C 1 γ = 0.632 (kg / cm) when ethanol was used as the immersion liquid, and the maximum pore diameter d (μm) was determined by substituting P (kg / cm 2 ) into the above equation.
［参考例２］ 抗体モノマーと凝集体の比率の測定
抗体モノマーと凝集体の比率はゲルろ過クロマトグラフィーによって評価した。島津製作所株式会社製クロマトグラフＬＣ−１０Ａシステムにゲルろ過カラムとして東ソー株式会社製ＴＳＫｇｅｌ Ｇ３０００ＳＷＸＬを取り付け、バッファーとして０．１Ｍリン酸＋０．２Ｍアルギニン（ｐＨ６．８）を用いて、２５℃において流速０．８ｍｌ／ｍｉｎでカラムに通液し、ここに評価サンプルを２０μＬ添加した。カラム透過後に抗体モノマーと凝集体はそれぞれ分離した溶出ピークを示し、得られた抗体モノマーと凝集体のピーク面積比から、溶液中でのそれぞれの存在比率を算出した。
[Reference Example 2] Measurement of ratio of antibody monomer to aggregate The ratio of antibody monomer to aggregate was evaluated by gel filtration chromatography. Tosoh Corporation TSKgel G3000SWXL was attached to the chromatograph LC-10A system manufactured by Shimadzu Corporation as a gel filtration column, and 0.1 M phosphoric acid + 0.2 M arginine (pH 6.8) was used as a buffer. The solution was passed through the column at 8 ml / min, and 20 μL of the evaluation sample was added thereto. After passing through the column, the antibody monomer and the aggregate showed elution peaks separated from each other, and the abundance ratio in the solution was calculated from the peak area ratio of the obtained antibody monomer and the aggregate.
［参考例３］ 抗体モノマーと凝集体を含む混合液の調整
ｐＨ６、７又はｐＨ８の２０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液を作成し、ここに塩化ナトリウム（ＮａＣｌ）を添加して、塩濃度が所定の値となるように調整した後、抗体タンパク質としてｈｕｍａｎ ＩｇＧ（田辺三菱製薬製、ヴェノグロブリンＩＨ）を２ｇ／Ｌとなるように添加し、夾雑物として凝集体を含む抗体モノマーの混合液を作成した。また、ｐＨ５及びｐＨ６の５０ｍＭ Ａｃｅｔａｔｅ−ＮａＯＨを作成し、同様にして、塩濃度が所定の値の抗体モノマーと凝集体を含む混合液を作成した。ここで、塩濃度は緩衝液調整時のＮａＣｌ添加重量により調整し、ｐＨは市販のｐＨメーター（東亜ディーケーケー（株）、ＨＭ−３０Ｓ）を用いて測定した。
[Reference Example 3] Preparation of mixed solution containing antibody monomer and aggregate A 20 mM Tris-HCl buffer solution of pH 6, 7 or pH 8 was prepared, sodium chloride (NaCl) was added thereto, and the salt concentration was a predetermined value. Then, human IgG (manufactured by Mitsubishi Tanabe Pharma Corporation, Venoglobulin IH) was added as an antibody protein so as to be 2 g / L, and a mixed solution of antibody monomers containing aggregates as a contaminant was prepared. In addition, 50 mM Acetate-NaOH having pH 5 and pH 6 was prepared, and similarly, a mixed solution containing antibody monomers and aggregates having a predetermined salt concentration was prepared. Here, the salt concentration was adjusted by the NaCl addition weight at the time of buffer adjustment, and the pH was measured using a commercially available pH meter (Toa DKK Co., Ltd., HM-30S).
［参考例４］ 抗体モノマー精製評価
上記の参考例３で作成した抗体モノマーと凝集体の混合液１０ｍＬを、参考例１で作成した評価モジュールに透過させた。その際、事前に評価モジュールには抗体モノマーと凝集体を含まない同じｐＨと塩濃度の緩衝液を２０ｍＬ通液し、評価モジュールを平衡化しておいた。また、混合液の透過後、平衡化に用いたものと同じ緩衝液４ｍＬをモジュール内に残存する抗体モノマーの洗浄液として通液し、これも透過液に加えて１４ｍＬの透過液を回収した。回収した透過液は参考例２の方法により、抗体モノマーと凝集体の比率を測定し、評価モジュールに通液する前の比率と比較した。溶液は評価モジュール内の中空糸多孔膜の内側から外側に向かって流速２ｍＬ／ｍｉｎにて通液した。評価はＧＥヘルスケアバイオサイエンス製ＡＫＴＡｅｘｐｌｏｒｅｒ１００を用い、評価液の２８０ｎｍのＵＶ吸光度を測定し、下記式（ＩＶ）に従って、ＵＶ吸光度から精製後の抗体モノマーの回収率を評価した。
回収率（％）＝（１４×Ｉ1）／（１０×Ｉ0）×１００・・・（ＩＶ）
式（ＩＶ）中、Ｉ0及びＩ1はそれぞれモジュール透過前の混合液及び回収した透過液の２８０ｎｍのＵＶ吸光度である。
Reference Example 4 Antibody Monomer Purification Evaluation 10 mL of the antibody monomer / aggregate mixture prepared in Reference Example 3 was passed through the evaluation module prepared in Reference Example 1. At that time, 20 mL of a buffer solution having the same pH and salt concentration not containing antibody monomers and aggregates was passed through the evaluation module in advance to equilibrate the evaluation module. After the permeation of the mixed solution, 4 mL of the same buffer solution used for equilibration was passed as a washing solution for the antibody monomer remaining in the module, and this was added to the permeated solution to recover 14 mL of the permeated solution. The recovered permeate was measured for the ratio of antibody monomer to aggregate by the method of Reference Example 2 and compared with the ratio before passing through the evaluation module. The solution was passed at a flow rate of 2 mL / min from the inside to the outside of the hollow fiber porous membrane in the evaluation module. For evaluation, AKTAexplorer100 manufactured by GE Healthcare Bioscience was used to measure the UV absorbance of the evaluation solution at 280 nm, and the recovery rate of the purified antibody monomer was evaluated from the UV absorbance according to the following formula (IV).
Recovery rate (%) = (14 × I 1 ) / (10 × I 0 ) × 100 (IV)
In formula (IV), I 0 and I 1 are the UV absorbance at 280 nm of the mixed solution before the module permeation and the collected permeated solution, respectively.
［実施例１］
参考例３で得られたｐＨ８．０及び塩濃度０Ｍの、抗体モノマーと凝集体を含む混合液中の、全抗体タンパク質中の凝集体比率を参考例２の方法により測定したところ、５．０９％であった。またこの混合液の２８０ｎｍのＵＶ吸光度は７５４ｍＡＵであった。参考例４に従い、参考例３で得られたｐＨ８．０及び塩濃度０Ｍの、抗体モノマーと凝集体を含む混合液を、参考例１で作成した評価モジュールに、１０ｍＬ通液し、洗浄液も含めて１４ｍＬを透過液として回収した。得られた透過液に含まれる全抗体タンパク質中の凝集体比率を参考例２の方法により測定したところ、０．１２％であった。また、透過液の２８０ｎｍのＵＶ吸光度は４５７ｍＡＵであり、全抗体モノマーの回収率は８５％であった。これにより、凝集体が除去された抗体モノマーが高い回収率で得られることが示された。
[Example 1]
When the ratio of aggregates in the total antibody protein in the mixed solution containing the antibody monomer and aggregates having a pH of 8.0 and a salt concentration of 0 M obtained in Reference Example 3 was measured by the method of Reference Example 2, it was 5.09. %Met. Further, the UV absorbance at 280 nm of this mixed solution was 754 mAU. In accordance with Reference Example 4, 10 mL of the mixed solution containing the antibody monomer and the aggregate having a pH of 8.0 and a salt concentration of 0 M obtained in Reference Example 3 was passed through the evaluation module created in Reference Example 1, and the cleaning liquid was also included. 14 mL was recovered as permeate. The aggregate ratio in the total antibody protein contained in the obtained permeate was measured by the method of Reference Example 2 and found to be 0.12%. Further, the UV absorbance at 280 nm of the permeate was 457 mAU, and the recovery rate of all antibody monomers was 85%. Thereby, it was shown that the antibody monomer from which the aggregate was removed was obtained with a high recovery rate.
［比較例１］
参考例１で作成した評価モジュールの代わりに市販のアニオン交換クロマトグラフィーカラム（ＧＥヘルスケア製、ＨｉＴｒａｐＤＥＡＥ ＦＦ １ｍＬ）を用い、通液速度を１ｍＬ／ｍｉｎにした以外は、実施例１と同様にして、抗体モノマー精製評価を行った。得られた透過液に含まれる全抗体タンパク質中の凝集体比率を参考例２の方法により測定したところ、３．８９％であり、通液速度を半分にしたにも関わらず、凝集体の除去率は極めて低かった。また、透過液の２８０ｎｍのＵＶ吸光度は２８９ｍＡＵであり、回収率は５４％であり、回収率も低かった。
[Comparative Example 1]
A commercially available anion exchange chromatography column (manufactured by GE Healthcare, HiTrapDEAE FF 1 mL) was used instead of the evaluation module created in Reference Example 1, and the liquid flow rate was set to 1 mL / min. Then, antibody monomer purification evaluation was performed. The ratio of aggregates in the total antibody protein contained in the obtained permeate was measured by the method of Reference Example 2. As a result, it was 3.89%. The rate was very low. Further, the UV absorbance at 280 nm of the permeated liquid was 289 mAU, the recovery rate was 54%, and the recovery rate was also low.
［実施例２］
参考例３で得られたｐＨ７．０及び塩濃度０Ｍの、抗体モノマーと凝集体を含む混合液中の、全抗体タンパク質中の凝集体比率を参考例２の方法により測定したところ、５．４７％であった。またこの混合液の２８０ｎｍのＵＶ吸光度は７７０ｍＡＵであった。参考例４に従い、参考例３で得られたｐＨ７．０及び塩濃度０Ｍの、抗体モノマーと凝集体を含む混合液を、参考例１で作成した評価モジュールに、１０ｍＬ通液し、洗浄液も含めて１４ｍＬを透過液として回収した。得られた透過液に含まれる全抗体タンパク質中の凝集体比率を参考例２の方法により測定したところ、０．４９％であった。また、透過液の２８０ｎｍのＵＶ吸光度は５０６ｍＡＵであり、回収率は９２％であった。これにより、凝集体が除去された抗体モノマーが高い回収率で得られることが示された。
[Example 2]
When the ratio of aggregates in the total antibody protein in the mixed solution containing the antibody monomer and aggregates having a pH of 7.0 and a salt concentration of 0 M obtained in Reference Example 3 was measured by the method of Reference Example 2, it was 5.47. %Met. Further, the UV absorbance at 280 nm of this mixed solution was 770 mAU. In accordance with Reference Example 4, 10 mL of the mixed solution containing the antibody monomer and the aggregate having a pH of 7.0 and a salt concentration of 0 M obtained in Reference Example 3 was passed through the evaluation module created in Reference Example 1, and the cleaning solution was also included. 14 mL was recovered as permeate. When the ratio of aggregates in the total antibody protein contained in the obtained permeate was measured by the method of Reference Example 2, it was 0.49%. Further, the UV absorbance at 280 nm of the permeate was 506 mAU, and the recovery rate was 92%. Thereby, it was shown that the antibody monomer from which the aggregate was removed was obtained with a high recovery rate.
［実施例３］
参考例３で得られたｐＨ８．０及び塩濃度０．０５Ｍの、抗体モノマーと凝集体を含む混合液中の、全抗体タンパク質中の凝集体比率を参考例２の方法により測定したところ、４．７１％であった。またこの混合液の２８０ｎｍのＵＶ吸光度は７３８ｍＡＵであった。参考例４に従い、参考例３で得られたｐＨ８．０及び塩濃度０．０５Ｍの、抗体モノマーと凝集体を含む混合液を、参考例１で作成した評価モジュールに、１０ｍＬ通液し、洗浄液も含めて１４ｍＬを透過液として回収した。得られた回収液に含まれる全抗体タンパク質中の凝集体比率を参考例２の方法により測定したところ、０．７７％であった。また、透過液の２８０ｎｍのＵＶ吸光度は４９６ｍＡＵであり、回収率は９４％であった。これにより、凝集体が除去された抗体モノマーが高い回収率で得られることが示された。
[Example 3]
When the aggregate ratio in the total antibody protein in the mixed solution containing the antibody monomer and the aggregate at pH 8.0 and salt concentration 0.05M obtained in Reference Example 3 was measured by the method of Reference Example 2, 4 was found. 71%. Further, the UV absorbance at 280 nm of this mixed solution was 738 mAU. In accordance with Reference Example 4, 10 mL of the mixed solution containing the antibody monomer and the aggregate having a pH of 8.0 and a salt concentration of 0.05 M obtained in Reference Example 3 was passed through the evaluation module created in Reference Example 1, and the washing solution 14 mL was collected as a permeate. The aggregate ratio in the total antibody protein contained in the obtained recovered liquid was measured by the method of Reference Example 2 and found to be 0.77%. Further, the UV absorbance at 280 nm of the permeate was 496 mAU, and the recovery rate was 94%. Thereby, it was shown that the antibody monomer from which the aggregate was removed was obtained with a high recovery rate.
［実施例４］
参考例３で得られたｐＨ６．０及び塩濃度０．５Ｍの、抗体モノマーと凝集体の混合液中の、全抗体タンパク質中の凝集体比率を参考例２の方法により測定したところ、３．８７％であった。またこの混合液の２８０ｎｍのＵＶ吸光度は７３２ｍＡＵであった。参考例４に従い、参考例３で得られたｐＨ６．０及び塩濃度０．５Ｍの、抗体モノマーと凝集体を含む混合液を、参考例１で作成した評価モジュールに、１０ｍＬ通液し、洗浄液も含めて１４ｍＬを透過液として回収した。得られた透過液に含まれる全抗体タンパク質中の凝集体比率を参考例２の方法により測定したところ、０．８６％であった。また、透過液の２８０ｎｍのＵＶ吸光度は５１３ｍＡＵであり、回収率は９８％であった。これにより、凝集体が除去された抗体モノマーが高い回収率で得られることが示された。
[Example 4]
When the ratio of aggregates in the total antibody protein in the mixed solution of antibody monomer and aggregates having a pH of 6.0 and a salt concentration of 0.5 M obtained in Reference Example 3 was measured by the method of Reference Example 2, 3. 87%. Further, the UV absorbance at 280 nm of this mixed solution was 732 mAU. In accordance with Reference Example 4, 10 mL of the mixed solution containing the antibody monomer and the aggregate having a pH of 6.0 and a salt concentration of 0.5 M obtained in Reference Example 3 was passed through the evaluation module created in Reference Example 1, and the washing solution 14 mL was collected as a permeate. When the ratio of aggregates in the total antibody protein contained in the obtained permeate was measured by the method of Reference Example 2, it was 0.86%. Further, the UV absorbance at 280 nm of the permeate was 513 mAU, and the recovery rate was 98%. Thereby, it was shown that the antibody monomer from which the aggregate was removed was obtained with a high recovery rate.
以上により、アニオン交換基が固定された多孔膜を用いることにより、抗体モノマーの精製が、高速でかつ簡便になされることが示された。 From the above, it was shown that the purification of the antibody monomer can be performed at high speed and simply by using the porous membrane on which the anion exchange group is fixed.
アニオン交換基が固定された多孔膜に、抗体モノマーと夾雑物を含む混合液を通液することによって、抗体モノマーの凝集体を含む夾雑物を容易に除去することができ、精製された抗体モノマーを得ることが可能となる。この方法は、従来のカラムクロマトグラフィーを用いる方法に比べて、より高い凝集体除去能を有し、高濃度の抗体モノマー及び夾雑物を含む混合液であっても高速での処理が可能であり、スケールアップも容易である。このことから、工業レベルで医薬品を製造する際の抗体モノマーの精製に適するという産業上の利用可能性を有する。 By passing a mixed solution containing antibody monomers and contaminants through a porous membrane on which an anion exchange group is fixed, contaminants including antibody monomer aggregates can be easily removed, and purified antibody monomers. Can be obtained. This method has a higher ability to remove aggregates than the conventional method using column chromatography, and even a mixed solution containing a high concentration of antibody monomer and impurities can be processed at high speed. Scale up is easy. From this, it has the industrial applicability that it is suitable for purification of the antibody monomer at the time of manufacturing a pharmaceutical at an industrial level.
Claims (8)
前記多孔膜に吸着させる工程において、夾雑物を多孔膜に吸着させる、請求項１〜４のいずれかに記載の抗体モノマーの精製方法。 The mixed solution has a pH of 6 to 9 and a salt concentration of 0 M to 0.2 M, or a pH of 4 to 7 and a salt concentration of 0.05 M to 2 M,
The method for purifying antibody monomers according to any one of claims 1 to 4, wherein in the step of adsorbing to the porous membrane, impurities are adsorbed to the porous membrane.
前記混合液のｐＨが６〜９及び塩濃度が０Ｍ〜０．１Ｍであり、
前記多孔膜に吸着させる工程において、抗体モノマーを多孔膜に吸着させ、
さらにその後、ｐＨが４〜９及び塩濃度が０．１Ｍ〜２Ｍの溶出液を前記多孔膜に通液し、前記多孔膜に吸着した抗体モノマーを溶出回収する工程を含む、請求項１〜４のいずれかに記載の抗体モノマーの精製方法。 The isoelectric point of the antibody monomer is less than 7.5;
The pH of the mixture is 6-9 and the salt concentration is 0M-0.1M,
In the step of adsorbing to the porous membrane, the antibody monomer is adsorbed to the porous membrane,
The method further comprises a step of passing an eluate having a pH of 4 to 9 and a salt concentration of 0.1 M to 2 M through the porous membrane, and eluting and recovering the antibody monomer adsorbed on the porous membrane. The method for purifying the antibody monomer according to any one of the above.
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