JP5931363B2 - Protein purification method - Google Patents
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- JP5931363B2 JP5931363B2 JP2011149458A JP2011149458A JP5931363B2 JP 5931363 B2 JP5931363 B2 JP 5931363B2 JP 2011149458 A JP2011149458 A JP 2011149458A JP 2011149458 A JP2011149458 A JP 2011149458A JP 5931363 B2 JP5931363 B2 JP 5931363B2
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- Peptides Or Proteins (AREA)
Description
本発明は精製技術に関し、特にタンパク質の精製方法に関する。 The present invention relates to a purification technique, and more particularly to a protein purification method.
食品及び医療などの分野において、様々な有用なタンパク質を効率的に精製する方法は重要な技術として認識されている。即ち、タンパク質はその性質によって特定の機能を示すため、目的とするタンパク質を単離し、精製する技術が各分野において求められている。一般に有用タンパク質は、ヒトを含む動物由来のものとして抽出される場合と、目的とするタンパク質の遺伝子を組み込んだプラスミドを導入した大腸菌、あるいは細胞培養により発現させて得られる場合と、に大別される。いずれの場合においても、目的とするタンパク質を単離、精製するためには、複数の工程に渡る複雑なプロセスを用いる必要がある。 In fields such as food and medicine, methods for efficiently purifying various useful proteins are recognized as important techniques. That is, since a protein exhibits a specific function depending on its property, a technique for isolating and purifying a target protein is required in each field. In general, useful proteins are broadly classified into cases where they are extracted as derived from animals including humans, and cases where they are obtained by expression in Escherichia coli into which a plasmid containing the target protein gene has been introduced or cell culture. The In any case, in order to isolate and purify the target protein, it is necessary to use a complicated process involving a plurality of steps.
動物由来の有用タンパク質として、代表的なものに、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モノクローナル抗体、及びポリクローナル抗体などの抗体タンパク質、さらには、ヒトから採血された血液から得られる、フィブリノゲン、血液凝固第ＶＩＩＩ因子、フォンビルブラント因子、免疫グロブリン、血清アルブミン、トロンビン、アンチトロンビン、及びトランスフェリンなどの、各種の血漿分画製剤がある。これら、動物細胞培養液から得られる抗体タンパク質は、遠心分離、及び精密ろ過膜による除濁の後に、クロマトグラフィー工程によって精製される（例えば、非特許文献１参照。）。ここで、クロマトグラフィー工程は、目的とするタンパク質を、吸着して回収する工程（Ｂｉｎｄ−ａｎｄ−Ｅｌｕｔｅ）と、不純物となるタンパク質などを吸着して、目的タンパク質を透過して回収する工程（Ｆｌｏｗｔｈｒｏｕｇｈ）と、に大別される。また、血漿分画製剤は、採血された血漿を集めたプール血漿に、エタノールや酸などを添加して、物理化学的条件を少しずつ変化させ、特定のタンパク質が沈殿しやすい条件を作ることにより、目的とするタンパク質を取り出す、１９４０年代に開発されたコーン分画法を基本として、ろ過やクロマトグラフィーなどの分離手法を組み合わせた方法により精製され、工業的にも当該精製方法が一般に用いられる（例えば、非特許文献２参照。）。 As useful proteins derived from animals, representative examples include monoclonal antibodies produced by cell culture using animal-derived cell lines, and antibody proteins such as polyclonal antibodies, and further obtained from blood collected from humans. There are various plasma fractionated preparations such as fibrinogen, blood coagulation factor VIII, von Willebrand factor, immunoglobulin, serum albumin, thrombin, antithrombin, and transferrin. These antibody proteins obtained from animal cell culture fluids are purified by a chromatography step after centrifugation and turbidity by a microfiltration membrane (see, for example, Non-Patent Document 1). Here, the chromatography step includes a step of binding and recovering the target protein (Bind-and-Elute) and a step of absorbing and recovering the target protein through the target protein (Flowthrough). ). In addition, plasma fractionation products are prepared by adding ethanol, acid, etc. to pooled plasma, which is a collection of collected blood plasma, and gradually changing the physicochemical conditions so that specific proteins are likely to precipitate. Based on the corn fractionation method developed in the 1940s for extracting the target protein, the protein is purified by a combination of separation methods such as filtration and chromatography, and the purification method is generally used industrially ( For example, refer nonpatent literature 2.).
特に血液凝固因子のような血中濃度の低い有用タンパク質を有効に単離、精製、濃縮するためには、分画法と、クロマトグラフィー技術と、の組み合わせが不可欠となる。例えば、特許文献１には、特定の緩衝液によって平衡化されたジエチルアミノエチル（ＤＥＡＥ）のアニオン交換基を有するクロマトグラフィーカラムにより、フォンビルブラント因子を精製、濃縮する方法が記載されている。また、非特許文献３にはアフィニティクロマトグラフィー、ウィルス除去膜及びアニオン交換クロマトグラフィーを組み合わせた工程により、血液凝固第ＶＩＩＩ因子を精製する方法が報告されている。さらに、特許文献２では特定のイオン交換クロマトグラフィーろ過タイプのメンブレンを用いて、血液凝固第ＶＩＩＩ因子又はフォンビルブラント因子を精製する方法が報告されている。 In particular, in order to effectively isolate, purify and concentrate useful proteins with low blood concentrations such as blood coagulation factors, a combination of fractionation and chromatographic techniques is indispensable. For example, Patent Document 1 describes a method of purifying and concentrating von Willebrand factor using a chromatography column having an anion exchange group of diethylaminoethyl (DEAE) equilibrated with a specific buffer. Non-Patent Document 3 reports a method of purifying blood coagulation factor VIII by a process combining affinity chromatography, virus removal membrane and anion exchange chromatography. Furthermore, Patent Document 2 reports a method of purifying blood coagulation factor VIII or von Willebrand factor using a specific ion exchange chromatography filtration type membrane.
上記に示したように、クロマトグラフィー工程は、細胞培養液などから不純物を除去して目的物を精製する目的に有用であり、また分画法とクロマトグラフィー法とを組み合わせる方法は、血液中での濃度の低い血液凝固因子などを濃縮、精製するために有用である。しかしながら、カラムクロマトグラフィーは吸着するタンパク質の分子量が大きいほど、吸着量が低下することが知られており（例えば、特許文献３参照。）、フロースルー（Ｆｌｏｗｔｈｒｏｕｇｈ）によって不純物を除去する場合に、高分子量の不純物タンパク質の除去が困難となる場合がある。さらに、第ＶＩＩＩ因子、フォンビルブラント因子、及びフィブリノゲンのような、高分子量タンパク質のＢｉｎｄ−ａｎｄ−Ｅｌｕｔｅによる精製にクロマトグラフィーを用いる場合、動的吸着容量が通常１０ｍｇ／ｍＬにも満たないために、低分子量タンパク質の精製に比べて精製効率が低下するという難点があり、特許文献１及び非特許文献２に開示された方法では、迅速な精製処理が容易ではない。また、特許文献２で開示されたイオン交換クロマトグラフィーろ過タイプのメンブレンにおいても、高分子量タンパク質の吸着量が十分ではないという課題は解決されていない。 As described above, the chromatography step is useful for the purpose of purifying the target product by removing impurities from the cell culture medium and the like, and the method combining the fractionation method and the chromatography method is performed in blood. It is useful for concentrating and purifying blood coagulation factors and the like having a low concentration. However, column chromatography is known to decrease the amount of protein adsorbed as the molecular weight of the adsorbed protein increases (see, for example, Patent Document 3). When impurities are removed by flow-through, the amount of adsorbed protein increases. Removal of molecular weight impurity proteins may be difficult. Furthermore, when chromatography is used to bind high molecular weight proteins such as Factor VIII, von Willebrand factor, and fibrinogen with Bind-and-Elute, the dynamic adsorption capacity is usually less than 10 mg / mL. However, there is a problem that the purification efficiency is lowered as compared with the purification of low molecular weight proteins, and the methods disclosed in Patent Document 1 and Non-Patent Document 2 are not easy for rapid purification treatment. Further, even in the ion exchange chromatography filtration type membrane disclosed in Patent Document 2, the problem that the adsorption amount of the high molecular weight protein is not sufficient has not been solved.
上記に記載したように、従来のカラムクロマトグラフィーを用いた方法では、高分子量の不純物タンパク質を含む、広い範囲の分子量分布を有する不純物を除去することが難しく、また、高分子量の有用タンパク質を吸着及び溶出して、効率的かつ迅速に精製することも容易ではない。 As described above, it is difficult to remove impurities having a wide range of molecular weight distribution, including high molecular weight impurity proteins, and adsorb high molecular weight useful proteins using conventional column chromatography methods. And it is not easy to elute and purify efficiently and quickly.
かかる状況に鑑み、本発明の解決しようとする課題は、効率的かつ迅速に高分子量の目的タンパク質を精製する方法を提供することである。 In view of this situation, the problem to be solved by the present invention is to provide a method for purifying a high molecular weight target protein efficiently and rapidly.
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、アニオン交換膜が、高分子量タンパク質に対して、特異的に高い吸着容量を有することを見出し、さらに、目的に応じて水素イオン指数（ｐＨ）が調整されたタンパク質溶液を、このアニオン交換膜に通液することにより、高分子量の不純物タンパク質の除去、及び高分子量の目的タンパク質の精製が有効になされることを見出し、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 an anion exchange membrane has a specifically high adsorption capacity for high molecular weight proteins, and further, depending on the purpose, By passing a protein solution with an adjusted ion index (pH) through this anion exchange membrane, it was found that removal of high molecular weight impurity protein and purification of high molecular weight target protein were made effective. Completed the invention.
すなわち、本発明の態様は、目的タンパク質と、分子量の異なる複数の不純物タンパク質と、を含有する溶液をアニオン交換膜に通液して、目的タンパク質を精製する方法であって、複数の不純物タンパク質の等電点の値に対し、溶液の水素イオン指数の値を高く調整する調整工程と、調整された溶液をアニオン交換膜に通液する通液工程と、目的タンパク質を回収する回収工程と、を有し、分子量の異なる複数の不純物タンパク質は、少なくとも分子量が１００ｋＤａ未満の成分と、分子量が１００ｋＤａ以上の成分と、を含み、通液工程を経た溶液中の１００ｋＤａ未満の不純物タンパク質の成分の体積あたりの質量濃度と、１００ｋＤａ以上の不純物タンパク質の成分の体積あたりの質量濃度と、が、通液前の溶液中の５％以下となる、方法であることを要旨とする。 That is, an aspect of the present invention is a method for purifying a target protein by passing a solution containing the target protein and a plurality of impurity proteins having different molecular weights through an anion exchange membrane. An adjustment step for adjusting the value of the hydrogen ion index of the solution higher than the isoelectric point value, a liquid passing step for passing the adjusted solution through the anion exchange membrane, and a recovery step for recovering the target protein. The plurality of impurity proteins having different molecular weights include at least a component having a molecular weight of less than 100 kDa and a component having a molecular weight of 100 kDa or more per volume of the component of the impurity protein having a molecular weight of less than 100 kDa in the solution passed through And the mass concentration per volume of the component of the impurity protein of 100 kDa or more is 5% or less in the solution before passing through. And summarized in that a method.
本発明の、アニオン交換膜を用いた精製方法によれば、動物細胞培養液などに含まれる、高分子量成分を含む、広い分子量範囲の不純物タンパク質を有効に除去することができる。さらに、第ＶＩＩＩ因子、フォンビルブラント因子、及びフィブリノゲンなどに代表される、高分子量タンパク質の吸着溶出による精製を、効率的かつ迅速に実施することが可能となる。 According to the purification method using an anion exchange membrane of the present invention, it is possible to effectively remove impurity proteins in a wide molecular weight range including a high molecular weight component contained in an animal cell culture medium or the like. Furthermore, purification by adsorption elution of high molecular weight proteins represented by factor VIII, von Willebrand factor, fibrinogen and the like can be carried out efficiently 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可能である。 Hereinafter, a mode for carrying out the present invention (hereinafter referred to as “the present embodiment”) will be described in detail. It should be noted that the present invention is not limited to the following embodiment, and can be implemented with various modifications within the scope of the gist.
本実施の形態に係るタンパク質の精製方法は、目的タンパク質と、分子量の異なる複数の不純物タンパク質と、を含有する溶液をアニオン交換膜に通液して、目的タンパク質を精製する方法であって、複数の不純物タンパク質の等電点の値に対し、溶液の水素イオン指数の値を高く調整する調整工程と、調整された溶液をアニオン交換膜に通液する通液工程と、目的タンパク質を回収する回収工程と、を有する。分子量の異なる複数の不純物タンパク質は、少なくとも分子量が１００ｋＤａ未満の成分と、分子量が１００ｋＤａ以上の成分と、を含む。通液工程を経た溶液中の１００ｋＤａ未満の不純物タンパク質の成分の体積あたりの質量濃度と、１００ｋＤａ以上の不純物タンパク質の成分の体積あたりの質量濃度と、は、通液前の溶液中の５％以下となる。 The protein purification method according to the present embodiment is a method for purifying a target protein by passing a solution containing the target protein and a plurality of impurity proteins having different molecular weights through an anion exchange membrane. Adjustment process for adjusting the hydrogen ion exponent of the solution to be higher than the isoelectric point value of the impurity protein, a flow process for passing the adjusted solution through the anion exchange membrane, and a recovery for recovering the target protein And a process. The plurality of impurity proteins having different molecular weights include at least a component having a molecular weight of less than 100 kDa and a component having a molecular weight of 100 kDa or more. The mass concentration per volume of the component of the impurity protein of less than 100 kDa in the solution passed through the liquid passing step and the mass concentration per volume of the component of the impurity protein of 100 kDa or higher are 5% or less of the solution before passing through It becomes.
本実施の形態で用いられる多孔膜状のアニオン交換膜は、高分子量タンパク質の吸着量が高く、分子量が１００ｋＤａ以上、好ましくは２００ｋＤａ以上、より好ましくは３００ｋＤａ以上のタンパク質の１０％破過の動的吸着容量が、好ましくは２０ｍｇ／ｍＬ以上、より好ましくは３０ｍｇ／ｍＬ以上である。なお、１０％破過とは、透過液中の物質濃度が、供給された物質含有溶液の濃度の１０％を超えた時点のことをいう。また、動的吸着容量Ａは、溶液の濃度をＱ、破過した時までに透過させた溶液の体積をＶB、アニオン交換膜の膜体積をＶMとして、下記式（１）で与えられる。
Ａ＝Ｑ×ＶB／ＶM ・・・（１）
一般に市販されているアニオン交換膜としては、旭化成メディカル製ＱｙｕＳｐｅｅｄ（登録商標）Ｄ、Ｐａｌｌ製ＭｕｓｔａｎｇＱ、Ｓａｒｔｏｒｉｕｓ製ＳａｒｔｏｂｉｎｄＱ、Ｎａｔｒｉｘ製ａｄｓｅｐｔＱ、Ｍｉｌｌｉｐｏｒｅ製ＣｈｒｏｍａＳｏｒｂなどが挙げられるが、これらの中でも、ポリエチレン多孔質基材にグラフト鎖を固定した形態を有する、旭化成メディカル製ＱｙｕＳｐｅｅｄ（登録商標）Ｄが、最も高分子量タンパク質の動的吸着容量が高く、好ましい。
The porous membrane-like anion exchange membrane used in the present embodiment has a high adsorption amount of high molecular weight protein and a dynamic of 10% breakthrough of a protein having a molecular weight of 100 kDa or more, preferably 200 kDa or more, more preferably 300 kDa or more. The adsorption capacity is preferably 20 mg / mL or more, more preferably 30 mg / mL or more. In addition, 10% breakthrough refers to a point in time when the substance concentration in the permeate exceeds 10% of the concentration of the supplied substance-containing solution. Also, the dynamic adsorption capacity A is the concentration of the solution Q, volume V B of the solution is transmitted through the time that breakthrough, the membrane volume of the anion exchange membrane as V M, is given by the following equation (1) .
A = Q × V B / V M (1)
Examples of commercially available anion exchange membranes include QyuSpeed (registered trademark) D manufactured by Asahi Kasei Medical, MustangQ manufactured by Pall, SartobindQ manufactured by Sartorius, adseptQ manufactured by Millix, ChromaSorb manufactured by Millipore, and the like. QyuSpeed (registered trademark) D manufactured by Asahi Kasei Medical, which has a form in which graft chains are fixed to the material, is preferred because it has the highest dynamic adsorption capacity for high molecular weight proteins.
アニオン交換膜の細孔径は、０．１μｍ以上０．８μｍ以下、好ましくは０．２μｍ以上０．８μｍ以下、さらに好ましくは０．２μｍ以上０．６μｍ以下である。細孔径が小さすぎると、溶液を通液する際の圧力が高くなる傾向にある。また細孔径が大きすぎると、高分子量タンパク質の吸着性能が低下する傾向にある。 The pore diameter of the anion exchange membrane is 0.1 μm or more and 0.8 μm or less, preferably 0.2 μm or more and 0.8 μm or less, and more preferably 0.2 μm or more and 0.6 μm or less. If the pore diameter is too small, the pressure at the time of passing the solution tends to increase. On the other hand, if the pore diameter is too large, the adsorption performance of the high molecular weight protein tends to decrease.
アニオン交換膜は、アニオン交換基を有する。アニオン交換基は例えば３級アミンである。アニオン交換膜の、体積あたりのリガンド密度に対応する、塩素イオン交換容量の値は、０．３ｍｍｏｌ／ｍＬ以上１．０ｍｍｏｌ／ｍＬ以下が好ましく、より好ましくは、０．４ｍｍｏｌ／ｍＬ以上０．８ｍｍｏｌ／ｍＬ以下である。イオン交換容量が高いほど、リガンド密度が高いため、高分子量タンパク質の吸着に有利であるが、高すぎると高分子量タンパク質の、アニオン交換膜のグラフト鎖への進入が阻害される傾向にある。 The anion exchange membrane has an anion exchange group. An anion exchange group is, for example, a tertiary amine. The value of the chloride ion exchange capacity corresponding to the ligand density per volume of the anion exchange membrane is preferably 0.3 mmol / mL or more and 1.0 mmol / mL or less, more preferably 0.4 mmol / mL or more and 0.8 mmol. / ML or less. The higher the ion exchange capacity, the higher the ligand density, which is advantageous for the adsorption of high molecular weight proteins. However, if the ion exchange capacity is too high, the high molecular weight proteins tend to be inhibited from entering the graft chains of the anion exchange membrane.
アニオン交換膜に溶液を通液する際の通液速度は、精製処理の効率化のためにより速いことが好ましい。通常のビーズカラムにタンパク質を吸着させる場合、通液速度が速くなると、吸着性能が低下することは、タンパク質精製に関する技術分野では公知であり、特に１分間当たりのカラム体積の５倍以上の体積の溶液を供給する速度で通液すると、タンパク質の吸着性能は著しく低下する。これに対し、アニオン交換膜はタンパク質の吸着性能は通液速度に殆ど依存しない。したがって、カラムを用いた場合に比べて明確に処理効率が向上するために、アニオン交換膜に溶液を通液する速度は、１分間当たりアニオン交換膜の体積の５倍以上の体積の溶液を供給することに相当する流速、即ち、５ＭＶ／ｍｉｎ以上が可能であり、より好ましくは１０ＭＶ／ｍｉｎ以上である。流速の上限は、用いる溶液の粘度及び濁度などの性状に依存するが、通液の際の膜間差圧を検知することにより、容易に設定することができる。ここで、溶液とは、タンパク質溶液、並びにアニオン交換膜の平衡化、洗浄及びタンパク質の溶出に用いる塩溶液のことを指す。 It is preferable that the flow rate at the time of passing the solution through the anion exchange membrane is faster in order to improve the efficiency of the purification treatment. It is well known in the technical field related to protein purification that when the protein is adsorbed on a normal bead column, the adsorption performance decreases as the liquid flow rate increases. In particular, the volume of the column is more than 5 times the column volume per minute. When the solution is fed at a rate at which the solution is supplied, the protein adsorption performance is significantly reduced. On the other hand, in the anion exchange membrane, the protein adsorption performance hardly depends on the liquid passing speed. Therefore, in order to improve the processing efficiency clearly compared to the case where a column is used, the solution passing through the anion exchange membrane is supplied at a rate of 5 times or more the volume of the anion exchange membrane per minute. It is possible to achieve a flow rate equivalent to that of 5 MV / min, more preferably 10 MV / min or more. The upper limit of the flow rate depends on properties such as the viscosity and turbidity of the solution to be used, but can be easily set by detecting the transmembrane pressure difference during the passage. Here, the solution refers to a protein solution and a salt solution used for equilibration of an anion exchange membrane, washing, and protein elution.
アニオン交換膜に通液するタンパク質溶液のｐＨは、宿主細胞由来タンパク質（ＨＣＰ： Ｈｏｓｔ Ｃｅｌｌ Ｐｒｏｔｅｉｎ）等の不純物タンパク質を膜に吸着させるため、不純物タンパク質の等電点（ｐＩ）より、高い値に調整する。ｐＨを不純物タンパク質のｐＩより高くすることにより、不純物タンパク質はアニオン交換膜に吸着される。タンパク質溶液が塩を含む場合、吸着が有効になされるためには、溶液のｐＨは不純物タンパク質のｐＩより、十分に高い値であることが好ましい。具体的には、細胞培養液と同程度の０．１ｍｏｌ／ＬのＮａＣｌを塩として含む溶液の場合には、ｐＨは吸着される不純物タンパク質のｐＩよりも、１以上高い値であることが好ましい。 The pH of the protein solution passing through the anion exchange membrane is adjusted to a value higher than the isoelectric point (pI) of the impurity protein in order to adsorb the impurity protein such as host cell protein (HCP) to the membrane. To do. By making the pH higher than the pI of the impurity protein, the impurity protein is adsorbed on the anion exchange membrane. When the protein solution contains a salt, it is preferable that the pH of the solution is sufficiently higher than the pI of the impurity protein so that the adsorption is effective. Specifically, in the case of a solution containing 0.1 mol / L NaCl equivalent to the cell culture solution as a salt, the pH is preferably one or more higher than the pI of the impurity protein to be adsorbed. .
また、アニオン交換膜に通液するタンパク質溶液のｐＨは、目的タンパク質を膜に吸着させる場合には、目的タンパク質のｐＩより、高い値に調整する。ｐＨを目的タンパク質のｐＩより高くすることにより、目的タンパク質はアニオン交換膜に吸着される。タンパク質溶液が塩を含む場合、吸着が有効になされるためには、溶液のｐＨは目的タンパク質のｐＩより、十分に高い値であることが好ましい。具体的には、細胞培養液と同程度の０．１ｍｏｌ／ＬのＮａＣｌを塩として含む溶液の場合には、ｐＨは吸着される目的タンパク質のｐＩよりも、１以上高い値であることが好ましい。 Further, the pH of the protein solution passing through the anion exchange membrane is adjusted to a value higher than the pI of the target protein when the target protein is adsorbed on the membrane. By setting the pH higher than the pI of the target protein, the target protein is adsorbed on the anion exchange membrane. When the protein solution contains a salt, it is preferable that the pH of the solution is sufficiently higher than the pI of the target protein so that the adsorption is effective. Specifically, in the case of a solution containing 0.1 mol / L NaCl equivalent to that of the cell culture solution as a salt, the pH is preferably one or more higher than the pI of the target protein to be adsorbed. .
目的タンパク質を含む溶液をアニオン交換膜に通液し、透過液として精製されたタンパク質を回収する場合には、溶液のｐＨは目的タンパク質のｐＩより低い値であることが好ましいが、溶液が塩を含む場合には、溶液のｐＨは目的タンパク質のｐＩより高い値であることも可能である。具体的には、細胞培養液と同程度の０．１ｍｏｌ／ＬのＮａＣｌを塩として含む溶液の場合には、ｐＨは透過する目的タンパク質のｐＩ（ｐＩｂ）に対して、ｐＩｂ＋１より低く調整されれば、ｐＩの値より高いｐＨであっても、目的タンパク質を透過液として回収することができる。 When the solution containing the target protein is passed through an anion exchange membrane and the purified protein is recovered as a permeate, the pH of the solution is preferably lower than the pI of the target protein. If included, the pH of the solution can be higher than the pI of the target protein. Specifically, in the case of a solution containing 0.1 mol / L NaCl equivalent to the cell culture solution as a salt, the pH is adjusted to be lower than pIb + 1 with respect to pI (pIb) of the target protein to be permeated. For example, the target protein can be recovered as a permeate even at a pH higher than the value of pI.
アニオン交換膜に吸着した目的タンパク質を溶出、回収する場合、塩を含む溶液、又は吸着されたタンパク質のｐＩ値よりもｐＨを低く調整した溶液を、溶出液として用いる。塩濃度は０．１ｍｏｌ／Ｌ以上２．０ｍｏｌ／Ｌ以下が好ましく、より好ましくは０．１５ｍｏｌ／Ｌ以上１．０ｍｏｌ／Ｌ以下である。塩濃度が低すぎると、十分な溶出が得られない傾向にあり、塩濃度が高すぎると、目的タンパク質溶液の脱塩に負荷がかかる傾向にある。ｐＨを調整した溶液のｐＨは、塩濃度が０．１ｍｏｌ／Ｌ以下の場合に、目的タンパク質のｐＩより低い値に調整することが好ましく、その範囲において具体的な値は目的タンパク質の種類に依存する。 When the target protein adsorbed on the anion exchange membrane is eluted and recovered, a solution containing a salt or a solution adjusted to have a pH lower than the pI value of the adsorbed protein is used as the eluent. The salt concentration is preferably from 0.1 mol / L to 2.0 mol / L, more preferably from 0.15 mol / L to 1.0 mol / L. If the salt concentration is too low, sufficient elution tends not to be obtained, and if the salt concentration is too high, the desalting of the target protein solution tends to be burdened. The pH of the adjusted solution is preferably adjusted to a value lower than the pI of the target protein when the salt concentration is 0.1 mol / L or less, and the specific value in this range depends on the type of the target protein. To do.
目的タンパク質を溶出、回収する場合の効率を示す、膜体積あたりの回収量は、精製処理が迅速かつ効率的に実施されるために、１０ｍｇ／ｍＬ以上であることが好ましく、より好ましくは２０ｍｇ／ｍＬ以上、さらに好ましくは３０ｍｇ／ｍＬ以上である。膜体積あたりの回収量は、用いるアニオン交換膜の目的とするタンパク質の動的吸着容量、及び供給量によって異なるが、これらの値が大きいほど大きくなる。したがって、目的とする高分子量タンパク質の動的吸着容量の大きなアニオン交換膜が用いられる。 The amount recovered per membrane volume, which indicates the efficiency in eluting and recovering the target protein, is preferably 10 mg / mL or more, more preferably 20 mg / mL, in order to carry out the purification process quickly and efficiently. mL or more, more preferably 30 mg / mL or more. The recovered amount per membrane volume varies depending on the target protein dynamic adsorption capacity and the supply amount of the anion exchange membrane to be used, but increases as these values increase. Therefore, an anion exchange membrane having a large dynamic adsorption capacity for the target high molecular weight protein is used.
本発明の目的タンパク質は特には限定しないが、分子量が１００ｋDa以上であると、アニオン交換膜への吸着容量がカラムに比べて優位に高いため、好ましい。高分子量の目的タンパク質は、分子量が１００ｋＤａ以上であれば特に限定されないが、血漿分画製剤として回収される、有用なタンパク質である免疫グロブリン（ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＥ、及びＩｇＤ）、血液凝固第ＶＩＩＩ因子、フォンビルブラント因子、フィブリノゲン、大腸菌あるいは細胞培養によって得られる酵素、並びに動物あるいは植物の組織から抽出される有用タンパク質が挙げられ、本実施の形態に係る精製方法は、これらのタンパク質の精製に有効である。 The target protein of the present invention is not particularly limited, but a molecular weight of 100 kDa or more is preferable because the adsorption capacity to the anion exchange membrane is significantly higher than that of the column. The target protein having a high molecular weight is not particularly limited as long as the molecular weight is 100 kDa or more. However, useful proteins such as immunoglobulins (IgG, IgM, IgA, IgE, and IgD) recovered as a plasma fractionation product, blood coagulation Examples include Factor VIII, von Willebrand Factor, fibrinogen, E. coli or enzymes obtained by cell culture, and useful proteins extracted from animal or plant tissues. The purification method according to the present embodiment uses these proteins. Effective for purification.
目的タンパク質と、分子量の異なる複数の不純物タンパク質と、を含有する溶液は、不純物タンパク質以外の不純物をさらに含みうる。不純物の種類は特に限定されないが、高分子量タンパク質を目的タンパク質とする場合に一般的な、有機溶剤、界面活性剤、脂質、さらには高ｐＩタンパク質などの、アニオン交換膜に吸着しにくい性質を有する不純物がある。アニオン交換膜に吸着しにくい不純物は、目的タンパク質をアニオン交換膜に吸着させる過程において、非吸着成分として透過するために、目的タンパク質を溶出して回収することにより、除去することができる。 The solution containing the target protein and a plurality of impurity proteins having different molecular weights may further contain impurities other than the impurity protein. The type of impurity is not particularly limited, but has a property that it is difficult to adsorb on an anion exchange membrane such as organic solvents, surfactants, lipids, and even high pI proteins, which are common when a high molecular weight protein is used as a target protein. There are impurities. Impurities that are difficult to adsorb on the anion exchange membrane can be removed by eluting and collecting the target protein in order to permeate it as a non-adsorbed component in the process of adsorbing the target protein on the anion exchange membrane.
また、アニオン交換膜に吸着する不純物は、溶出条件を制御することによって除去することができる。例えば、目的タンパク質ではない低分子量の不純物タンパク質は、一般に高分子量タンパク質を溶出するための溶液の塩濃度より低い塩濃度の溶液で溶出される傾向にある。そのため、例えば、予め０．１ｍｏｌ／Ｌ以下の低塩濃度の溶出液で低分子量タンパク質を溶出し、次いで０．１ｍｏｌ／Ｌ以上の高塩濃度の溶出液で精製された目的タンパク質を溶出し、回収する。 Impurities adsorbed on the anion exchange membrane can be removed by controlling the elution conditions. For example, a low molecular weight impurity protein that is not the target protein generally tends to be eluted in a solution having a salt concentration lower than the salt concentration of the solution for eluting the high molecular weight protein. Therefore, for example, the low molecular weight protein is eluted with a low salt concentration eluate of 0.1 mol / L or less in advance, and then the purified target protein is eluted with a high salt concentration eluate of 0.1 mol / L or more, to recover.
以下、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、本実施の形態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 examples and comparative examples (also simply referred to as “examples” in the present specification). The scope of the present embodiment is as follows. It is not limited to examples only.
（目的タンパク質と広い分子量範囲の不純物タンパク質を含む混合液のアニオン交換膜への通液）
２０ｍｍｏｌ／ＬのＴｒｉｓ−ＨＣｌ（ｐＨ７．５）緩衝液に、目的タンパク質として、キモトリプシノーゲン（α−ｃｈｙｍｏｔｒｙｐｓｉｎｏｇｅｎ−Ａ、分子量２５ｋＤａ、ｐＩ９．２、Ｓｉｇｍａ−Ａｌｄｒｉｃｈ製）０．２５ｇ／Ｌを含む溶液に、不純物タンパク質として、表１に示すように、チログロブリン（ＴＨＹ）（分子量６６０ｋＤａ、Ｓｉｇｍａ−Ａｌｄｒｉｃｈ製）、β−ラクトグロブリン（ＢＬＧ）（分子量１８ｋＤａ、Ｓｉｇｍａ−Ａｌｄｒｉｃｈ製）、オブアルブミン（ＯＶＡ）（分子量４３ｋＤａ、Ｓｉｇｍａ−Ａｌｄｒｉｃｈ製）、及びウシ血清アルブミン（ＢＳＡ）（分子量６５ｋＤａ、Ｓｉｇｍａ−Ａｌｄｒｉｃｈ製）の分子量の異なる４種類のタンパク質をそれぞれの濃度が、０．２５ｇ／Ｌとなるように添加し、溶解した後、ザルトリウス社製の精密ろ過膜ミニザルト（最大細孔径０．２２μｍ）を用いてろ過し、評価に用いる、目的タンパク質と高分子量のタンパク質を含む４種類の不純物タンパク質の混合溶液を調製した。表１に示すように、少なくとも分子量が１００ｋＤａ未満であるＢＳＡの等電点と、分子量が１００ｋＤａ以上のチログロブリンの等電点と、の差は、１以上であった。
In a solution containing 0.25 g / L of chymotrypsinogen (α-chymotrypsinogen-A, molecular weight 25 kDa, pI9.2, Sigma-Aldrich) as a target protein in a 20 mmol / L Tris-HCl (pH 7.5) buffer. As shown in Table 1, as an impurity protein, thyroglobulin (THY) (molecular weight 660 kDa, manufactured by Sigma-Aldrich), β-lactoglobulin (BLG) (molecular weight 18 kDa, manufactured by Sigma-Aldrich), ovalbumin (OVA) ( The molecular weight of 43 kDa, Sigma-Aldrich), and bovine serum albumin (BSA) (molecular weight of 65 kDa, Sigma-Aldrich) are different from each other in the concentration of 0.25 g / L. After adding and dissolving, it is filtered using Sartorius microfiltration membrane mini-salt (maximum pore size 0.22 μm), and mixed with 4 types of impurity proteins including target protein and high molecular weight protein used for evaluation A solution was prepared. As shown in Table 1, the difference between the isoelectric point of BSA having a molecular weight of less than 100 kDa and the isoelectric point of thyroglobulin having a molecular weight of 100 kDa or more was 1 or more.
グラフト鎖を有し、アニオン交換基として３級アミンを有する多孔膜状のアニオン交換膜として、旭化成メディカル社製ＱｙｕＳｐｅｅｄ（登録商標）Ｄ （ＱＳＤ） ０．６ｍＬ （細孔径０．２−０．３μｍ、イオン交換容量０．５５−０．６ｍｍｏｌ／ｍＬ）を用意し、２０ｍｍｏｌ／ＬのＴｒｉｓ−ＨＣｌ（ｐＨ７．５）緩衝液を２０ｍＬ通液して平衡化した後、膜体積の１４０倍の溶液体積（１４０ＭＶ）に相当する８４ｍＬの、タンパク質混合溶液を透過させ、１０ＭＶごとの透過フラクションを採取した。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８ｍＬ／ｍｉｎにて通液した。この流速は、毎分膜体積の１３．３倍の溶液を供給しており、規格化した流速１３．３ＭＶ／ｍｉｎと記載する。 As a porous membrane-shaped anion exchange membrane having a graft chain and a tertiary amine as an anion exchange group, QyuSpeed (registered trademark) D (QSD) 0.6 mL (pore diameter 0.2-0.3 μm) manufactured by Asahi Kasei Medical Co., Ltd. And an ion exchange capacity of 0.55-0.6 mmol / mL), and 20 mL of 20 mmol / L Tris-HCl (pH 7.5) buffer solution was allowed to equilibrate, and then the solution was 140 times the membrane volume. 84 mL of the protein mixed solution corresponding to the volume (140 MV) was permeated, and a permeate fraction was collected every 10 MV. The solution was passed at a flow rate of 8 mL / min from the inside to the outside of the hollow fiber porous membrane in the evaluation module. This flow rate supplies a solution of 13.3 times the membrane volume per minute and is described as a normalized flow rate of 13.3 MV / min.
得られた透過フラクションに含まれるタンパク質を評価するために、１０−２０ＭＶ、４０−５０ＭＶ、７０−８０ＭＶ、１００−１１０ＭＶ及び１３０−１４０ＭＶの透過フラクションについて、ＳＤＳ−ＰＡＧＥを用いて解析した。分析に用いる透過液１０μＬを同量のサンプル処理液（第一化学薬品株式会社製、トリスＳＤＳ β ＭＥサンプル処理液）と混合し、１００℃で５分間熱処理した。得られたサンプルを、マイクロピペットを用いて電気泳動用ゲルプレート（第一化学薬品株式会社製、マルチゲルＩＩミニ８／１６）に１ウェルにつき１０μＬを適用した。次に、泳動用緩衝液（第一化学薬品株式会社製、ＳＤＳ−トリス−グリシン泳動緩衝液を１０倍希釈して使用）を満たした電気泳動槽（和光純薬株式会社製、ＥａｓｙＳｅｐａｒａｔｏｒ（登録商標））に電気泳動用ゲルプレートを挿入し、３０ｍＡの定電流で１時間泳動させ、透過液中のタンパク質を分離した。泳動後、図１に示すように、ゲルプレートを染色試薬（フナコシ株式会社製、ＩｎｓｔａｎｔＢｌｕｅ）で染色した。 In order to evaluate the protein contained in the obtained permeation fraction, the permeation fractions of 10-20 MV, 40-50 MV, 70-80 MV, 100-110 MV, and 130-140 MV were analyzed using SDS-PAGE. 10 μL of the permeate used for analysis was mixed with the same amount of sample processing solution (Tris SDS β ME sample processing solution, manufactured by Daiichi Chemical Co., Ltd.) and heat-treated at 100 ° C. for 5 minutes. Using the obtained sample, 10 μL per well was applied to a gel plate for electrophoresis (manufactured by Daiichi Chemical Co., Ltd., Multigel II Mini 8/16) using a micropipette. Next, an electrophoresis tank (manufactured by Wako Pure Chemical Industries, Ltd., EasySeparator (registered trademark)) filled with a buffer for electrophoresis (Daiichi Chemical Co., Ltd., using SDS-Tris-Glycine electrophoresis buffer diluted 10-fold). )), An electrophoresis gel plate was inserted, and electrophoresis was performed at a constant current of 30 mA for 1 hour to separate proteins in the permeate. After the electrophoresis, as shown in FIG. 1, the gel plate was stained with a staining reagent (Funakoshi Co., Ltd., InstantBlue).
図１において、レーン１は分子量マーカー、レーン２は透過前のタンパク質溶液、レーン３からレーン７は、それぞれ１０−２０ＭＶ、４０−５０ＭＶ、７０−８０ＭＶ、１００−１１０ＭＶ及び１３０−１４０ＭＶの透過フラクションの泳動結果である。この結果から、透過量５０ＭＶまでは全分子量の不純物タンパク質が除去されて、分子量２５ｋＤａの目的タンパク質であるキモトリプシノーゲンのみが透過液中に含まれ、透過量８０ＭＶ以降で不純物タンパク質が徐々に漏れ始めていている事が示された。 In FIG. 1, lane 1 is a molecular weight marker, lane 2 is a protein solution before permeation, lane 3 to lane 7 are permeation fractions of 10-20 MV, 40-50 MV, 70-80 MV, 100-110 MV and 130-140 MV, respectively. It is an electrophoresis result. From this result, the impurity protein with the total molecular weight was removed until the permeation amount was 50 MV, and only the chymotrypsinogen, which is the target protein with a molecular weight of 25 kDa, was contained in the permeate. It was shown that
［比較例１］
（目的タンパク質と広い分子量範囲の不純物タンパク質を含むタンパク質混合液のアニオン交換カラムへの通液）
実施例１で用いたものと同じ、目的タンパク質と異なる分子量の４種類の不純物タンパク質の混合溶液を、アニオン交換カラムに通液し、透過液中に含まれる各タンパク質の濃度を評価した。透過フラクションはカラム体積の１０倍に相当する体積（１０ＣＶ）ごとに採取した。アニオン交換カラムとして、ＧＥヘルスケア製、ＨｉＴｒａｐＱ ＨＰ＿１ｍｌ（粒子径９０μｍ、イオン交換容量０．１８−０．２６ｍｍｏｌ／ｍＬ）を用い、流速２ｍＬ／ｍｉｎ（２ＣＶ／ｍｉｎ、あるいは３００ｃｍ／ｈｒ）で、実施例１と同様の方法で通液し、その透過フラクションをＳＤＳ−ＰＡＧＥを用いて評価した。結果を実施例１と同じく図１に示す。レーン８は透過前のタンパク質溶液、レーン９からレーン１３は、それぞれ１０−２０ＣＶ、４０−５０ＣＶ、７０−８０ＣＶ、１００−１１０ＣＶ及び１３０−１４０ＣＶの透過フラクションの泳動結果である。この結果から、アニオン交換カラムを用いた場合、最初の透過フラクションから、高分子量の不純物タンパク質が含まれ、実施例１に比べて目的タンパク質の十分な精製が実現されない事が示された。
[Comparative Example 1]
(Passing the protein mixture containing the target protein and impurity protein in a wide molecular weight range through the anion exchange column)
A mixed solution of four types of impurity proteins having the same molecular weight as that of the target protein, as used in Example 1, was passed through an anion exchange column, and the concentration of each protein contained in the permeate was evaluated. The permeated fraction was collected for each volume (10 CV) corresponding to 10 times the column volume. As an anion exchange column, HI Healthcare's HiTrapQ HP — 1 ml (particle diameter 90 μm, ion exchange capacity 0.18-0.26 mmol / mL) was used, and the flow rate was 2 mL / min (2 CV / min or 300 cm / hr). The liquid was passed in the same manner as in Example 1, and the permeated fraction was evaluated using SDS-PAGE. The results are shown in FIG. Lane 8 is the protein solution before permeation, and lane 9 to lane 13 are the electrophoresis results of the permeated fractions of 10-20 CV, 40-50 CV, 70-80 CV, 100-110 CV and 130-140 CV, respectively. From this result, it was shown that, when an anion exchange column was used, the first permeation fraction contained a high molecular weight impurity protein, and the target protein could not be sufficiently purified as compared with Example 1.
（タンパク質混合液のアニオン交換膜への通液）
表２に示すように、２０ｍｍｏｌ／ＬのＴｒｉｓ−ＨＣｌ（ｐＨ７．５）緩衝液に、チログロブリン（ＴＨＹ）（分子量６６０ｋＤａ、Ｓｉｇｍａ−Ａｌｄｒｉｃｈ製）、β−ラクトグロブリン（ＢＬＧ）（分子量１８ｋＤａ、Ｓｉｇｍａ−Ａｌｄｒｉｃｈ製）、オブアルブミン（ＯＶＡ）（分子量４３ｋＤａ、Ｓｉｇｍａ−Ａｌｄｒｉｃｈ製）、及びウシ血清アルブミン（ＢＳＡ）（分子量６５ｋＤａ、Ｓｉｇｍａ−Ａｌｄｒｉｃｈ製）の分子量の異なる４種類のタンパク質をそれぞれの濃度が、０．２５ｇ／Ｌとなるように添加し、溶解した後、ザルトリウス社製の精密ろ過膜ミニザルト（最大細孔径０．２２μｍ）を用いてろ過し、評価に用いる、高分子量のタンパク質を含む４種類のタンパク質の混合溶液を調製した。表１に示すように、少なくとも分子量が１００ｋＤａ未満であるＢＳＡの等電点と、分子量が１００ｋＤａ以上のチログロブリンの等電点と、の差は、１以上であった。
As shown in Table 2, in a 20 mmol / L Tris-HCl (pH 7.5) buffer solution, thyroglobulin (THY) (molecular weight: 660 kDa, manufactured by Sigma-Aldrich), β-lactoglobulin (BLG) (molecular weight: 18 kDa, Sigma) -Aldrich), ovalbumin (OVA) (molecular weight 43 kDa, Sigma-Aldrich), and bovine serum albumin (BSA) (molecular weight 65 kDa, Sigma-Aldrich) with different concentrations of four proteins, After adding and dissolving so that it may become 0.25 g / L, it filters using the microfiltration membrane minisalt (maximum pore diameter 0.22 micrometer) made from Sartorius, and is used for evaluation. A mixed solution of proteins was prepared. As shown in Table 1, the difference between the isoelectric point of BSA having a molecular weight of less than 100 kDa and the isoelectric point of thyroglobulin having a molecular weight of 100 kDa or more was 1 or more.
グラフト鎖を有し、アニオン交換基として３級アミンを有する多孔膜状のアニオン交換膜として、旭化成メディカル社製ＱｙｕＳｐｅｅｄ（登録商標）Ｄ （ＱＳＤ） ０．６ｍＬ （細孔径０．２−０．３μｍ、イオン交換容量０．５５−０．６ｍｍｏｌ／ｍＬ）を用意し、２０ｍｍｏｌ／ＬのＴｒｉｓ−ＨＣｌ（ｐＨ７．５）緩衝液を２０ｍＬ通液して平衡化した後、破過が開始するまで４種類のタンパク質の混合溶液を透過させた。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８ｍＬ／ｍｉｎにて通液した。この流速は、毎分膜体積の１３．３倍の溶液を供給しており、規格化した流速１３．３ＭＶ／ｍｉｎと記載する。 As a porous membrane-shaped anion exchange membrane having a graft chain and a tertiary amine as an anion exchange group, QyuSpeed (registered trademark) D (QSD) 0.6 mL (pore diameter 0.2-0.3 μm) manufactured by Asahi Kasei Medical Co., Ltd. , Ion exchange capacity 0.55-0.6 mmol / mL), and 20 mL of 20 mmol / L Tris-HCl (pH 7.5) buffer was allowed to equilibrate and then 4 until the breakthrough started. A mixed solution of various proteins was permeated. The solution was passed at a flow rate of 8 mL / min from the inside to the outside of the hollow fiber porous membrane in the evaluation module. This flow rate supplies a solution of 13.3 times the membrane volume per minute and is described as a normalized flow rate of 13.3 MV / min.
評価はＧＥヘルスケアバイオサイエンス製ＡＫＴＡｅｘｐｌｏｒｅｒ１００を用いて実施し、同装置に設置されているＵＶ吸光度計により、透過液の２８０ｎｍのＵＶ吸光度をモニターした。膜体積の６０倍の溶液体積（６０ＭＶ）に相当する、３６ｍＬの通液量を超えるまで、透過液のＵＶ吸光度は検出されず、４種類総てのタンパク質がアニオン交換膜に吸着し、４種類総てのタンパク質が除去された透過液が得られた。 The evaluation was carried out using an AKTA explorer 100 manufactured by GE Healthcare Bioscience, and the UV absorbance of the permeated solution at 280 nm was monitored with a UV absorbance meter installed in the apparatus. The UV absorbance of the permeate is not detected until 36 mL, which corresponds to a solution volume 60 times the membrane volume (60 MV), and all four types of proteins are adsorbed on the anion exchange membrane. A permeate from which all proteins were removed was obtained.
また、２０ＭＶずつの透過液中に含まれる、各タンパク質の濃度を、ゲルろ過クロマトグラフィーにより評価した。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、０．１ｍｏｌ／Ｌのリン酸及び０．２ｍｏｌ／Ｌのアルギニン（ｐＨ６．８）を含むバッファーを用いて、３０℃において流速０．６ｍｌ／ｍｉｎでカラムに通液し、ここに評価サンプルを１０μＬ添加し、ゲルろ過カラムを透過して得られる、リテンションタイム（保持時間）ごとの２８０ｎｍのＵＶ吸光度を、システムに付属のＵＶ吸光度計により計測した。 Further, the concentration of each protein contained in each 20 MV permeate was evaluated by gel filtration chromatography. A TSKgel G3000SWXL manufactured by Tosoh Corporation is attached as a gel filtration column to the chromatograph LC-10A system manufactured by Shimadzu Corporation, and a buffer containing 0.1 mol / L phosphoric acid and 0.2 mol / L arginine (pH 6.8) is provided. The sample was passed through the column at a flow rate of 0.6 ml / min at 30 ° C., 10 μL of the evaluation sample was added thereto, and the UV absorbance at 280 nm for each retention time (retention time) obtained by permeation through the gel filtration column. Was measured with a UV absorbance meter attached to the system.
まず、各タンパク質単独の溶液をゲルろ過クロマトグラフィーによって評価することにより、各タンパク質のリテンションタイムを計測すると共に、ピーク面積とタンパク質濃度との検量線を作成し、この結果をもとに、アニオン交換膜の透過液に含まれる、各タンパク質の濃度を求めた。図２に透過液量と、その透過液に含まれる、各タンパク質の濃度と、をプロットした。ここで、タンパク質濃度は、供給液濃度ｃ０に対する透過液中の濃度ｃの比、ｃ／ｃ０で表している。図２より、ＱＳＤを透過することにより、供給液に含まれる異なる分子量の全タンパク質が、透過液量６０ＭＶまで、透過液中から完全に除去されていることが示された。 First, the solution of each protein alone is evaluated by gel filtration chromatography to measure the retention time of each protein and to create a calibration curve between peak area and protein concentration. Based on this result, anion exchange The concentration of each protein contained in the membrane permeate was determined. FIG. 2 plots the amount of permeate and the concentration of each protein contained in the permeate. Here, the protein concentration is represented by c / c0, which is the ratio of the concentration c in the permeate to the supply liquid concentration c0. From FIG. 2, it was shown that all the proteins having different molecular weights contained in the feed solution were completely removed from the permeate up to a permeate amount of 60 MV by permeating QSD.
［比較例２］
（タンパク質混合液のアニオン交換カラムへの通液）
実施例２で用いたものと同じ、異なる分子量の４種類のタンパク質の混合溶液を、アニオン交換カラムに通液し、透過液中に含まれる各タンパク質の濃度を評価した。アニオン交換カラムとして、ＧＥヘルスケア製、ＨｉＴｒａｐＱ ＨＰ＿１ｍｌ（粒子径９０μｍ、イオン交換容量０．１８−０．２６ｍｍｏｌ／ｍＬ）を用い、流速２ｍＬ／ｍｉｎ（２ＣＶ／ｍｉｎ、あるいは３００ｃｍ／ｈｒ）で、実施例２と同様の方法で通液した。実施例２のアニオン交換膜からの透過液と異なり、アニオン交換カラムでは、最初の透過フラクションからＵＶ吸光度が検出され、透過液にタンパク質が含まれていることが示された。
[Comparative Example 2]
(Flow of protein mixture through anion exchange column)
A mixed solution of four types of proteins having the same molecular weight as that used in Example 2 was passed through an anion exchange column, and the concentration of each protein contained in the permeate was evaluated. As an anion exchange column, HI Healthcare's HiTrapQ HP — 1 ml (particle diameter 90 μm, ion exchange capacity 0.18-0.26 mmol / mL) was used, and the flow rate was 2 mL / min (2 CV / min or 300 cm / hr). The liquid was passed in the same manner as in Example 2. Unlike the permeate from the anion exchange membrane of Example 2, in the anion exchange column, UV absorbance was detected from the first permeate fraction, indicating that the permeate contained protein.
カラム体積の２０倍に相当する体積（２０ＣＶ）ごとの、透過液に含まれる各タンパク質の濃度を、実施例２と同様にしてゲルろ過クロマトグラフィーにより評価した。図３にアニオン交換カラムの透過液量と、その透過液に含まれる、各タンパク質の濃度をプロットした。図３より、最初の透過フラクションの段階から透過液中にタンパク質が含まれており、特に分子量１００ｋＤａ以上の高分子量のタンパク質が、吸着されずに透過することが確認された。 The concentration of each protein contained in the permeate for each volume (20 CV) corresponding to 20 times the column volume was evaluated by gel filtration chromatography in the same manner as in Example 2. FIG. 3 plots the permeate amount of the anion exchange column and the concentration of each protein contained in the permeate. From FIG. 3, it was confirmed that proteins were contained in the permeate from the first permeation fraction stage, and in particular, high molecular weight proteins having a molecular weight of 100 kDa or more permeate without being adsorbed.
（目的タンパク質の回収）
実施例２で調製した、４種類のタンパク質を不純物タンパク質とし、当該４種類のタンパク質を含む混合溶液に、塩濃度０．１ｍｏｌ／ＬとなるようにＮａＣｌを添加し、さらに目的タンパク質として抗体ＩｇＧタンパク質（株式会社ベネシス製、献血ヴェノグロブリン−ＩＨ、分子量約１７０ｋＤａ、ｐＩ約７〜８）を２ｍｇ／ｍＬとなるように添加した後、ｐＨを、抗体ＩｇＧタンパク質のｐIの値と、１と、の和で得られる値以下の８．０に調整した。得られた溶液を実施例１と同様にしてアニオン交換膜ＱＳＤに、６０ＭＶに相当する体積だけ通液し、透過液を回収した。
(Recovery of target protein)
Four types of proteins prepared in Example 2 are used as impurity proteins, NaCl is added to a mixed solution containing the four types of proteins so that the salt concentration is 0.1 mol / L, and antibody IgG protein is used as a target protein. (Benesis Co., Ltd., blood donation-venoglobulin-IH, molecular weight of about 170 kDa, pI of about 7-8) was added to 2 mg / mL, and then the pH was adjusted to the value of pI of the antibody IgG protein and 1. It adjusted to 8.0 below the value obtained by the sum. The obtained solution was passed through the anion exchange membrane QSD by a volume corresponding to 60 MV in the same manner as in Example 1, and the permeate was collected.
得られた透過液を、実施例２と同様にして、ゲルろ過クロマトグラフィーにより評価したところ、ＩｇＧであることを示す分子量１７０ｋＤａのみのピークが確認され、不純物である広範囲の分子量に渡る４種類のタンパク質が除去され、目的タンパク質のみが回収されたことが示された。また、ＩｇＧタンパク質溶液の濃度と２８０ｎｍでのＵＶ吸光度から得られる検量線から、透過液中のＩｇＧタンパク質の濃度を評価したところ、１．９ｍｇ／ｍＬであった。これより、目的タンパク質であるＩｇＧの回収率は９５％であった。 The obtained permeate was evaluated by gel filtration chromatography in the same manner as in Example 2. As a result, a peak with only a molecular weight of 170 kDa indicating IgG was confirmed, and four kinds of impurities over a wide range of molecular weights as impurities were observed. It was shown that the protein was removed and only the target protein was recovered. Further, the concentration of IgG protein in the permeate was evaluated from a calibration curve obtained from the concentration of the IgG protein solution and the UV absorbance at 280 nm and found to be 1.9 mg / mL. From this, the recovery rate of the target protein IgG was 95%.
（タンパク質混合溶液からの高分子量タンパク質（ＴＨＹ）の回収）
実施例２と同様にして、ＴＨＹ、β−ラクトグロブリン、オブアルブミン、ＢＳＡをそれぞれ０．２５ｇ／ｍＬ溶解した混合タンパク質溶液を作成した。ただし、実施例４においては、ＴＨＹを高分子量の目的タンパク質とし、β−ラクトグロブリン、オブアルブミン、ＢＳＡを目的としない低分子量のタンパク質とする。この混合溶液をＱＳＤの膜体積の６０倍に相当する３６ｍＬを、ＱＳＤに通液し、緩衝液で洗浄した。次に、目的としない低分子量のタンパク質を溶出するために０．１ｍｏｌ／ＬのＮａＣｌを含む、前記緩衝液を２０ｍＬ通液し、その後、目的とするＴＨＹを溶出するために０．２ｍｏｌ／ＬのＮａＣｌを含む緩衝液を同様に１０ｍＬ通液し、この溶出液を回収した。
(Recovery of high molecular weight protein (THY) from protein mixed solution)
In the same manner as in Example 2, a mixed protein solution was prepared by dissolving 0.25 g / mL of THY, β-lactoglobulin, ovalbumin, and BSA. However, in Example 4, THY is a high molecular weight target protein, and β-lactoglobulin, ovalbumin, and BSA are low molecular weight proteins not intended. 36 mL of this mixed solution corresponding to 60 times the membrane volume of QSD was passed through QSD and washed with a buffer solution. Next, 20 mL of the buffer solution containing 0.1 mol / L NaCl is passed to elute the low molecular weight protein which is not the target, and then 0.2 mol / L is used to elute the target THY. Similarly, 10 mL of a buffer solution containing NaCl was passed, and this eluate was recovered.
回収した０．２ｍｏｌ／ＬのＮａＣｌを含む緩衝液よりなる溶出液に含まれる、各タンパク質の濃度を実施例１と同様にして、ゲルろ過クロマトグラフィーにより評価したところ、目的タンパク質であるＴＨＹは０．７６ｍｇ／ｍＬ、目的としないタンパク質であるβ−ラクトグロブリン、オブアルブミン、及びＢＳＡはそれぞれ、０．０１ｍｇ／ｍＬ、０．０１ｍｇ／ｍＬ、０．０１２ｍｇ／ｍＬであった。また、ＴＨＹの回収率は８４％、膜体積あたりの回収量は１２．６ｍｇ／ｍＬ−ａｄであった。この結果から、溶出条件を制御することにより、目的とするタンパク質を濃縮、精製して回収可能なことが示された。 When the concentration of each protein contained in the recovered eluate comprising 0.2 mol / L NaCl-containing buffer was evaluated by gel filtration chromatography in the same manner as in Example 1, the target protein THY was 0. .76 mg / mL, β-lactoglobulin, ovalbumin, and BSA, which were not intended proteins, were 0.01 mg / mL, 0.01 mg / mL, and 0.012 mg / mL, respectively. Moreover, the recovery rate of THY was 84%, and the recovery amount per membrane volume was 12.6 mg / mL-ad. From this result, it was shown that the target protein can be concentrated and purified and recovered by controlling the elution conditions.
（目的タンパク質（ＦＶＩＩＩ）からの不純物界面活性剤の除去と濃縮）
血液凝固第ＶＩＩＩ因子（日本赤十字社製、ＣＲＯＳＳ ＥＩＧＨＴ Ｍ（登録商標））０．０１ｇ、２０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）緩衝液９５０ｍＬ、界面活性剤、Ｔｗｅｅｎ２０（ポリオキシエチレンソルビタンモノラウラート、及び東京化成工業より購入）１０ｍＬを混合し、０．１％の界面活性剤を含み、血液凝固第ＶＩＩＩ因子の濃度が０．０１ｇ／Ｌの希薄溶液を作成した。この溶液の全量を、実施例１と同様の方法で、アニオン交換膜モジュール、旭化成メディカル社製ＱｙｕＳｐｅｅｄ Ｄ（ＱＳＤ） ０．６ｍＬに通液して血液凝固第ＶＩＩＩ因子を同モジュールに吸着させ、２０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）緩衝液１５ｍＬを通液して、モジュールを洗浄した後、１．０ｍｏｌ／Ｌ ＮａＣｌを含む、２０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）緩衝液１０ｍＬを通液して、吸着した血液凝固第ＶＩＩＩ因子を溶出、回収した。
(Removal and concentration of impurity surfactant from target protein (FVIII))
Blood coagulation factor VIII (Japan Red Cross, CROSS EIGHT M (registered trademark)) 0.01 g, 20 mmol / L Tris-HCl (pH 8.0) buffer 950 mL, surfactant, Tween 20 (polyoxyethylene sorbitan monolaur 10 ml of Lato and purchased from Tokyo Chemical Industry Co., Ltd.) were mixed, and a dilute solution containing 0.1% surfactant and a blood coagulation factor VIII concentration of 0.01 g / L was prepared. The whole amount of this solution was passed through 0.6 mL of an anion exchange membrane module, QyuSpeed D (QSD) manufactured by Asahi Kasei Medical Corporation in the same manner as in Example 1 to adsorb blood coagulation factor VIII to the module, 20 mmol After passing 15 mL of / L Tris-HCl (pH 8.0) buffer and washing the module, 10 mL of 20 mmol / L Tris-HCl (pH 8.0) buffer containing 1.0 mol / L NaCl was passed through. The adsorbed blood coagulation factor VIII was eluted and collected.
得られた回収液の回収率を実施例２と同様の方法によりゲルろ過クロマトグラフィーの方法で評価したところ、回収液中の血液凝固第ＶＩＩＩ因子の濃度は、０．９７ｇ／ｍＬであり、回収率は９７％であった。また、回収液に界面活性剤は含まれていなかった。これらの結果より、不純物である界面活性剤が除去され、精製及び濃縮された血液凝固第ＶＩＩＩ因子が得られることが確認された。 When the recovery rate of the obtained recovered liquid was evaluated by gel filtration chromatography in the same manner as in Example 2, the concentration of blood coagulation factor VIII in the recovered liquid was 0.97 g / mL. The rate was 97%. Further, the recovered liquid contained no surfactant. From these results, it was confirmed that the surfactant, which is an impurity, was removed, and purified and concentrated blood coagulation factor VIII was obtained.
（細胞培養液からの不純物タンパク質の除去）
Ｉｎｖｉｔｒｏｇｅｎ社製、ＣＤ ＯｐｔｉＣＨＯ無血清培地を用いて得られた細胞培養液を、ザルトリウス社製の精密ろ過膜ミニザルト（最大細孔径０．２２μｍ）を用いてろ過し、評価に用いる不純物溶液を得た。また、この不純物溶液６０ｍＬをアニオン交換膜であるＱＳＤに通液し、膜体積の１００倍（１００ＭＶ）に相当する体積の、ｐＨ７．５及び電気伝導度１０ｍＳ／ｃｍの不純物溶液の透過液を得た。体積２ｍＬの得られた不純物溶液、及びその透過液に、それぞれ３倍量の体積（６ｍＬ）の、−２０℃に冷却したアセトンを添加し、−２０℃にて７２時間放置後、遠心分離機を用いて沈殿物を回収することにより、それぞれの溶液に含まれていた、塩及び各種夾雑物が除去された不純物タンパク質を得た。
(Removal of impurity proteins from cell culture)
The cell culture solution obtained using CD OptiCHO serum-free medium manufactured by Invitrogen was filtered using a microfiltration membrane mini-salt (maximum pore size 0.22 μm) manufactured by Sartorius, and an impurity solution used for evaluation was obtained. . Further, 60 mL of this impurity solution was passed through QSD as an anion exchange membrane to obtain a permeate of an impurity solution having a volume corresponding to 100 times the membrane volume (100 MV) and having a pH of 7.5 and an electric conductivity of 10 mS / cm. It was. 3 mL volume (6 mL) of acetone cooled to −20 ° C. was added to the obtained impurity solution having a volume of 2 mL and the permeate, and allowed to stand at −20 ° C. for 72 hours, and then centrifuged. By collecting the precipitate using, an impurity protein contained in each solution from which salts and various impurities were removed was obtained.
得られたそれぞれの不純物タンパク質の分子量及びｐＩの分布を調べるために、Ｉｎｖｉｔｏｒｏｇｅｎ社製ＺＯＯＭ ＩＰＧ Ｒｕｎｎｅｒを用いて、２次元電気泳動を実施した。ここで、ＺＯＯＭ ＩＰＧ Ｒｕｎｎｅｒとは、評価に必要な泳動槽及び試薬類を含む、装置一式の名称である。それぞれの、得られた不純物タンパク質に、１３０μＬの泳道用サンプル調製液（８．４ｍｏｌ／Ｌ Ｕｒｅａ，２．４ｍｏｌ／Ｌ Ｔｈｉｏｕｒｅａ， ４．８％ＣＨＡＰＳをソニケーターと撹拌にて溶解し、０．２２ｕｍフィルターにて濾過後、分注し−２０℃にて保管した後、室温に戻したもの）、１．５μＬのＩｎｖｉｔｏｒｏｇｅｎ社製 ＺＯＯＭキャリア アンフォライト（ｐＨ３−１０）、１．５μＬの０．２％ＢＰＢ（ブロモフェノールブルー、Ｓｉｇｍａ−Ａｌｄｒｉｃｈ社製）水溶液を添加し、さらに精製水を加えて１５５μＬとして溶解し、泳動用サンプルを調製した。得られたサンプル１５５μＬを、１次元目のストリップゲル（Ｉｎｖｉｔｏｒｏｇｅｎ社製 ＺＯＯＭストリップゲルｐＨ３−１０（ｎｏｎ−Ｌｉｎｅａｒ））に添加し、パワーサプライ（Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ（現ＧＥヘルスケアバイオサイエンス）社製 Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ Ｐｏｗｅｒ Ｓｕｐｐｌｙ−ＥＰＳ ３５００ＸＬ）を使用して、１７５Ｖにて２０分間、次いで１７５Ｖから２０００Ｖまで昇圧しながら ４５分間、さらに２０００Ｖにて６０分間の電気勾配をかけて泳動を行った。 In order to examine the molecular weight and pI distribution of each impurity protein obtained, two-dimensional electrophoresis was performed using a ZOOM IPG Runner manufactured by Invitrogen. Here, ZOOM IPG Runner is the name of a set of apparatuses including a migration tank and reagents necessary for evaluation. In each of the obtained impurity proteins, 130 μL of sample preparation solution for swimming road (8.4 mol / L Urea, 2.4 mol / L Thiourea, 4.8% CHAPS was dissolved by stirring with a sonicator, and a 0.22 μm filter. After filtration at -20 ° C., the solution was dispensed and returned to room temperature, 1.5 μL of Invitrogen's ZOOM carrier ampholite (pH 3-10), 1.5 μL of 0.2% BPB An aqueous solution (bromophenol blue, manufactured by Sigma-Aldrich) was added, and purified water was further added to dissolve as 155 μL to prepare a sample for electrophoresis. 155 μL of the obtained sample was added to a first-dimensional strip gel (ZOOM strip gel pH3-10 (non-Linear) manufactured by Invitrogen), and Electrophoresis Power manufactured by Power Supply (Pharmacia Biotech (now GE Healthcare Bioscience)). Using Supply-EPS 3500XL), electrophoresis was performed by applying an electric gradient of 175 V for 20 minutes, then increasing pressure from 175 V to 2000 V for 45 minutes, and further 2000 V for 60 minutes.
一次元目の電気泳動終了後のストリップゲルは、室温で緩やかに振とうしながらＳＤＳ処理を１５分間行った。ＳＤＳ処理液として、ストリップゲル１本に対してＩｎｖｉｔｏｒｏｇｅｎ社製 ＬＤＳ Ｓａｍｐｌｅ Ｂｕｆｆｅｒ （４ｘ） １．２５ｍＬを精製水で５ｍＬに希釈したものを用いた。二次元目の電気泳動では、Ｉｎｖｉｔｏｒｏｇｅｎ社製 ＮｕＰａｇｅ ４−１２％ Ｂｉｓ−Ｔｒｉｓ ＺＯＯＭ ｇｅｌｓ（１０ｍｍ・ＩＰＧｗｅｌｌ） にＳＤＳ処理を行ったストリップゲルを添加し、泳動用緩衝液にはＩｎｖｉｔｏｒｏｇｅｎ社製ＭＯＰＳ ＳＤＳ Ｒｕｎｎｉｎｇ Ｂｕｆｆｅｒ（２０ｘ）を精製水にて１Ｘに希釈したものを使用した。また、分子量サイズマーカーには、Ｉｎｖｉｔｏｒｏｇｅｎ社製 Ｎｏｖｅｘ Ｓｈａｒｐ Ｕｎｓｔａｉｎｅｄ Ｐｒｏｔｅｉｎ Ｓｔａｎｄａｒｄ を用い、２００Ｖにて５５分間、定電圧で泳動を行った。二次元目の泳動を終了したゲルは、メタノール５０％・酢酸１０％を精製水に溶解した固定液に浸し、室温で一晩静置した。固定したゲルは、コスモ・バイオ株式会社製２Ｄ−銀染色試薬・ＩＩを用いて、銀染色を行い、分子量及びｐＩ分布を示す、２次元電気泳動の結果を得た。 The strip gel after the completion of the first-dimensional electrophoresis was subjected to SDS treatment for 15 minutes while gently shaking at room temperature. As the SDS treatment solution, a solution obtained by diluting 1.25 mL of LDS Sample Buffer (4x) manufactured by Invitrogen to 5 mL with one strip gel was used. In the second-dimensional electrophoresis, strip gel treated with SDS is added to NuPage 4-12% Bis-Tris ZOOM gels (10 mm IPGwell) manufactured by Invitrogen, and MOPS SDS Running from Invitrogen is added to the electrophoresis buffer. A buffer (20 ×) diluted 1 × with purified water was used. In addition, Novex Sharp Unstained Protein Standard manufactured by Invitrogen was used as the molecular weight size marker, and electrophoresis was performed at 200 V for 55 minutes at a constant voltage. The gel after the second dimensional migration was immersed in a fixative solution in which 50% methanol and 10% acetic acid were dissolved in purified water, and left at room temperature overnight. The immobilized gel was subjected to silver staining using 2D-silver staining reagent II manufactured by Cosmo Bio, and the result of two-dimensional electrophoresis showing molecular weight and pI distribution was obtained.
図４に得られた２次元電気泳動の結果を示す。ここで、ａ）は細胞培養液から得られた不純物溶液に含まれていたタンパク質、ｂ）は不純物溶液のアニオン交換膜ＱＳＤの透過液に含まれていたタンパク質の、それぞれの２次元電気泳動パターンである。図４ａ）の結果から、細胞培養液に含まれる不純物タンパク質は、約３００ｋＤａまでの分子量、及び少なくとも３から９．５までの範囲に渡るｐＩの、広い範囲の分布を有していることが示される。これに対し、図４ｂ）の結果から、アニオン交換膜ＱＳＤの透過液に含まれるタンパク質は、ｐＩが４以下の全分子量範囲のタンパク質が除去されたことが確認された。よって、ｐＩの差が１以上の複数の不純物タンパク質を除去可能なことが示された。 FIG. 4 shows the obtained two-dimensional electrophoresis result. Here, a) is the protein contained in the impurity solution obtained from the cell culture solution, and b) is the two-dimensional electrophoresis pattern of the protein contained in the permeate of the anion exchange membrane QSD of the impurity solution. It is. The results of FIG. 4a) show that the impurity proteins contained in the cell culture medium have a broad range distribution with a molecular weight of up to about 300 kDa and a pI ranging from at least 3 to 9.5. It is. On the other hand, from the result of FIG. 4b), it was confirmed that the protein contained in the permeated liquid of the anion exchange membrane QSD was removed from the whole molecular weight range having a pI of 4 or less. Therefore, it was shown that a plurality of impurity proteins having a pI difference of 1 or more can be removed.
［比較例３］
アニオン交換膜ＱＳＤの換わりに、アニオン交換カラムとして、ＧＥヘルスケア製、ＨｉＴｒａｐＱ ＨＰ＿１ｍｌを用い、流速２ｍＬ／ｍｉｎ（２ＣＶ／ｍｉｎ、あるいは３００ｃｍ／ｈｒ）にて通液する以外は、実施例６と全く同様にして、細胞培養液に含まれる不純物溶液の透過液を回収し、２次元電気泳動法を用いて、透過液中のタンパク質の分子量及びｐＩの分布を評価した。図５に得られた２次元電気泳動の結果を示す。これより、やや不純物タンパク質の除去が認められるものの、広い範囲の分子量分布のタンパク質が除去されるｐＩ領域はなかった。
[Comparative Example 3]
Instead of the anion exchange membrane QSD, GE Healthcare's HiTrapQ HP — 1 ml was used as the anion exchange column, and the flow rate was 2 mL / min (2 CV / min or 300 cm / hr). Similarly, the permeated solution of the impurity solution contained in the cell culture solution was collected, and the molecular weight and pI distribution of the protein in the permeated solution were evaluated using a two-dimensional electrophoresis method. FIG. 5 shows the result of the two-dimensional electrophoresis obtained. As a result, although removal of the impurity protein was somewhat recognized, there was no pI region from which proteins having a wide range of molecular weight distribution were removed.
Claims (11)
前記複数の不純物タンパク質の等電点の値に対し、前記溶液の水素イオン指数の値を高く調整する調整工程と、
調整された前記溶液を前記アニオン交換膜に通液する通液工程と、
前記目的タンパク質を回収する回収工程と、
を有し、
前記分子量の異なる複数の不純物タンパク質は、少なくとも分子量が１００ｋＤａ未満の成分と、分子量が１００ｋＤａ以上の成分と、を含み、
前記アニオン交換膜が、ポリエチレン多孔質基材と、前記ポリエチレン多孔質基材に結合されたグラフト鎖と、を備え、前記ポリエチレン多孔質基材に設けられた孔の最大孔径が０．１μｍ以上０．８μｍ以下であり、
前記通液工程を経た溶液中の前記１００ｋＤａ未満の成分の体積あたりの質量濃度と、前記１００ｋＤａ以上の成分の体積あたりの質量濃度と、が、通液前の溶液中の５％以下となる、
方法。 In a method for purifying the target protein by passing a solution containing the target protein and a plurality of impurity proteins having different molecular weights through an anion exchange membrane,
An adjustment step of adjusting the value of the hydrogen ion index of the solution higher than the value of the isoelectric point of the plurality of impurity proteins;
A liquid passing step of passing the adjusted solution through the anion exchange membrane;
A recovery step of recovering the target protein;
Have
The plurality of impurity proteins having different molecular weights include at least a component having a molecular weight of less than 100 kDa and a component having a molecular weight of 100 kDa or more,
The anion exchange membrane comprises a polyethylene porous substrate and a graft chain bonded to the polyethylene porous substrate, and the maximum pore diameter of the holes provided in the polyethylene porous substrate is 0.1 μm or more and 0 .8 μm or less,
The mass concentration per volume of the component of less than 100 kDa in the solution passed through the liquid passing step and the mass concentration per volume of the component of 100 kDa or higher become 5% or less in the solution before passing through.
Method.
前記通液工程において、前記アニオン交換膜に、前記溶液を通液して、前記目的タンパク質を透過させ、
前記回収工程において、前記アニオン交換膜を透過した溶液を採取して、前記目的タンパク質を回収する、
請求項１又は２に記載の方法。 In the adjusting step, with respect to the isoelectric point of the target protein, the value of the hydrogen ion index of the solution is adjusted to be equal to or less than the value obtained by adding the isoelectric point of the target protein and 1.
In the liquid passing step, the solution is passed through the anion exchange membrane to allow the target protein to permeate,
In the recovery step, the solution that has permeated the anion exchange membrane is collected to recover the target protein.
The method according to claim 1 or 2.
前記通液工程において、前記アニオン交換膜に、前記溶液を通液して、前記目的タンパク質と、前記不純物タンパク質と、の両方を、前記アニオン交換膜に吸着させ、
前記回収工程において、塩濃度、及び／又は、水素イオン濃度を調整した溶出液を前記アニオン交換膜に通液して、前記目的タンパク質を溶出して回収する、
請求項１又は２に記載の方法。 In the adjustment step, with respect to the isoelectric point of the target protein, the value of the hydrogen ion index of the solution is adjusted high,
In the liquid passing step, the solution is passed through the anion exchange membrane to adsorb both the target protein and the impurity protein to the anion exchange membrane,
In the recovery step, an eluate adjusted in salt concentration and / or hydrogen ion concentration is passed through the anion exchange membrane, and the target protein is eluted and recovered.
The method according to claim 1 or 2.
前記多孔膜状のアニオン交換膜の単位体積あたりの塩素イオンの交換容量であるイオン交換容量が０．３ｍｍｏｌ／ｍＬ以上、１．０ｍｍｏｌ／ｍＬ以下である、請求項１乃至４のいずれか１項に記載の方法。 The anion exchange membrane has an anion exchange group of a tertiary amine;
The ion exchange capacity, which is an exchange capacity of chlorine ions per unit volume of the porous membrane-like anion exchange membrane, is 0.3 mmol / mL or more and 1.0 mmol / mL or less. The method described in 1.
前記通液工程において、前記不純物が前記アニオン交換膜を透過する、
請求項１乃至８のいずれか１項に記載の方法。 The solution includes at least one impurity selected from the group consisting of an organic solvent, a surfactant, and a lipid;
In the liquid passing step, the impurities permeate the anion exchange membrane.
9. A method according to any one of claims 1 to 8.
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