JP2010187615A - Method for purifying nucleic acid by using porous membrane having anion-exchanging group immobilized thereon - Google Patents
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Abstract
Description
本発明は、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を用いた核酸の精製方法に関する。 The present invention relates to a method for purifying a nucleic acid using a porous membrane in which an anion exchange group is immobilized via a graft chain.
遺伝子治療に代表されるような、医薬品への核酸の使用量が増加していることにより、大規模で高速処理が可能な核酸の精製方法が望まれている。ここで核酸とは、デオキシリボ核酸（ＤＮＡ）およびリボ核酸（ＲＮＡ）を指し、このうち、特にプラスミドＤＮＡはポリヌレオチドワクチンおよび遺伝子治療プロトコルにとって、極めて重要である。ポリヌレオチドワクチンは、特定の疾病に対して防御免疫を誘導するための有効な方法であり、中和抗体を生成するとともに、より好ましい細胞媒介性免疫反応を活性化する。より詳細には、目的の抗原をコードする遺伝子と哺乳類動物細胞中で活性なプロモーターとを含むプラスミドＤＮＡが対象動物に投与され、筋肉細胞によって細胞内部に取り込まれる。抗原ＤＮＡは転写および翻訳され、発現したタンパク質は、Ｔ細胞への提示のために細胞表面へと輸送される。疾病モデルでのポリヌクレオチドワクチンの前臨床免疫原性や有効性は、多数の感染性疾患に対して実証されている。プラスミドＤＮＡはさらに、機能的遺伝子の体内への投与、標的細胞への前記遺伝子の送達および疾病状態を選択的に調節するための治療用産物を包含する遺伝子治療処置について効果が認められている。 Due to an increase in the amount of nucleic acid used in pharmaceuticals, as typified by gene therapy, a method for purifying a nucleic acid capable of high-speed processing on a large scale is desired. Nucleic acid herein refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), of which plasmid DNA is particularly important for polynucleotide vaccines and gene therapy protocols. Polynucleotide vaccines are an effective way to induce protective immunity against specific diseases, generate neutralizing antibodies and activate more favorable cell-mediated immune responses. More specifically, plasmid DNA containing a gene encoding the antigen of interest and a promoter active in mammalian cells is administered to the subject animal and taken up inside the cells by muscle cells. The antigenic DNA is transcribed and translated, and the expressed protein is transported to the cell surface for presentation to T cells. The preclinical immunogenicity and effectiveness of polynucleotide vaccines in disease models have been demonstrated against a number of infectious diseases. Plasmid DNA is further recognized for gene therapy treatments, including administration of functional genes into the body, delivery of the genes to target cells, and therapeutic products for selectively modulating disease states.
通常、大腸菌（Ｅ．ｃｏｌｉ）が核酸培養の宿主として広く使われるが、酵母菌あるいは哺乳類または昆虫の細胞もまた、宿主細胞として用いることができる。これらの宿主を用いた核酸培養で得られた核酸には、宿主細胞壁およびその破片、さらにエンドトキシンなどが不純物として含まれている。このような不純物が最終製品である医薬品内に混入し、投与の際に患者の体内に取り込まれると、重大な症状、例えば発熱、血管の炎症、血液の凝固、免疫システムによる抗原の発生などが引き起こされることから、不純物を除去し、核酸を精製することは、核酸の医薬品としての使用において極めて重要となる。 Usually, E. coli is widely used as a host for nucleic acid culture, but yeast or mammalian or insect cells can also be used as host cells. Nucleic acids obtained by nucleic acid culture using these hosts contain host cell walls and fragments thereof, and endotoxins as impurities. If such impurities are mixed into the final drug product and taken into the patient's body during administration, serious symptoms such as fever, vascular inflammation, blood clotting, and generation of antigens by the immune system may occur. As a result, removing impurities and purifying nucleic acids is extremely important in the use of nucleic acids as pharmaceuticals.
核酸の精製は、実験室レベルでは一般にリボヌクレアーゼなどの酵素やリゾチウムによって宿主細胞を分解した後、有機溶剤、セシウム塩によって宿主細胞破片を凝集させ、遠心分離によって清澄化する方法が用いられている。この方法は大量の精製には不向きであり、さらに人体に有害なフェノールやセシウム塩などを用いることから、医薬品向けの核酸の精製には好ましくない。 At the laboratory level, nucleic acid purification is generally performed by degrading host cells with an enzyme such as ribonuclease or lysozyme, aggregating host cell debris with an organic solvent or a cesium salt, and clarifying the cells by centrifugation. This method is not suitable for large-scale purification, and further uses phenols or cesium salts that are harmful to the human body, which is not preferable for the purification of nucleic acids for pharmaceuticals.
工業的に核酸を精製する方法としては、核酸を含む宿主細胞の培養液を遠心分離によって清澄化した後、クロマトグラフィーカラムによる工程を用いることが一般的である。例えば、特許文献１にはアニオン交換クロマトグラフィーとゲルろ過クロマトグラフィーを用いたプラスミドＤＮＡの精製法が記載されている。ここで、最初の工程でアニオン交換クロマトグラフィーによりプラスミドＤＮＡを吸着後溶出回収し、第２の工程でサイズ分画により最終精製を行い、エンドトキシンなどのサイズの小さな不純物を除去する。特許文献２には第１工程としてアニオン交換クロマトグラフィーを、第２工程として逆相クロマトグラフィーを用いて、核酸を精製する方法が記載されている。特許文献３にはアニオン交換クロマトグラフィーのみの一工程によって、核酸を精製する方法が記載されている。このように工業的な核酸の大量精製において、カラムクロマトグラフィーを用いることは一般に行われている。 As a method for industrially purifying nucleic acids, it is common to use a step using a chromatography column after clarifying a culture solution of host cells containing nucleic acids by centrifugation. For example, Patent Document 1 describes a method for purifying plasmid DNA using anion exchange chromatography and gel filtration chromatography. Here, plasmid DNA is adsorbed and collected by anion exchange chromatography in the first step, and finally purified by size fractionation in the second step to remove small-sized impurities such as endotoxin. Patent Document 2 describes a method of purifying a nucleic acid using anion exchange chromatography as a first step and reverse phase chromatography as a second step. Patent Document 3 describes a method for purifying a nucleic acid by a single step using only anion exchange chromatography. As described above, column chromatography is generally used in mass purification of industrial nucleic acids.
また、特許文献４はカラムクロマトグラフィーでなく、弱アニオン交換基が直接表面に固定されている多孔膜を用いて、核酸とエンドトキシンとの混合液から、核酸を高濃度で分離する方法を開示する。さらに、特許文献５には清澄化され宿主細胞破片を除去した、核酸とエンドトキシンを含む水溶液から、イオン交換基を有しない、カットオフ分子量が１〜１０００ｋＤａの限外ろ過膜を用いて、核酸を精製する方法が記載されている。この方法ではサイズの大きな核酸は限外ろ過膜を透過せず、エンドトキシンのようなサイズの小さな分子は膜を透過することにより、核酸を精製する。 Patent Document 4 discloses a method for separating nucleic acid at a high concentration from a mixed solution of nucleic acid and endotoxin using a porous membrane in which a weak anion exchange group is directly fixed on the surface, instead of column chromatography. . Further, Patent Document 5 discloses a nucleic acid obtained from an aqueous solution containing nucleic acid and endotoxin, which has been clarified and removed from host cell debris, using an ultrafiltration membrane having an ion exchange group and a cutoff molecular weight of 1 to 1000 kDa. A method of purification is described. In this method, a nucleic acid having a large size does not permeate the ultrafiltration membrane, and a small molecule such as endotoxin permeates the membrane to purify the nucleic acid.
上記特許文献１に開示された方法においては、十分な精製度を得るためには、核酸の溶出における高い分解能が要求されるため、処理速度が遅くなるという問題がある。上記特許文献２に開示された方法においては、第２工程において有害な有機溶剤を用いるという問題がある。上記特許文献３に開示された方法においては、高い精製度で核酸を回収するためには、高分解能での溶出条件が必要であり、さらに処理速度が低下するという問題がある。これらのカラムクロマトグラフィーを用いた方法では、核酸をアニオン交換カラムクロマトグラフィーに吸着させる際、一般にその動的吸着容量は最大でも５ｇ／Ｌ（カラム体積）程度しか得られない。カラムクロマトグラフィーでの吸着はカラムを構成する樹脂の細孔に目的物が拡散により浸透し、吸着することに基づくが、一般に核酸の分子量は数十万から数千万と、他のタンパク質などの目的物に比べて極めて大きく、そのためビーズの細孔に吸着される量が少ないのである。さらに、カラムクロマトグラフィーでは高流速での通液ができないため、高速な精製処理が望めないという重大な問題もある。 The method disclosed in Patent Document 1 requires a high resolution in elution of nucleic acids in order to obtain a sufficient degree of purification, and thus has a problem that the processing speed becomes slow. The method disclosed in Patent Document 2 has a problem that a harmful organic solvent is used in the second step. The method disclosed in Patent Document 3 requires elution conditions with high resolution in order to recover nucleic acids with a high degree of purification, and further has a problem that the processing speed decreases. In these methods using column chromatography, when a nucleic acid is adsorbed on anion exchange column chromatography, the dynamic adsorption capacity is generally only about 5 g / L (column volume) at the maximum. Adsorption in column chromatography is based on the diffusion and adsorption of the target substance into the pores of the resin that constitutes the column. Generally, the molecular weight of nucleic acids ranges from several hundreds of thousands to tens of millions, such as other proteins. It is very large compared to the target product, and therefore, the amount adsorbed in the pores of the beads is small. Furthermore, since column chromatography cannot pass liquid at a high flow rate, there is a serious problem that high-speed purification treatment cannot be expected.
また、上記特許文献４に開示された方法はカラムクロマトグラフィーを用いる方法に比べると、迅速な処理が可能であるが、核酸とエンドトキシンとの多孔膜への選択的な吸着性の差が小さいために、核酸の回収率が低く、さらに核酸回収液中のエンドトキシンの残存量も高いという問題がある。弱アニオン交換基が直接表面に固定されている多孔膜への、核酸の動的吸着容量は８ｇ／Ｌ（膜体積）程度しか得られないことも、核酸の精製度が低いことの理由と考えられる。さらに、上記特許文献５に開示された方法も高速処理には不適であり、かつ溶液交換のために大量の供給液が必要という問題がある。また核酸やエンドトキシンの分子が膜の表面に堆積し、通液速度を著しく低下させる。 In addition, the method disclosed in Patent Document 4 can be processed more quickly than the method using column chromatography, but the difference in selective adsorptivity between the nucleic acid and endotoxin on the porous membrane is small. In addition, the nucleic acid recovery rate is low, and the remaining amount of endotoxin in the nucleic acid recovery solution is also high. The fact that only about 8 g / L (membrane volume) of dynamic adsorption capacity of nucleic acid can be obtained on a porous membrane in which a weak anion exchange group is directly fixed on the surface is considered to be a reason for the low purity of nucleic acid. It is done. Furthermore, the method disclosed in Patent Document 5 is also unsuitable for high-speed processing, and there is a problem that a large amount of supply liquid is required for solution exchange. In addition, nucleic acid and endotoxin molecules are deposited on the surface of the membrane, and the flow rate is significantly reduced.
このように、これまでの核酸精製方法では宿主細胞破片やエンドトキシン等を不純物として含む核酸含有溶液から、高速処理で効率的に、かつ医薬的に安全な方法で核酸を精製することは困難である。また、医薬品として有用なレベルにまで核酸を精製するためには、遠心分離工程に続いて複数のプロセスを併用することが必要である。 As described above, it is difficult to purify nucleic acids from nucleic acid-containing solutions containing host cell debris, endotoxin, and the like as impurities by a high-speed process and in a pharmaceutically safe manner using conventional nucleic acid purification methods. . In order to purify nucleic acids to a level useful as a pharmaceutical product, it is necessary to use a plurality of processes in combination following the centrifugation step.
かかる状況から、本発明が解決しようとする課題は、簡便で、かつ高速処理が可能なプロセスによって、核酸を医薬品レベルにまで精製することが可能であり、しかもスケールアップが容易な、核酸の精製方法を提供することである。 Under such circumstances, the problem to be solved by the present invention is that nucleic acid can be purified to a pharmaceutical level by a process that is simple and capable of high-speed processing, and that can be easily scaled up. Is to provide a method.
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、グラフト鎖を介して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、核酸の精製において、高速処理により、工業レベルで不純物を除去することに有効であることを見出し、本発明を完成させた。 As a result of intensive studies to solve the above problems, the present inventors have used a porous membrane in which an anion exchange group is immobilized on the surface via a graft chain. The present invention has been completed by finding it effective for removing impurities at an industrial level.
すなわち、本発明は、以下の工程を含む、核酸の精製方法：（ｐ）アニオン交換基がグラフト鎖を介して表面に固定されている多孔膜に、核酸含有溶液を通液し、前記多孔膜へ前記核酸を吸着させる工程；および（ｑ）前記多孔膜に吸着した前記核酸を、溶出液を通液して溶出回収する工程、に関する。
本発明はまた、前記核酸含有溶液が、前記多孔膜の細孔径より大きなサイズの不純物および前記多孔膜の細孔径より小さな不純物を含む、上記の核酸の精製方法に関する。
本発明はまた、前記工程（ｐ）における核酸含有溶液の電気伝導度が２０ｍＳ／ｃｍ〜６０ｍＳ／ｃｍであり、前記工程（ｑ）における溶出液の電気伝導度が９０ｍＳ／ｃｍ〜１６０ｍＳ／ｃｍである、上記の核酸の精製方法に関する。
本発明はまた、前記工程（ｐ）の後であって、前記工程（ｑ）に先立って、さらに以下の工程：（ｍ）前記多孔膜に不純物溶出液を通液して不純物を溶出する工程；を含む、請求項１または２に記載の核酸の精製方法であって、前記工程（ｍ）における不純物溶出液の電気伝導度が２０ｍＳ／ｃｍ〜６０ｍＳ／ｃｍであり、前記工程（ｑ）における溶出液の電気伝導度が９０ｍＳ／ｃｍ〜１６０ｍＳ／ｃｍである、上記の核酸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、上記の核酸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、上記の核酸の精製方法に関する。
本発明はまた、前記多孔膜が、０．１μｍ〜１．０μｍの最大細孔径を有する、上記の核酸の精製方法に関する。
That is, the present invention includes a nucleic acid purification method comprising the following steps: (p) passing a nucleic acid-containing solution through a porous membrane having an anion exchange group immobilized on the surface via a graft chain, and said porous membrane And (q) a step of eluting and collecting the nucleic acid adsorbed on the porous membrane by passing an eluate through the eluate.
The present invention also relates to the method for purifying a nucleic acid as described above, wherein the nucleic acid-containing solution contains impurities having a size larger than the pore size of the porous membrane and impurities smaller than the pore size of the porous membrane.
The present invention also provides that the electric conductivity of the nucleic acid-containing solution in the step (p) is 20 mS / cm to 60 mS / cm, and the electric conductivity of the eluate in the step (q) is 90 mS / cm to 160 mS / cm. The present invention relates to a method for purifying the nucleic acid.
The present invention also includes the following step after the step (p) and prior to the step (q): (m) a step of eluting impurities by passing an impurity eluent through the porous membrane. The method for purifying nucleic acid according to claim 1 or 2, wherein the impurity eluate in step (m) has an electric conductivity of 20 mS / cm to 60 mS / cm, and in step (q) The present invention relates to the method for purifying a nucleic acid as described above, wherein the electric conductivity of the eluate is 90 mS / cm to 160 mS / cm.
The present invention also relates to the above-described method for purifying a nucleic acid, wherein the anion exchange group is a diethylamino group.
The present invention also relates to the method for purifying a nucleic acid as described above, wherein the graft chain contains a polymer of glycidyl methacrylate.
The present invention also relates to the method for purifying a nucleic acid as described above, wherein the porous membrane has a maximum pore size of 0.1 μm to 1.0 μm.
本発明の核酸の精製方法を用いることにより、宿主細胞培養液のような、不純物を含む核酸含有溶液から核酸の回収および精製を有効にかつ迅速に実施することができる。 By using the nucleic acid purification method of the present invention, it is possible to effectively and rapidly recover and purify nucleic acid from a nucleic acid-containing solution containing impurities such as a host cell culture solution.
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、核酸とともに、宿主細胞破片、エンドトキシンなどの不純物を含む核酸含有溶液から核酸を精製する方法であって、アニオン交換基がグラフト鎖を介して表面に固定されている多孔膜を用いて、該核酸含有溶液のろ過を行って、多孔膜に核酸を吸着し、吸着した核酸を溶出液で溶出回収することによる核酸の精製方法である。核酸含有溶液中の不純物のうち、多孔膜に非吸着な不純物は透過液中に回収され、多孔膜に吸着する不純物は一旦多孔膜に吸着した後に不純物溶出液を通液することにより回収され、多孔膜の細孔径よりサイズの大きな宿主細胞破片等の不純物は細孔を透過せず、核酸含有溶液から除去される。 This embodiment is a method for purifying a nucleic acid from a nucleic acid-containing solution containing impurities such as host cell debris and endotoxin together with the nucleic acid, wherein a porous membrane having an anion exchange group immobilized on the surface via a graft chain is used. It is a method for purifying a nucleic acid by filtering the nucleic acid-containing solution, adsorbing the nucleic acid to a porous membrane, and eluting and collecting the adsorbed nucleic acid with an eluent. Among the impurities in the nucleic acid-containing solution, impurities that are not adsorbed to the porous membrane are collected in the permeate, and impurities that are adsorbed to the porous membrane are once adsorbed to the porous membrane and then collected by passing the impurity eluate, Impurities such as host cell debris larger than the pore diameter of the porous membrane do not permeate the pores and are removed from the nucleic acid-containing solution.
本実施の形態における「核酸」とは、ＤＮＡ、ＲＮＡ全般を示す。医薬品の工業的生産という観点からは、Ｅ．ｃｏｌｉなどから産生されるプラスミドＤＮＡが挙げられるがこれに限定されない。また、その分子量も特に限定されないが、本実施の形態における精製方法の行い易さという観点からは、高分子量の核酸（例えば分子量２０万以上）がより好ましい場合がある。 “Nucleic acid” in the present embodiment refers to DNA and RNA in general. From the viewpoint of industrial production of pharmaceuticals, E.I. Examples include, but are not limited to, plasmid DNA produced from E. coli. Moreover, although the molecular weight is not particularly limited, a high molecular weight nucleic acid (for example, a molecular weight of 200,000 or more) may be more preferable from the viewpoint of ease of performing the purification method in the present embodiment.
本実施の形態における「核酸含有溶液」に含まれる不純物としては、核酸以外の成分であれば特に限定されない。例えば核酸が前記プラスミドＤＮＡの場合には、宿主細胞破片およびそれに由来する不純物、例えば、ＨＣＰ（宿主細胞タンパク質）を含む各種タンパク質、ウイルスおよびエンドトキシンなどが挙げられるが、これらに限定されない。 The impurities contained in the “nucleic acid-containing solution” in the present embodiment are not particularly limited as long as they are components other than nucleic acids. For example, when the nucleic acid is the plasmid DNA, host cell debris and impurities derived therefrom, for example, various proteins including HCP (host cell protein), viruses, endotoxins, and the like, are not limited thereto.
本実施の形態において用いられる「アニオン交換基がグラフト鎖を介して表面に固定されている多孔膜」とは、基材となる多孔質体およ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にアニオン交換基が固定されている多孔膜である。アニオン交換基がグラフト鎖を介して、多孔膜表面に固定されていることで、核酸の吸着量が著しく増加する。 The “porous membrane in which the anion exchange group is fixed to the surface via the graft chain” used in the present embodiment means that the graft chain is fixed to the surface of the porous body serving as a base material and the pores thereof, Furthermore, it is a porous membrane in which an anion exchange group is fixed to the graft chain. Since the anion exchange group is immobilized on the surface of the porous membrane via the graft chain, the amount of adsorbed nucleic acid is remarkably increased.
多孔膜の基材は、特に限定されないが、機械的性質の保持という観点から、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、ブチレンおよ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、または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およびクロロトリフルオロエチレンなどが挙げられる。 The substrate of the porous film is not particularly limited, but is preferably composed of a polyolefin polymer from the viewpoint of maintaining mechanical properties. 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、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、ポリエチレンまた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 the material is particularly excellent in mechanical strength and can obtain a high adsorption capacity.
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表面に結合した、基材と同種または異種の分子鎖である。多孔膜の表面および細孔に、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 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また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また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、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、より高い吸着容量および多孔膜の耐久性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. From the viewpoint of ensuring both higher adsorption capacity and durability of the porous membrane, the binding rate of the graft chain to the porous membrane (graft rate) can be measured using the method described in Examples and the like described later. Preferably they are 20%-200%, More preferably, they are 20%-150%, More preferably, they are 30%-70%.
アニオン交換基は、核酸含有溶液中の核酸を吸着することができれば特に制限されない。例えば、アニオン交換基としては、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が同一また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（グラフト鎖に導入されたトリメチルアンモニウム基、Ｍｅ3Ｎ+−）などが挙げられる。多孔膜に導入されたグラフト鎖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およびＱが好ましく、ＤＥＡがより好ましい。 The anion exchange group is not particularly limited as long as it can adsorb the nucleic acid in the nucleic acid-containing solution. For example, the anion exchange group is not particularly limited, but includes a diethylamino group (DEA, Et 2 N-), a quaternary ammonium group (Q, R 3 N + -), a quaternary aminoethyl group (QAE, R 3 N +). - (CH 2) 2 -) , diethylaminoethyl group (DEAE, Et 2 N- (CH 2) 2 -), diethylaminopropyl group (DEAP, Et 2 N- (CH 2) 3 -) , and the like. 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 (a trimethylammonium group introduced into a graft chain, Me 3 N + -). From the viewpoint of easy chemical fixation to the graft chain introduced into 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およびジエチルアンモニウムまた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 the 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、核酸含有溶液中の不純物を除去し、かつ高い透過流速を得るために、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。 The maximum pore diameter of the porous membrane is preferably 0.1 μm to 1.0 μm, more preferably 0.1 μm to 0.8 μm in order to remove impurities in the nucleic acid-containing solution and obtain a high permeation flow rate. And more preferably 0.2 μm to 0.6 μm.
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%.
上記細孔径および空孔率の測定は、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。 The pore diameter and the porosity can be measured by methods known to those skilled in the art as described in “Membrane Technology” by Marcel Mulder (IPC Co., Ltd.) and the like. Examples thereof include measurement methods such as observation with an electron microscope, bubble point method, mercury intrusion method, and transmittance method.
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、円板または円筒状などの形態が挙げられる。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また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、液体または気体が透過する性質を有する多孔膜を意味する。中空糸の外径および内径は、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 the outer layer of the hollow fiber are continuous by pores that are through-holes. Means a porous film 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.
本実施の形態による核酸の精製方法は、上記の多孔膜に核酸含有溶液を通液し、多孔膜に核酸を吸着する工程（ｐ）を含む。該工程（ｐ）においては、核酸含有溶液中の不純物であって多孔膜の孔径より大きな不純物の除去も行われる。ここで、核酸含有溶液のｐＨは４〜９．５が好ましく、ｐＨ５〜９がより好ましい。 The method for purifying nucleic acid according to the present embodiment includes a step (p) of passing a nucleic acid-containing solution through the porous membrane and adsorbing the nucleic acid to the porous membrane. In the step (p), impurities which are impurities in the nucleic acid-containing solution and larger than the pore diameter of the porous membrane are also removed. Here, the pH of the nucleic acid-containing solution is preferably 4 to 9.5, more preferably pH 5 to 9.
核酸の等電位点（ｐＩ）は通常３以下であるため、広いｐＨ範囲および比較的高い塩濃度(電気伝導度)の核酸含有溶液を上記多孔膜に通液すると、多孔膜のアニオン交換基に核酸が吸着する。これに対し、宿主細胞破片に代表される不純物のｐＩは３〜８の範囲にあるため、核酸に比べて、上記多孔膜のアニオン交換基に不純物が吸着するための、通液させる溶液のｐＨおよび塩濃度(電気伝導度)の範囲はより限定される。これは不純物がアルブミン、グロブリンなどの一般的な生体タンパク質の場合も同様である。 Since the equipotential point (pI) of nucleic acid is usually 3 or less, when a nucleic acid-containing solution having a wide pH range and a relatively high salt concentration (electrical conductivity) is passed through the porous membrane, it becomes an anion exchange group of the porous membrane. Nucleic acids are adsorbed. On the other hand, since the pI of impurities typified by host cell debris is in the range of 3 to 8, the pH of the solution to be passed for allowing the impurities to adsorb to the anion exchange groups of the porous membrane as compared with nucleic acids And the range of salt concentration (electrical conductivity) is more limited. This is the same when the impurities are general biological proteins such as albumin and globulin.
また、核酸はその分子量が数十万から数千万と極めて高いために、アニオン交換基に吸着する際の吸着点が、分子量の小さな不純物（例えばエンドトキシン）よりも多い。このため、核酸とエンドトキシンのｐＩが同程度であっても、核酸の方がより高い塩濃度(電気伝導度)の核酸含有溶液の通液で、多孔膜のアニオン交換基に吸着可能であるという性質がある。 Moreover, since the molecular weight of the nucleic acid is extremely high, from several hundred thousand to several tens of million, the number of adsorption points when adsorbing to the anion exchange group is larger than that of impurities having a small molecular weight (for example, endotoxin). For this reason, even if the pI of the nucleic acid and endotoxin are about the same, the nucleic acid can be adsorbed to the anion exchange group of the porous membrane by passing the nucleic acid-containing solution having a higher salt concentration (electrical conductivity). There is a nature.
さらに、アニオン交換基への吸着は、カラムクロマトグラフィーの樹脂への吸着のような、微細孔への拡散浸透による吸着ではないため、核酸のような分子サイズの大きな目的物を吸着する場合には、アニオン交換基が固定された多孔膜は、より優れた吸着性を示す。 Furthermore, since adsorption to anion exchange groups is not adsorption by diffusion and penetration into micropores, such as adsorption to resin in column chromatography, when adsorbing a target object with a large molecular size such as nucleic acid. The porous membrane on which the anion exchange group is fixed exhibits better adsorptivity.
これらの特性は、アニオン交換基がグラフト鎖を介して、多孔膜の細孔表面に固定されている場合に特に顕著となり、核酸の多孔膜への動的吸着容量は３０ｇ／Ｌ（膜体積）を超える値が得られる。これはグラフト鎖により、核酸分子へのアニオン交換基の吸着点の数がより増加することによると考えられる。 These characteristics are particularly remarkable when the anion exchange group is immobilized on the pore surface of the porous membrane via a graft chain, and the dynamic adsorption capacity of nucleic acid to the porous membrane is 30 g / L (membrane volume). A value greater than is obtained. This is considered to be due to the fact that the number of adsorption points of the anion exchange group to the nucleic acid molecule is further increased by the graft chain.
上記の工程（ｐ）において多孔膜に吸着した核酸を、溶出液を通液して溶出回収する工程（ｑ）を行うことにより、核酸を精製することができる。溶出液は、不純物を含まないものであればその組成は特に制限されず、技術分野で通常用いられる組成の溶出液を用いることができる。例えば高塩濃度の溶液や、水酸化ナトリウム溶液のような高ｐＨの溶液を用いることもできる。溶出液の電気伝導度は、好ましくは９０ｍＳ／ｃｍ〜１６０ｍＳ／ｃｍ（ＮａＣｌでは約１．０Ｍ〜２．２Ｍ）であり、より好ましくは１００ｍＳ／ｃｍ〜１５０ｍＳ／ｃｍ（ＮａＣｌでは約１．２Ｍ〜２．０Ｍ）である。 The nucleic acid can be purified by carrying out the step (q) in which the nucleic acid adsorbed on the porous membrane in the step (p) is passed through the eluate and eluted and recovered. The composition of the eluate is not particularly limited as long as it does not contain impurities, and an eluate having a composition usually used in the technical field can be used. For example, a solution having a high salt concentration or a solution having a high pH such as a sodium hydroxide solution can be used. The electric conductivity of the eluate is preferably 90 mS / cm to 160 mS / cm (about 1.0 M to 2.2 M for NaCl), more preferably about 100 MS / cm to 150 mS / cm (about 1.2 M to about NaCl). 2.0M).
上記の工程（ｐ）および（ｑ）を含む核酸の精製方法は、大別すれば：
（ａ）核酸含有溶液の塩濃度(電気伝導度)を、不純物が多孔膜に吸着しない程度にまで増加させ、多孔膜へ核酸を吸着させると同時に、ろ液に含まれる非吸着成分として不純物を除去する工程（ｐ）の後、溶出液を通液して核酸を溶出回収する工程（ｑ）を行う方法；および
（ｂ）核酸含有溶液の塩濃度(電気伝導度)を、不純物の少なくとも一部および核酸がともに多孔膜に吸着する濃度として、不純物および核酸をともに多孔膜に吸着させ、吸着しない不純物成分をろ液として除去する工程（ｐ）の後、工程（ｐ）で多孔膜に吸着した不純物のみが溶出する塩濃度(電気伝導度)の不純物溶出液を通液することにより、不純物を溶出し、ろ液として除去する工程（ｍ）を行い、その後、核酸が溶出する塩濃度(電気伝導度)の溶出液を通液して核酸を溶出回収する工程（ｑ）を行う方法；
の２種類に分けられる。（ａ）の方法は、例えば核酸よりも大きな等電位点を有する宿主細胞破片などの除去に特に有効であり得る。（ｂ）の方法は、例えば核酸とより近い等電位点を有するエンドトキシンなどの除去に特に有効であり得る。
The nucleic acid purification method including the above steps (p) and (q) can be broadly classified as follows:
(A) The salt concentration (electrical conductivity) of the nucleic acid-containing solution is increased to such an extent that the impurities are not adsorbed to the porous membrane, and at the same time, the nucleic acids are adsorbed to the porous membrane, and at the same time A method of performing a step (q) of eluting and collecting a nucleic acid by passing an eluate after the removing step (p); and (b) a salt concentration (electrical conductivity) of the nucleic acid-containing solution at least one of impurities. As a concentration at which both the nucleic acid and the nucleic acid are adsorbed on the porous membrane, after the step (p) in which both the impurities and the nucleic acid are adsorbed on the porous membrane and the impurity components not adsorbed are removed as a filtrate, adsorbed on the porous membrane in the step (p) The step (m) is carried out by eluting impurities and removing them as filtrate by passing through an impurity eluate having a salt concentration (electrical conductivity) at which only the impurities are eluted, and then the salt concentration at which nucleic acids are eluted ( (Electrical conductivity) Method of performing step (q) eluting recovered;
It is divided into two types. The method (a) can be particularly effective in removing, for example, host cell debris having an isoelectric point larger than that of the nucleic acid. The method (b) can be particularly effective for removing endotoxin having an equipotential point closer to that of a nucleic acid, for example.
（ａ）の方法の場合、工程（ｐ）における核酸含有溶液の塩濃度は、好ましくは電気伝導度で２０ｍＳ／ｃｍ〜６０ｍＳ／ｃｍの範囲（ＮａＣｌでは約０．２Ｍ〜０．６Ｍの範囲）であり、より好ましくは電気伝導度で３０ｍＳ／ｃｍ〜５０ｍＳ／ｃｍの範囲（ＮａＣｌでは約０．３Ｍ〜０．５Ｍの範囲）である。 In the case of the method (a), the salt concentration of the nucleic acid-containing solution in step (p) is preferably in the range of 20 mS / cm to 60 mS / cm in terms of electric conductivity (in the range of about 0.2 M to 0.6 M for NaCl). More preferably, the electric conductivity is in the range of 30 mS / cm to 50 mS / cm (in the range of about 0.3 M to 0.5 M for NaCl).
（ｂ）の方法の場合、工程（ｐ）における核酸含有溶液の塩濃度は、好ましくは電気伝導度で０ｍＳ／ｃｍ〜３０ｍＳ／ｃｍの範囲（ＮａＣｌでは約０Ｍから０．３Ｍの範囲）であり、より好ましくは電気伝導度で１ｍＳ／ｃｍ〜２０ｍＳ／ｃｍの範囲（ＮａＣｌでは約０Ｍから０．２Ｍの範囲）である。また、工程（ｍ）における不純物溶出液の塩濃度は、好ましくは電気伝導度で２０ｍＳ／ｃｍ〜６０ｍＳ／ｃｍの範囲（ＮａＣｌでは約０．２Ｍ〜０．６Ｍの範囲）であり、より好ましくは電気伝導度で３０ｍＳ／ｃｍ〜５０ｍＳ／ｃｍの範囲（ＮａＣｌでは約０．３Ｍ〜０．５Ｍの範囲）である。工程（ｍ）における不純物溶出液の組成は特に制限されず、技術分野で通常用いられる組成の溶出液を用いることができる。 In the case of the method (b), the salt concentration of the nucleic acid-containing solution in the step (p) is preferably in the range of 0 mS / cm to 30 mS / cm in electrical conductivity (in the range of about 0 M to 0.3 M for NaCl). More preferably, the electric conductivity is in the range of 1 mS / cm to 20 mS / cm (in the range of about 0 M to 0.2 M for NaCl). Moreover, the salt concentration of the impurity eluate in the step (m) is preferably in the range of 20 mS / cm to 60 mS / cm in terms of electrical conductivity (in the range of about 0.2 M to 0.6 M for NaCl), more preferably The electric conductivity is in the range of 30 mS / cm to 50 mS / cm (in the range of about 0.3 M to 0.5 M for NaCl). The composition of the impurity eluate in the step (m) is not particularly limited, and an eluate having a composition usually used in the technical field can be used.
このように、多孔膜に通液する溶液の電気伝導度を制御し、また、異なる電気伝導度の溶液を通液する順序を制御することによって、核酸を簡便かつ高い精製度で精製することができる。本実施の形態において、溶液の電気伝導度の調節方法は当業者に公知の手法を用いて行うことができ、典型的には溶液中の塩濃度を変化させることにより行うことができる。電気伝導度の測定方法は例えば市販の導電率計を用いて、当業者に公知の手法を用いて行うことができる。 Thus, by controlling the electrical conductivity of the solution passing through the porous membrane and controlling the order of passing the solutions having different electrical conductivities, the nucleic acid can be purified easily and with a high degree of purification. it can. In the present embodiment, the method for adjusting the electrical conductivity of the solution can be performed using a technique known to those skilled in the art, and can be typically performed by changing the salt concentration in the solution. For example, a commercially available conductivity meter can be used to measure the electrical conductivity using techniques known to those skilled in the art.
上記に記載した方法で核酸を溶出回収することにより、精製された目的の核酸を得ることができる。また、動物細胞培養液からの抗体の精製プロセスのような、核酸が不純物となるプロセスにおいても、本実施の形態の方法および多孔膜を用い、本明細書の記載を参照することにより、容易に核酸のみを除去することが可能である。 By purifying and recovering the nucleic acid by the method described above, a purified target nucleic acid can be obtained. Also, in a process in which nucleic acid becomes an impurity, such as a process for purifying an antibody from animal cell culture fluid, the method of the present embodiment and the porous membrane can be used to easily refer to the description in this specification. Only nucleic acids can be removed.
以下、参考例、実施例および比較例（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］ アニオン交換基がグラフト鎖を介して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、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およ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 a porous membrane module in which an anion exchange group is fixed on the surface via a graft chain (i) Introduction of graft chain into hollow fiber porous membrane, outer diameter 3.0 mm, inner diameter 2.0 mm, A polyethylene hollow fiber porous membrane having a maximum pore size 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、そのオリゴマーおよ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。 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 liquid, 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%.
このグラフト率から、下記式（ＩＩＩ）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
・・・（ＩＩＩ）
From this graft ratio, using the following formula (III), the number of moles of introduced GMA (molecular weight 142) relative to the number of moles of CH 2 groups (molecular weight 14), which is the backbone 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
... (III)
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、グラフト反応後の中空糸多孔膜に対して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を有する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 composed of a mixed solution of 50 parts by volume of diethylamine and 50 parts by volume of pure water was placed in a glass reaction tube at 20 parts by mass with respect to the hollow fiber porous membrane after the graft reaction, and adjusted to 30 ° C. A hollow fiber porous membrane with a graft chain introduced therein was inserted and allowed to stand for 210 minutes to replace the epoxy group of the graft chain with a diethylamino group, thereby obtaining a hollow fiber porous membrane having a diethylamino group as an anion exchange group. It was. 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Ｎ2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（ＩＶ）を用いて算出した。
Ｔ＝１００×Ｎ1／Ｎ2
＝１００×｛（ｗ2−ｗ1）／Ｍ1｝／｛ｗ1（ｄｇ／（ｄｇ＋１００））／Ｍ2｝
・・・（ＩＶ）
式（ＩＶ）中、Ｍ1はジエチルアンモニウムの分子量（７３．１４）、ｗ1はグラフト重合反応後の中空糸多孔膜の重量、ｗ2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 (IV) as the number of moles N 1 substituted with a diethylamino group out of the number of moles N 2 of the epoxy group.
T = 100 × N 1 / N 2
= 100 × {(w 2 −w 1 ) / M 1 } / {w 1 (dg / (dg + 100)) / M 2 }
... (IV)
In formula (IV), M 1 is the molecular weight of diethylammonium (73.14), w 1 is the weight of the hollow fiber porous membrane after the graft polymerization reaction, w 2 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）アニオン交換基がグラフト鎖を介して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をグラフト鎖を介して固定した中空糸多孔膜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、アニオン交換基がグラフト鎖を介して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 via a graft chain Three hollow fiber porous membranes obtained in (ii) with a diethylamino group fixed as an anion exchange group via a graft chain A hollow fiber porous membrane module in which both ends are fixed to a polysulfonic acid module case with an epoxy potting agent so that the hollow portion of the hollow fiber porous membrane is not blocked, and an anion exchange group is fixed via a graft chain Was made. 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（Ｖ）に従って、中空糸多孔膜の最大細孔径を算出した。
ｄ＝Ｃ1γ／Ｐ・・・（Ｖ）
式（Ｖ）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 (V), where d is the maximum pore diameter and γ is the surface tension.
d = C 1 γ / P (V)
In formula (V), 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］ 電気伝導度の異なるＤＮＡ溶液の調製
和光純薬製ＤＮＡ（デオキシリボ核酸ナトリウム、サケ精巣由来、粉末、分子量３０万〜９００万）０．２ｇを２０００ｍＬの２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）緩衝液に溶解し、０．１ｇ／Ｌの塩を含まないｐＨ８．０のＤＮＡ溶液２０００ｍＬを調整した。この溶液の電気伝導度は１．１５ｍＳ／ｃｍであった。このＤＮＡ溶液２００ｍＬを小分けし、ここにＮａＣｌ(和光純薬製)１７．５３ｇを添加して塩濃度１．５ＭのＤＮＡ溶液を調整した。この溶液の電気伝導度は１１４．１ｍＳ／ｃｍであった。同様にして、２００ｍＬに小分けした塩を含まないＤＮＡ溶液を５本用意し、そのうち４本にはＮａＣｌを１４．０２ｇ、１０．５２ｇ、７．０１ｇまたは３．５１ｇを添加して、それぞれ塩濃度が１．２Ｍ、０．９Ｍ、０．６Ｍおよび０．３Ｍの溶液を調整した。これらの電気伝導度はそれぞれ９５．２ｍＳ／ｃｍ、７４．９５ｍＳ／ｃｍ、６２．７ｍＳ／ｃｍおよび２８．５ｍＳ／ｃｍであった。このようにして電気伝導度が１．１５ｍＳ／ｃｍ〜１１４．１ｍＳ／ｃｍの０．１ｇ／ＬのＤＮＡ溶液を得た。
[Reference Example 2] Preparation of DNA solutions having different electric conductivities 0.2 g of DNA (manufactured by sodium deoxyribonucleic acid, derived from salmon testis, powder, molecular weight 300,000 to 9000 million) manufactured by Wako Pure Chemical Industries, Ltd. was added to 2000 mL of 20 mM Tris-HCl (pH 8. 0) Dissolved in a buffer solution to prepare 2000 mL of a pH 8.0 DNA solution containing no 0.1 g / L salt. The electrical conductivity of this solution was 1.15 mS / cm. 200 mL of this DNA solution was subdivided, and 17.53 g of NaCl (manufactured by Wako Pure Chemical Industries, Ltd.) was added thereto to prepare a DNA solution having a salt concentration of 1.5 M. The electrical conductivity of this solution was 114.1 mS / cm. Similarly, prepare 5 DNA solutions containing no salt divided into 200 mL, 4 of which were added 14.02 g, 10.52 g, 7.01 g or 3.51 g of NaCl, Prepared 1.2M, 0.9M, 0.6M and 0.3M solutions. Their electrical conductivities were 95.2 mS / cm, 74.95 mS / cm, 62.7 mS / cm and 28.5 mS / cm, respectively. Thus, a 0.1 g / L DNA solution having an electric conductivity of 1.15 mS / cm to 114.1 mS / cm was obtained.
［参考例３］ ｐＨの異なるＤＮＡ溶液の調整
上記参考例２で調製した２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）、０．１ｇ／ＬのＤＮＡ溶液を２００ｍＬに小分けしたものを２本用意し、これらに１Ｎ ＮａＯＨ水溶液を攪拌しながら滴下して、それぞれｐＨ９．０およびｐＨ８．５に調整した。同様にして２００ｍＬに小分けしたＤＮＡ溶液を更に２本用意し、それぞれここに塩酸を攪拌しながら滴下して、ｐＨ７．５およびｐＨ７．０に調整した。
[Reference Example 3] Preparation of DNA solutions having different pHs Two 20 mM Tris-HCl (pH 8.0) prepared in Reference Example 2 and 0.1 g / L DNA solution divided into 200 mL were prepared. 1N NaOH aqueous solution was added dropwise with stirring to pH 9.0 and pH 8.5, respectively. Similarly, two DNA solutions subdivided into 200 mL were prepared, and hydrochloric acid was added dropwise thereto while stirring to adjust the pH to 7.5 and 7.0.
和光純薬製ＤＮＡ（デオキシリボ核酸ナトリウム、サケ精巣由来、粉末）０．１ｇを１０００ｍＬの２５ｍＭ Ｃｉｔｒａｔｅ−ＮａＯＨ（ｐＨ５．０）緩衝液に溶解し、０．１ｇ／Ｌの塩を含まないｐＨ５．０のＤＮＡ溶液２０００ｍＬを調整した。この溶液を２００ｍＬ小分けしたものを２本用意し、これらに１Ｎ ＮａＯＨ水溶液を攪拌しながら滴下して、それぞれｐＨ５．５およびｐＨ６．０に調整した。
このようにしてｐＨ５．０〜ｐＨ９．０の０．１ｇ／ＬのＤＮＡ溶液を得た。
Wako Pure Chemical Industries, Ltd. DNA (Deoxyribonucleic acid sodium, derived from salmon testis, powder) 0.1 g was dissolved in 1000 mL of 25 mM Citrate-NaOH (pH 5.0) buffer solution, and pH 5.0 without 0.1 g / L salt was added. Of DNA solution was prepared. Two 200 mL aliquots of this solution were prepared, and a 1N NaOH aqueous solution was dropped into them while stirring to adjust the pH to 5.5 and pH 6.0, respectively.
In this way, a 0.1 g / L DNA solution having a pH of 5.0 to 9.0 was obtained.
［参考例４］ ＤＮＡ動的吸着容量の測定
上記の参考例２および３で得られた、電気伝導度およびｐＨの異なる、０．１ｇ／ＬのＤＮＡ溶液を、参考例１で作成した評価モジュールにそれぞれ透過させた。ＤＮＡ溶液の濃度Ｑ、評価モジュールが破過した時点までに透過させたＤＮＡ溶液の体積ＶD、および評価モジュール内の中空糸多孔膜ＶMの体積から、下記式（ＶＩ）を用いて動的吸着容量Ａを算出した。
Ａ＝Ｑ×ＶB／ＶM ・・・（ＶＩ）
[Reference Example 4] Measurement of DNA dynamic adsorption capacity Evaluation module prepared in Reference Example 1 using 0.1 g / L DNA solutions obtained in Reference Examples 2 and 3 and having different electrical conductivity and pH. Each was permeated. The concentration of the DNA solution Q, from the volume of the hollow fiber porous membrane V M in the volume V D and evaluation module, the DNA solution was transmitted by the time evaluation module has breakthrough, dynamic using the following formula (VI) The adsorption capacity A was calculated.
A = Q × V B / V M (VI)
なお、中空糸多孔膜の体積とは、中空部分を除いた膜体積であり、モジュール内の中空糸多孔膜の体積ＶMは、中空糸多孔膜の外径をＯＤ、内径をＩＤ、有効長をＬ、モジュール内の中空糸多孔膜の本数をｎとしたとき、以下の式（ＶＩＩ）で算出される。
ＶM＝π×（ＯＤ2−ＩＤ2）×Ｌ×ｎ／４・・・（ＶＩＩ）
Note that the volume of the hollow fiber porous membrane is a membrane volume excluding hollow portion, the volume V M of the hollow fiber porous membrane in the module, the outer diameter of the hollow fiber porous membrane OD, inner diameter ID, effective length Is L, and the number of hollow fiber porous membranes in the module is n, it is calculated by the following formula (VII).
V M = π × (OD 2 −ID 2 ) × L × n / 4 (VII)
評価モジュールが破過した時点とは、モジュール透過後の透過液中のＤＮＡ濃度が、モジュール透過前のＤＮＡ溶液の濃度の１０％である０．０１ｇ／Ｌを超えた時点のことをいう。溶液の透過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ＵＶ吸光度および電気伝導度を測定した。 The time when the evaluation module broke through refers to the time when the DNA concentration in the permeate after permeation of the module exceeded 0.01 g / L, which is 10% of the concentration of the DNA solution before permeation of the module. The solution permeated 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, AK Healthcare Bioscience AKTAexplorer100 was used, and UV absorbance and electrical conductivity of the evaluation liquid were measured.
［参考例５］ 不純物を含むＤＮＡ溶液の作成
上記参考例２で得られたｐＨ８．０、電気伝導度１．１５ｍＳ／ｃｍ、濃度０．１ｇ／ＬのＤＮＡ溶液２００ｍＬに、不純物としてＢＳＡ（ＳＩＧＭＡ製、Ａｌｂｕｍｉｎ ｆｒｏｍ ｂｏｖｉｎｅ ｓｅｒｕｍ）を０．２ｇ添加し、ＤＮＡ濃度が０．１ｇ／Ｌ、不純物であるＢＳＡ濃度が１ｇ／Ｌの溶液を作成した。ＢＳＡの通常の分子量は、６７５００である。
[Reference Example 5] Preparation of DNA Solution Containing Impurities To 200 mL of the DNA solution having a pH of 8.0, an electric conductivity of 1.15 mS / cm, and a concentration of 0.1 g / L obtained in Reference Example 2, BSA (SIGMA) was added as an impurity. 0.2 g (Albumin from bovine serum) was added to prepare a solution having a DNA concentration of 0.1 g / L and an impurity BSA concentration of 1 g / L. The normal molecular weight of BSA is 67500.
［参考例６］ ＳＤＳ−ＰＡＧＥによるタンパク質の分析
溶液中のタンパク質を分析するためにＳＤＳ−ＰＡＧＥを用いた。分析に用いる溶液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サンプル処理液またはトリスＳＤＳβＭＥ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）に１ウェルにつき１０μＬ適用し、泳動用バッファー（第一化学薬品株式会社製、ＳＤＳ−トリス−グリシン泳動バッファーを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、ＥａｓｙＳｅｐａｒａｔｏｒTM）に挿入した。３０ｍＡの定電流で１時間泳動させて、溶液中のタンパク質を分離した。泳動後のゲルプレートは染色試薬（フナコシ株式会社製、ＩｎｓｔａｎｔＢｌｕｅ、または第一化学薬品株式会社製、２Ｄ−銀染色試薬−ＩＩ）を用いて染色し、タンパク質のバンドを確認した。
[Reference Example 6] Analysis of proteins by SDS-PAGE SDS-PAGE was used to analyze proteins in solution. 10 μL of the solution used for analysis was mixed with the same amount of sample processing solution (Tris SDS sample processing solution or Tris SDS βME sample processing solution, manufactured by Daiichi Chemicals Co., Ltd.), and heat-treated at 100 ° C. for 5 minutes. The obtained sample was applied to a gel plate for electrophoresis (manufactured by Daiichi Chemicals Co., Ltd., Multigel II Mini) using a micropipette, and 10 μL per well was applied to the electrophoresis buffer (Daiichi Chemicals Co., Ltd., SDS). -Inserted into an electrophoresis tank (Easy Separator ™ manufactured by Wako Pure Chemical Industries, Ltd.) filled with Tris-Glycine running buffer diluted 10 times. Electrophoresis was performed for 1 hour at a constant current of 30 mA to separate proteins in the solution. The gel plate after electrophoresis was stained with a staining reagent (Funakoshi Co., Ltd., InstantBlue, or Daiichi Chemicals Co., Ltd., 2D-silver staining reagent-II) to confirm the protein band.
［実施例１］
参考例２で得られたｐＨ８．０、電気伝導度１．１５ｍＳ／ｃｍ、濃度０．１ｇ／ＬのＤＮＡ溶液を、参考例４の方法に従って、参考例１の評価モジュールに通液してチャージした。１８９ｍＬ通液したところでＤＮＡの破過が確認されたため通液を停止した。これより得られた動的吸着容量は３６ｍｇ／ｍＬであった。ＤＮＡ溶液の通液停止後、２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）緩衝液２０ｍＬを評価モジュールに通液して非吸着のＤＮＡを洗浄した後、電気伝導度１４８ｍＳ／ｃｍの２Ｍ ＮａＣｌ水溶液１０ｍＬを通液して、吸着したＤＮＡを溶出回収した。溶出液中のＤＮＡ濃度をｉｎｖｉｔｒｏｇｅｎ製、Ｑｕａｎｔ−ｉＴTM ｄｓＤＮＡ ＨＳ Ａｓｓａｙ Ｋｉｔを用いて処理した後、ＱｕｂｉｔTMフルオロメーターを用いて測定したところ、２．０ｍｇ／ｍＬであり、核酸が高濃度で回収された。
[Example 1]
Charge the DNA solution obtained in Reference Example 2 having a pH of 8.0, an electric conductivity of 1.15 mS / cm, and a concentration of 0.1 g / L through the evaluation module of Reference Example 1 according to the method of Reference Example 4. did. When 189 mL was passed, DNA breakthrough was confirmed, and the flow was stopped. The dynamic adsorption capacity obtained from this was 36 mg / mL. After stopping the flow of the DNA solution, 20 mL of 20 mM Tris-HCl (pH 8.0) buffer was passed through the evaluation module to wash the non-adsorbed DNA, and then 10 mL of 2 M NaCl aqueous solution having an electric conductivity of 148 mS / cm was passed through. The adsorbed DNA was eluted and collected. The DNA concentration in the eluate was measured with a Qubit ™ fluorometer after treatment with Invitrogen's Quant-iT ™ dsDNA HS Assay Kit, and it was 2.0 mg / mL, and the nucleic acid was recovered at a high concentration. It was done.
［実施例２］
参考例５で得られたｐＨ８．０、電気伝導度１．１５ｍＳ／ｃｍ、濃度０．１ｇ／ＬのＤＮＡと濃度１．０ｇ／ＬのＢＳＡを含む溶液に、ＮａＣｌを３．５１ｇ添加して、塩濃度０．３Ｍ、電気伝導度２８．５ｍＳ／ｃｍの溶液を調整し、これを実施例１と同様に評価モジュールに１５０ｍＬ通液してチャージした。通液中のろ過液のＵＶ吸光度の値は１５０ｍＡＵであり、これは１．０ｇ／ＬのＢＳＡ溶液の吸光度に等しいことから、ＢＳＡはモジュールに非吸着でろ液中に含まれ、ＤＮＡはモジュールに全吸着していると考えることができる。溶液の通液停止後、２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）緩衝液２０ｍＬを評価モジュールに通液して洗浄した後、電気伝導度１４８ｍＳ／ｃｍの２Ｍ ＮａＣｌ水溶液１０ｍＬを通液して、吸着したＤＮＡを溶出回収した。得られた溶出液を参考例６に従ってＳＤＳ−ＰＡＧＥにより評価したところ、ＢＳＡのバンドは見られなかったことから、溶出液中のＤＮＡが精製されていることが確認された。溶出液中のＤＮＡ濃度を実施例１と同様にして測定したところ、１．４ｍｇ／ｍＬであり、核酸が高濃度で回収され、回収率は９３％だったことが示された。
[Example 2]
To a solution containing DNA of pH 8.0, electrical conductivity of 1.15 mS / cm, concentration of 0.1 g / L and BSA of concentration of 1.0 g / L obtained in Reference Example 5, 3.51 g of NaCl was added. Then, a solution having a salt concentration of 0.3 M and an electric conductivity of 28.5 mS / cm was prepared, and 150 mL of the solution was passed through the evaluation module and charged in the same manner as in Example 1. The UV absorbance value of the filtrate in the flow-through is 150 mAU, which is equal to the absorbance of 1.0 g / L BSA solution, so that BSA is not adsorbed on the module and is contained in the filtrate, and DNA is contained in the module. It can be considered that all are adsorbed. After stopping the flow of the solution, 20 mL of 20 mM Tris-HCl (pH 8.0) buffer solution was passed through the evaluation module for washing, and then 10 mL of 2 M NaCl aqueous solution having an electric conductivity of 148 mS / cm was passed through and adsorbed. DNA was eluted and collected. When the obtained eluate was evaluated by SDS-PAGE according to Reference Example 6, no BSA band was observed, confirming that the DNA in the eluate was purified. When the DNA concentration in the eluate was measured in the same manner as in Example 1, it was 1.4 mg / mL, indicating that the nucleic acid was recovered at a high concentration and the recovery rate was 93%.
［実施例３］
参考例５で得られたｐＨ８．０、電気伝導度１．１５ｍＳ／ｃｍ、濃度０．１ｇ／ＬのＤＮＡと濃度１．０ｇ／ＬのＢＳＡを含む溶液を実施例２と同様にして評価モジュールに１５０ｍＬ通液してチャージした。通液中のろ過液のＵＶ吸光度の値は通液約５０ｍＬまでは０ｍＡＵであったが、その後１５０ｍＡＵにまで上昇した。１５０ｍＡＵの値は１．０ｇ／ＬのＢＳＡ溶液の吸光度に等しいことから、通液約５０ｍＬまではＢＳＡおよびＤＮＡ共に評価モジュールに全吸着し、その後破過したＢＳＡが非吸着でろ液中に含まれ、ＤＮＡは全吸着していると考えることができる。通液５０ｍＬから１５０ｍＬまでのろ過液を採集し、ろ過液サンプルとした。溶液の通液停止後、２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）緩衝液２０ｍＬを評価モジュールに通液して洗浄した後、電気伝導度２９ｍＳ／ｃｍの０．３Ｍ ＮａＣｌ水溶液２０ｍＬを通液して、溶出液１を回収した。再度２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）緩衝液２０ｍＬを評価モジュールに通液して洗浄した後、電気伝導度１４８ｍＳ／ｃｍの２Ｍ ＮａＣｌ水溶液１０ｍＬを通液して、溶出液２を回収した。得られたろ過液サンプルおよび溶出液１を参考例６に従ってＳＤＳ−ＰＡＧＥにより評価したところ、ＢＳＡのバンドのみが見られた。これより、チャージ中にはＤＮＡは全吸着し、ＢＳＡは破過後にろ液中に排出されていること、および溶出液１中にはＢＳＡのみが含まれていることが確認された。また、同様にして参考例６に従って溶出液２をＳＤＳ−ＰＡＧＥにより評価したところ、ＢＳＡのバンドは見られず、ＤＮＡのバンドのみが見られた。これより、溶出液２中には精製されたＤＮＡのみが含まれることが確認された。溶出液２中のＤＮＡ濃度を実施例１と同様にして測定したところ、１．３５ｍｇ／ｍＬであり、核酸が高濃度で回収され、回収率は９０％であったことが示された。
[Example 3]
Evaluation solution was obtained in the same manner as in Example 2 except that the solution obtained in Reference Example 5 containing pH 8.0, electrical conductivity 1.15 mS / cm, DNA having a concentration of 0.1 g / L and BSA having a concentration of 1.0 g / L was used. The solution was charged by passing 150 mL. The value of the UV absorbance of the filtrate during the passage was 0 mAU up to about 50 mL, but then increased to 150 mAU. Since the value of 150 mAU is equal to the absorbance of the 1.0 g / L BSA solution, both the BSA and DNA are completely adsorbed to the evaluation module up to about 50 mL, and then the breakthrough BSA is not adsorbed and is contained in the filtrate. It can be considered that DNA is completely adsorbed. Filtrate from 50 mL to 150 mL of liquid flow was collected and used as a filtrate sample. After stopping the flow of the solution, 20 mL of 20 mM Tris-HCl (pH 8.0) buffer solution was passed through the evaluation module for washing, and then 20 mL of 0.3 M NaCl aqueous solution having an electric conductivity of 29 mS / cm was passed through. Eluate 1 was collected. 20 mL of 20 mM Tris-HCl (pH 8.0) buffer solution was again passed through the evaluation module for washing, and then 10 mL of 2 M NaCl aqueous solution having an electric conductivity of 148 mS / cm was passed through to recover the eluate 2. When the obtained filtrate sample and eluate 1 were evaluated by SDS-PAGE according to Reference Example 6, only the BSA band was observed. From this, it was confirmed that DNA was completely adsorbed during charging, BSA was discharged into the filtrate after breakthrough, and that only BSA was contained in eluate 1. Similarly, when the eluate 2 was evaluated by SDS-PAGE according to Reference Example 6, no BSA band was observed, and only a DNA band was observed. From this, it was confirmed that eluate 2 contains only purified DNA. When the DNA concentration in the eluate 2 was measured in the same manner as in Example 1, it was 1.35 mg / mL, indicating that the nucleic acid was recovered at a high concentration and the recovery rate was 90%.
［実施例４］
（２）で得られた異なる電気伝導度の、ｐＨ８．０、濃度０．１ｇ／ＬのＤＮＡ溶液を実施例１と同様にして評価モジュールに通液してチャージした。電気伝導度が９５．２ｍＳ／ｃｍ、７４．９５ｍＳ／ｃｍ、６２．７ｍＳ／ｃｍおよび２８．５ｍＳ／ｃｍのＤＮＡ溶液を通液して、破過した際の通液量はそれぞれ、１．６ｍＬ、３．５ｍＬ、１５２．４ｍＬおよび１７５．１ｍＬであった。これより、電気伝導度が９５．２ｍＳ／ｃｍおよび７４．９５ｍＳ／ｃｍの溶液では、ＤＮＡは殆ど吸着しておらず、６２．７ｍＳ／ｃｍおよび２８．５ｍＳ／ｃｍの溶液でのＤＮＡの動的吸着容量は、それぞれ２９ｍｇ／ｍＬおよび３３ｍｇ／ｍＬであることが示された。これらの結果から、電気伝導度が約６０ｍＳ／ｃｍ以下の溶液であれば、ＤＮＡは評価モジュールに動的吸着容量が約３０ｍｇ／ｍＬで吸着することが確認された。
[Example 4]
The DNA solutions with different electrical conductivities obtained in (2) and having a pH of 8.0 and a concentration of 0.1 g / L were passed through the evaluation module and charged in the same manner as in Example 1. When DNA solutions having electrical conductivities of 95.2 mS / cm, 74.95 mS / cm, 62.7 mS / cm and 28.5 mS / cm were passed through and broke through, the flow rate was 1.6 mL, respectively. 3.5 mL, 152.4 mL and 175.1 mL. Thus, almost no DNA was adsorbed in the solutions having electrical conductivities of 95.2 mS / cm and 74.95 mS / cm, and the dynamics of DNA in the solutions of 62.7 mS / cm and 28.5 mS / cm. The adsorption capacities were shown to be 29 mg / mL and 33 mg / mL, respectively. From these results, it was confirmed that DNA was adsorbed to the evaluation module with a dynamic adsorption capacity of about 30 mg / mL in a solution having an electric conductivity of about 60 mS / cm or less.
［実施例５］
参考例３で得られた異なるｐＨの、濃度０．１ｇ／ＬのＤＮＡ溶液を実施例１と同様にして、評価モジュールに通液してチャージした。ｐＨがｐＨ５．０、ｐＨ５．５、ｐＨ６．０、ｐＨ７．０、ｐＨ７．５、ｐＨ８．５およびｐＨ９．０のＤＮＡ溶液を通液して、破過した際の通液量はそれぞれ、１７２ｍＬ、１７３ｍＬ、１９８ｍＬ、１７７ｍＬ、１５７ｍＬ、１８２．６ｍＬおよび１４２．５ｍＬであった。これより、それぞれのｐＨ値の溶液でのＤＮＡの動的吸着容量は、それぞれ３２ｍｇ／ｍＬ、３２ｍｇ／ｍＬ、３５ｍｇ／ｍＬ、３１ｍｇ／ｍＬ、２９ｍｇ／ｍＬ、３４ｍｇ／ｍＬおよび２７ｍｇ／ｍＬであることが示された。これらの結果から、少なくともｐＨ値が５〜９の範囲の溶液であれば、ＤＮＡは評価モジュールに動的吸着容量が約３０ｍｇ／ｍＬで吸着することが確認された。
[Example 5]
In the same manner as in Example 1, the DNA solutions having different pHs obtained in Reference Example 3 and having a concentration of 0.1 g / L were passed through the evaluation module and charged. When DNA solutions having pH of 5.0, pH 5.5, pH 6.0, pH 7.0, pH 7.5, pH 8.5, and pH 9.0 were passed through and passed through, the flow rates were 172 mL, respectively. 173 mL, 198 mL, 177 mL, 157 mL, 182.6 mL and 142.5 mL. From this, the dynamic adsorption capacity of DNA in each pH value solution is 32 mg / mL, 32 mg / mL, 35 mg / mL, 31 mg / mL, 29 mg / mL, 34 mg / mL and 27 mg / mL, respectively. It has been shown. From these results, it was confirmed that DNA was adsorbed to the evaluation module at a dynamic adsorption capacity of about 30 mg / mL when the solution had a pH value in the range of 5 to 9.
［比較例１］
カラムクロマトグラフィーによるＤＮＡのカラムへの吸着量を評価するために、ＧＥヘルスケアバイオサイエンス製のアニオン交換カラム、ＨｉＴｒａｐＱ ＦＦ１ｍＬを用い、これに参考例２で得られたｐＨ８．０、電気伝導度１．１５ｍＳ／ｃｍ、濃度０．１ｇ／ＬのＤＮＡ溶液を、流速１ｍＬ／ｍｉｎで実施例１と同様にしてカラムに通液してチャージした。その結果、５．０ｍＬまで通液したところで破過が確認され、動的吸着容量は０．５ｍｇ／ｍＬ以下であった。この結果から、カラムクロマトグラフィーを用いた場合のＤＮＡのカラムへの吸着量は、実施例のアニオン交換基がグラフト鎖を介して中空糸多孔膜を用いた場合に比べて極めて低く、大量の核酸精製プロセスには不向きであることが示された。
[Comparative Example 1]
In order to evaluate the amount of DNA adsorbed to the column by column chromatography, an anion exchange column manufactured by GE Healthcare Bioscience, HiTrapQ FF 1 mL was used, and the pH 8.0 obtained in Reference Example 2 and electric conductivity 1 were used. A DNA solution having a concentration of 15 mS / cm and a concentration of 0.1 g / L was charged by passing through a column in the same manner as in Example 1 at a flow rate of 1 mL / min. As a result, breakthrough was confirmed when the liquid was passed up to 5.0 mL, and the dynamic adsorption capacity was 0.5 mg / mL or less. From this result, the amount of DNA adsorbed to the column when using column chromatography is extremely low compared to the case where the anion exchange group of the example uses a hollow fiber porous membrane via a graft chain, and a large amount of nucleic acid It was shown to be unsuitable for the purification process.
アニオン交換基がグラフト鎖を介して固定した多孔膜に、核酸を含む溶液を通液する方法によって、容易に核酸を精製することができる。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、より高い核酸の吸着量と高流速での溶液の通液が可能なため、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とから、工業レベルで医薬品を製造する際の核酸の精製にも適するという産業上の利用可能性を有する。 Nucleic acids can be easily purified by a method of passing a solution containing nucleic acids through a porous membrane in which an anion exchange group is fixed via a graft chain. Compared with the conventional method using column chromatography, this method allows higher nucleic acid adsorption and solution flow at a higher flow rate, enabling high-speed processing and easier scale-up. Therefore, it has industrial applicability that it is suitable for the purification of nucleic acids when producing pharmaceuticals at an industrial level.
Claims (7)
（ｐ）アニオン交換基がグラフト鎖を介して表面に固定されている多孔膜に、核酸含有溶液を通液し、前記多孔膜へ前記核酸を吸着させる工程；および
（ｑ）前記多孔膜に吸着した前記核酸を、溶出液を通液して溶出回収する工程。 Nucleic acid purification method comprising the following steps:
(P) passing a nucleic acid-containing solution through a porous membrane having an anion exchange group immobilized on the surface via a graft chain, and adsorbing the nucleic acid to the porous membrane; and (q) adsorbing to the porous membrane A step of eluting and recovering the nucleic acid obtained by passing the eluate through.
（ｍ）前記多孔膜に不純物溶出液を通液して不純物を溶出する工程；
を含む、請求項１または２に記載の核酸の精製方法であって、
前記工程（ｍ）における不純物溶出液の電気伝導度が２０ｍＳ／ｃｍ〜６０ｍＳ／ｃｍであり、前記工程（ｑ）における溶出液の電気伝導度が９０ｍＳ／ｃｍ〜１６０ｍＳ／ｃｍである、請求項１または２に記載の核酸の精製方法。 After the step (p) and prior to the step (q), the following steps:
(M) a step of eluting impurities by passing an impurity eluate through the porous membrane;
A method for purifying a nucleic acid according to claim 1 or 2, comprising
The electrical conductivity of the impurity eluate in the step (m) is 20 mS / cm to 60 mS / cm, and the electrical conductivity of the eluate in the step (q) is 90 mS / cm to 160 mS / cm. Or the method for purifying a nucleic acid according to 2.
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| JP2012112930A (en) * | 2011-07-05 | 2012-06-14 | Asahi Kasei Medical Co Ltd | Method for purifying polymeric material |
| JP2014532718A (en) * | 2011-11-02 | 2014-12-08 | ジェネンテック， インコーポレイテッド | Overload / elution chromatography |
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| JPN6013057821; Ｊｏｕｒｎａｌ ｏｆ Ｃｈｒｏｍａｔｏｇｒａｐｈｙ Ａ，２００８，１２０７，ｐ．８４-９３ * |
| JPN6013057823; Ｊ．Ｉｏｎ Ｅｘｃｈａｎｇｅ，１９９６，７（２），ｐ．１３０-４１ * |
| JPN6013057825; Ｊ．Ｉｏｎ Ｅｘｃｈａｎｇｅ，２００７，１８（４），ｐ．４９２-７ * |
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| JP2012112930A (en) * | 2011-07-05 | 2012-06-14 | Asahi Kasei Medical Co Ltd | Method for purifying polymeric material |
| JP2014532718A (en) * | 2011-11-02 | 2014-12-08 | ジェネンテック， インコーポレイテッド | Overload / elution chromatography |
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