JP5691161B2 - Protein purification method - Google Patents
Protein purification method Download PDFInfo
- Publication number
- JP5691161B2 JP5691161B2 JP2009264400A JP2009264400A JP5691161B2 JP 5691161 B2 JP5691161 B2 JP 5691161B2 JP 2009264400 A JP2009264400 A JP 2009264400A JP 2009264400 A JP2009264400 A JP 2009264400A JP 5691161 B2 JP5691161 B2 JP 5691161B2
- Authority
- JP
- Japan
- Prior art keywords
- protein
- fine particles
- physiologically active
- magnetic fine
- active protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- Peptides Or Proteins (AREA)
Description
本発明は、磁性微粒子を用いた生理活性タンパク質の精製方法に関する。 The present invention relates to a method for purifying a physiologically active protein using magnetic fine particles.
遺伝子組換え技術の発達により、種々の生理活性タンパク質が安定した供給量で提供されるようになった。特に、抗体医薬分野は、治療効果が高く副作用が少ないなどの効果があり、多様な薬剤をターゲットにできることや、テーラーメード医療への適用が期待されており、製薬分野で注目されている（例えば、非特許文献１参照）。 With the development of gene recombination technology, various physiologically active proteins have been provided in a stable supply amount. In particular, the antibody drug field has an effect such as a high therapeutic effect and few side effects, and can be targeted to various drugs, and is expected to be applied to tailor-made medicine. Non-patent document 1).
このような遺伝子組換え技術によって産生された生理活性タンパク質の精製においては、宿主ＤＮＡ、目的の生理活性タンパク質以外のタンパク質、固形物およびフェノールレッドのような材料特異的な不純物等の夾雑物を除去する必要がある。一般には、宿主細胞から得られる生理活性タンパク質を含有するサンプルを、陰イオン交換クロマトグラフィー、ハイドロキシアパタイトクロマトグラフィー、またはこれらの組合せで処理することにより夾雑物を除去している。 In the purification of bioactive protein produced by such genetic recombination technology, host DNA, proteins other than the target bioactive protein, solids, and impurities such as material-specific impurities such as phenol red are removed. There is a need to. In general, contaminants are removed by treating a sample containing a physiologically active protein obtained from a host cell with anion exchange chromatography, hydroxyapatite chromatography, or a combination thereof.
特に、生理活性タンパク質が哺乳動物細胞を宿主として得られた抗体であるときには、プロテインＡまたはプロテインＧを固定化した担体やアパタイト担体をカラムに充填したカラム法により精製している（例えば、特許文献１および２参照）。 In particular, when the physiologically active protein is an antibody obtained using mammalian cells as a host, the protein is purified by a column method in which a carrier having protein A or protein G immobilized thereon or an apatite carrier is packed in the column (for example, patent documents) 1 and 2).
しかしながら、このようなカラム法は、サンプルの清澄化（硫安沈殿、カプリル酸沈殿、遠心およびフィルターろ過等）、サンプルの脱塩およびバッファー交換といった前処理が必要であり、かつ当該前処理には時間、労力、コストを要する。また、抗体を大量に精製する場合、巨大なカラムや巨大なサンプラーを必要とするため広い作業スペースが必要であり、かつ多大な時間を要するという問題点があった。 However, such column methods require pretreatment such as sample clarification (e.g. ammonium sulfate precipitation, caprylic acid precipitation, centrifugation and filter filtration), sample desalting and buffer exchange, and the pretreatment requires time. , Labor and cost. In addition, when purifying antibodies in large quantities, a large column and a large sampler are required, so that a large work space is required and a long time is required.
また、近年、リガンドが固定された磁性微粒子を目的のタンパク質を含むサンプルに添加し、該リガンドに結合する目的のタンパク質を吸着した後、磁性微粒子を回収し、目的物質を磁性微粒子から分離、回収する方法が知られている。例えば、ビオチン及びアビジンから選ばれた１種以上が下限臨界溶液温度（ＬＣＳＴ）を有するポリマーを介して磁性微粒子に固定された温度応答性磁性微粒子と磁石の磁力を用いた生体物質の精製方法が開発されている（例えば、特許文献３参照）。しかし、磁性微粒子を用いた精製方法では、大量のタンパク質、特に抗体を、簡便かつ低コストで精製することは困難であった。 In recent years, magnetic fine particles with ligands immobilized are added to a sample containing the target protein, the target protein that binds to the ligand is adsorbed, the magnetic fine particles are recovered, and the target substance is separated and recovered from the magnetic fine particles. How to do is known. For example, there is a method for purifying a biological material using temperature-responsive magnetic fine particles fixed to magnetic fine particles via a polymer having at least one selected from biotin and avidin and having a lower critical solution temperature (LCST) and the magnetic force of the magnet. It has been developed (see, for example, Patent Document 3). However, with the purification method using magnetic fine particles, it has been difficult to purify a large amount of proteins, particularly antibodies, simply and at low cost.
したがって、より高収率で実施コストの低い、生理活性タンパク質、特に抗体を精製する方法が望まれていた。 Therefore, there has been a demand for a method for purifying bioactive proteins, particularly antibodies, with higher yields and lower implementation costs.
本発明は、上記課題を解決するためになされたものであって、従来と比較して、格段に簡便、省スペース、低工数で大量の生理活性タンパク質、特に抗体を精製する技術を提供することを課題とする。 The present invention has been made to solve the above-mentioned problems, and provides a technique for purifying a large amount of physiologically active proteins, particularly antibodies, with much simpler, space-saving, and less man-hours than conventional ones. Is an issue.
本発明者らは、上記課題を解決するために鋭意検討を重ねた。その結果、以下の構成を採用することにより、本発明の課題を解決することを見出し、この知見に基づいて本発明を完成させた。
すなわち本発明は以下のとおりである。
１．以下の工程（１）〜（３）を少なくとも含み、工程（１）〜（３）の所要時間が４時間以下である生理活性タンパク質の精製方法。
（１）生理活性タンパク質を１０−１８〜１０−２ｍｏｌ／ｌの濃度で含み、かつ前処理していない試料と、生理活性タンパク質に対する親和性物質が結合した磁性微粒子とを混合し、混合液とする工程
（２）工程（１）で得られた混合液から磁力により磁性微粒子を分離する工程
（３）工程（２）で分離した磁性微粒子から生理活性タンパク質を溶出させる工程
２．生理活性タンパク質の収率が６０〜９９．９％である前項１に記載の方法。
３．工程（１）において、生理活性タンパク質を含み、かつ前処理していない試料の容量が１５ｍｌ以上である前項１または２に記載の方法。
４．磁性微粒子の平均粒子径が０．３〜１０μｍである前項１〜３のいずれか１項に記載の方法。
５．磁性微粒子１ｇ当りの生理活性タンパク質の精製量が５０〜１０００μｇである前項１〜４のいずれか１項に記載の方法。
６．生理活性タンパク質が抗体である前項１〜５のいずれか１項に記載の方法。
７．前処理が、細胞の分離、硫安沈殿、フェノールレッド除去、脱塩、陰イオン交換および陽イオン交換からなる群から選ばれる少なくとも１種である前項１〜６のいずれか１項に記載の方法。
８．生理活性タンパク質に対する親和性物質が、プロテインＡまたはプロテインＧである前項１〜７のいずれか１項に記載の方法。
The present inventors have made extensive studies to solve the above problems. As a result, the inventors have found that the problems of the present invention can be solved by adopting the following configurations, and have completed the present invention based on this finding.
That is, the present invention is as follows.
1. A method for purifying a physiologically active protein comprising at least the following steps (1) to (3), wherein the time required for steps (1) to (3) is 4 hours or less.
(1) A sample containing a biologically active protein at a concentration of 10 −18 to 10 −2 mol / l and not pretreated with magnetic fine particles to which an affinity substance for the physiologically active protein is bound, Step (2) Step of separating magnetic fine particles by magnetic force from the mixed solution obtained in Step (1) (3) Step of eluting physiologically active protein from the magnetic fine particles separated in Step (2) 2. The method according to item 1 above, wherein the yield of the physiologically active protein is 60 to 99.9%.
3. 3. The method according to item 1 or 2, wherein in the step (1), the volume of the sample containing a physiologically active protein and not pretreated is 15 ml or more.
4). 4. The method according to any one of items 1 to 3, wherein the magnetic fine particles have an average particle size of 0.3 to 10 μm.
5. 5. The method according to any one of items 1 to 4, wherein the purified amount of the physiologically active protein per 1 g of magnetic fine particles is 50 to 1000 μg.
6). 6. The method according to any one of items 1 to 5, wherein the physiologically active protein is an antibody.
7). 7. The method according to any one of items 1 to 6, wherein the pretreatment is at least one selected from the group consisting of cell separation, ammonium sulfate precipitation, phenol red removal, desalting, anion exchange, and cation exchange.
8). 8. The method according to any one of the preceding items 1 to 7, wherein the affinity substance for the physiologically active protein is protein A or protein G.
本発明の生理活性タンパク質の精製方法は、磁性微粒子を用いた精製方法であるため、カラム法に必要な前処理が不要であり、生理活性タンパク質の大量精製をする場合にカラム法のような大きな装置が不要である、という利点がある。また、本発明の生理活性タンパク質の精製方法は、従来の磁性微粒子を用いた方法と比較して、大量の生理活性タンパク質、特に抗体を短時間で精製できる、という利点がある。 Since the purification method of the bioactive protein of the present invention is a purification method using magnetic fine particles, the pretreatment necessary for the column method is not required. There is an advantage that an apparatus is unnecessary. In addition, the method for purifying a physiologically active protein of the present invention has an advantage that a large amount of physiologically active protein, in particular, an antibody can be purified in a short time as compared with the conventional method using magnetic fine particles.
以下、本発明について詳細に説明する。
本発明の方法は、以下の工程（１）〜（３）を少なくとも含み、工程（１）〜（３）の所要時間が４時間以下である生理活性タンパク質の精製方法である。
（１）生理活性タンパク質を１０−１８〜１０−２ｍｏｌ／ｌの濃度で含み、かつ前処理していない試料と、生理活性タンパク質に対する親和性物質が結合した磁性微粒子とを混合し、混合液とする工程
（２）工程（１）で得られた混合液から磁力により磁性微粒子を分離する工程
（３）工程（２）で分離した磁性微粒子から生理活性タンパク質を溶出させる工程
Hereinafter, the present invention will be described in detail.
The method of the present invention is a method for purifying a physiologically active protein comprising at least the following steps (1) to (3), wherein the time required for steps (1) to (3) is 4 hours or less.
(1) A sample containing a biologically active protein at a concentration of 10 −18 to 10 −2 mol / l and not pretreated with magnetic fine particles to which an affinity substance for the physiologically active protein is bound, (2) Step of separating magnetic fine particles by magnetic force from the mixture obtained in step (1) (3) Step of eluting physiologically active protein from the magnetic fine particles separated in step (2)
以下、各工程について説明する。
（１）生理活性タンパク質を１０−１８〜１０−２ｍｏｌ／ｌの濃度で含み、且つ前処理していない試料と、生理活性タンパク質に対する親和性物質が結合した磁性微粒子とを混合し、混合液とする工程
この工程は、生理活性タンパク質を含む試料と磁性微粒子とを混合し、生理活性タンパク質（タグ融合タンパク質を含む。）に対する親和性物質（以下、単に「リガンド」ということがある。）と生理活性タンパク質との親和性を利用して、生理活性タンパク質を磁性微粒子に結合させる工程である。
Hereinafter, each step will be described.
(1) A sample containing a biologically active protein at a concentration of 10 −18 to 10 −2 mol / l and not pretreated with magnetic fine particles bound with an affinity substance for the physiologically active protein In this step, a sample containing a physiologically active protein and magnetic fine particles are mixed, and an affinity substance (hereinafter sometimes simply referred to as “ligand”) for the physiologically active protein (including a tag fusion protein). This is a step of binding the physiologically active protein to the magnetic fine particles by utilizing the affinity with the physiologically active protein.
混合操作は、適当なバッファー中で生理活性タンパク質と磁性微粒子が接触し得るならば、制限はない。例えば、生理活性タンパク質を含む試料および磁性微粒子が供されたチューブを軽く転倒攪拌または振とうさせる程度で十分であり、例えば市販のボルテックスミキサー等を用いて混合する操作が挙げられる。 The mixing operation is not limited as long as the bioactive protein and the magnetic fine particles can be contacted in an appropriate buffer. For example, it is sufficient to lightly incline or shake the sample containing the bioactive protein and the tube provided with the magnetic fine particles, and examples thereof include an operation of mixing using a commercially available vortex mixer.
生理活性タンパク質とは、哺乳動物、特にヒトの生理活性タンパク質と実質的に同じ生物学的活性を有するものであり、天然由来のもの、および遺伝子組換え法により得られるものが含まれる。遺伝子組換え法によって得られるタンパク質には天然タンパク質とアミノ酸配列が同じであるもの、または該アミノ酸配列の１若しくは複数を欠失、置換、または付加したもので前記生物学的活性を有するものが含まれる。 The biologically active protein has substantially the same biological activity as that of a mammal, particularly a human biologically active protein, and includes those derived from nature and those obtained by gene recombination methods. Proteins obtained by genetic recombination include those having the same amino acid sequence as the natural protein, or those having one or more of the amino acid sequences deleted, substituted, or added and having the above biological activity It is.
生理活性タンパク質としては、例えば、ポリクローナル抗体、モノクローナル抗体、顆粒球コロニー刺激因子（Ｇ−ＣＳＦ）、成長ホルモン、インシュリンおよびプロラクチン等のタンパク質ホルモン、顆粒球マクロファージコロニー刺激因子(ＧＭ−ＣＳＦ)、エリスロポエチン(ＥＰＯ)およびトロンボポエチン等の造血因子、インターフェロン、ＩＬ−１およびＩＬ−６等のサイトカイン、組織プラスミノーゲン活性化因子(ｔＰＡ)、ウロキナーゼ、血清アルブミン、血液凝固第ＶＩＩＩ因子、レプチン、幹細胞成長因子(ＳＣＦ)、インスリン並びに副甲状腺ホルモンが挙げられるが、これらに限定されない。中でも、ポリクローナル抗体およびモノクローナル抗体がより好ましい。 Examples of physiologically active proteins include polyclonal antibodies, monoclonal antibodies, granulocyte colony stimulating factor (G-CSF), protein hormones such as growth hormone, insulin and prolactin, granulocyte macrophage colony stimulating factor (GM-CSF), erythropoietin ( EPO) and hematopoietic factors such as thrombopoietin, cytokines such as interferon, IL-1 and IL-6, tissue plasminogen activator (tPA), urokinase, serum albumin, blood coagulation factor VIII, leptin, stem cell growth factor ( SCF), insulin, and parathyroid hormone. Of these, polyclonal antibodies and monoclonal antibodies are more preferable.
タグ融合タンパク質とは、遺伝子工学技術などを利用して人為的に導入した標識を持つタンパク質である。タグ融合タンパク質は、例えば、目的のタンパク質分子の一部に、特定のアミノ酸配列を持つペプチド鎖、または酵素活性および／若しくは特定の物質に対する結合能を持つタンパク質を標識として導入することで、作製することができる。目的のタンパク質に当該標識を導入することで、該タンパク質の分離や検出が容易になる。 A tag fusion protein is a protein having a label artificially introduced using genetic engineering techniques. A tag fusion protein is produced, for example, by introducing a peptide chain having a specific amino acid sequence or a protein having an enzyme activity and / or a binding ability to a specific substance as a label into a part of a target protein molecule. be able to. By introducing the label into the target protein, the protein can be easily separated and detected.
タグ融合タンパク質のタグとしては、例えば、ポリヒスチジンタグ（Ｈｉｓタグ）、ＧＳＴタグ、ＨＡタグ、Ｃ−Ｍｙｃタグ、Ｖ５タグ、ＶＳＶ−Ｇタグ、ＨＳＶタグ、チオレドキシンタグ、アルカリフォスファターゼタグおよびリン酸化タンパク質タグが挙げられるが、これらに限定されない。 Examples of tags of the tag fusion protein include polyhistidine tag (His tag), GST tag, HA tag, C-Myc tag, V5 tag, VSV-G tag, HSV tag, thioredoxin tag, alkaline phosphatase tag and phosphorylated protein Include, but are not limited to, tags.
生理活性タンパク質に対する親和性物質としてプロテインＡまたはプロテインＧを用いる場合は、生理活性タンパク質としては、ポリクローナル抗体およびモノクローナル抗体が好ましい。ポリクローナル抗体およびモノクローナル抗体としては、ＩｇＧが好ましい。なお、ヒトＩｇＧには、ＩｇＧ１、ＩｇＧ２、ＩｇＧ３およびＩｇＧ４のサブクラスがあり、マウスＩｇＧには、ＩｇＧ１、ＩｇＧ２ａ、ＩｇＧ２ｂおよびＩｇＧ３のサブクラスがある。本発明では、これらを使用することがより好ましい。 When protein A or protein G is used as an affinity substance for a physiologically active protein, the physiologically active protein is preferably a polyclonal antibody or a monoclonal antibody. As a polyclonal antibody and a monoclonal antibody, IgG is preferable. Human IgG has IgG1, IgG2, IgG3 and IgG4 subclasses, and mouse IgG has IgG1, IgG2a, IgG2b and IgG3 subclasses. In the present invention, it is more preferable to use these.
生理活性タンパク質が糖鎖を有するタンパク質である場合、糖鎖の由来としては、特に制限はないが、哺乳動物細胞に付加される糖鎖が好ましい。哺乳動物細胞としては、例えば、チャイニーズハムスター卵巣細胞（ＣＨＯ細胞）、ベビーハムスター腎臓細胞（ＢＨＫ細胞）、アフリカミドリザル腎臓由細胞（ＣＯＳ細胞）およびヒト由来の細胞が挙げられる。 When the physiologically active protein is a protein having a sugar chain, the sugar chain is not particularly limited, but a sugar chain added to a mammalian cell is preferable. Examples of mammalian cells include Chinese hamster ovary cells (CHO cells), baby hamster kidney cells (BHK cells), African green monkey kidney cells (COS cells), and human-derived cells.
生理活性タンパク質がモノクローナル抗体である場合には、モノクローナル抗体はいかなる方法で製造されたものでもよい。モノクローナル抗体は、基本的には公知技術を使用し、抗原に感作させた免疫細胞を通常の細胞融合法によってミエローマ細胞と融合させて作成したハイブリドーマから生産することができる。 When the physiologically active protein is a monoclonal antibody, the monoclonal antibody may be produced by any method. A monoclonal antibody can basically be produced from a hybridoma prepared by fusing immune cells sensitized to an antigen with myeloma cells by a conventional cell fusion method using known techniques.
また、モノクローナル抗体は、ハイブリドーマが産生するモノクローナル抗体に限られるものではなく、ヒトに対する異種抗原性を低下させること等を目的として人為的に改変されたキメラ抗体を含む。さらに、トランスジェニック動物（ある特定の遺伝子を個体レベルで付加した遺伝子操作動物のこと。）およびファージディスプレイ等によって作製されたヒト抗体も好ましい。 In addition, the monoclonal antibody is not limited to the monoclonal antibody produced by the hybridoma, and includes a chimeric antibody artificially modified for the purpose of reducing the heterologous antigenicity to humans. Furthermore, human antibodies produced by a transgenic animal (a genetically engineered animal to which a specific gene has been added at the individual level), phage display, and the like are also preferred.
生理活性タンパク質を含む試料としては、例えば、生理活性タンパク質を含むＣＨＯ細胞などの哺乳動物細胞の培養培地およびこれに部分的精製などの一定の処理を施したもの、血清、血漿、腹水、リンパ液および尿が挙げられる。 Examples of the sample containing a physiologically active protein include a culture medium of mammalian cells such as CHO cells containing a physiologically active protein, and those subjected to a certain treatment such as partial purification, serum, plasma, ascites, lymph and Urine.
本発明の生理活性タンパク質の精製方法によれば、生理活性タンパク質を１０−１８〜１０−２ｍｏｌ／ｌ、好ましくは１０−５〜１０−２ｍｏｌ／ｌという高濃度で含む試料から、磁性微粒子を用いて生理活性タンパク質を精製することができる。生理活性タンパク質の濃度は、例えば、ブラッドフォード法、ＢＣＡ法、２８０ｎｍの吸光度の測定および電気泳動により、測定することができる。 According to the method for purifying a physiologically active protein of the present invention, magnetic fine particles are obtained from a sample containing the physiologically active protein at a high concentration of 10 −18 to 10 −2 mol / l, preferably 10 −5 to 10 −2 mol / l. Can be used to purify a physiologically active protein. The concentration of the physiologically active protein can be measured by, for example, Bradford method, BCA method, absorbance measurement at 280 nm, and electrophoresis.
磁性微粒子と混合する生理活性タンパク質を含む試料の容量は、１ｍｌ以上が好ましく、１５ｍｌ以上がより好ましい。また、５０ｌ以下が好ましく、１０ｌ以下がより好ましい。この範囲とすることで、４時間以下の生理活性タンパク質の精製が可能になる。 The volume of the sample containing the physiologically active protein mixed with the magnetic fine particles is preferably 1 ml or more, more preferably 15 ml or more. Moreover, 50 l or less is preferable and 10 l or less is more preferable. By setting it within this range, it becomes possible to purify a physiologically active protein in 4 hours or less.
工程（１）に供する生理活性タンパク質を含む試料は前処理していない試料である。生理活性タンパク質を含む試料の前処理としては、例えば、カラム法によりタンパク質を精製する際に必要である前処理が挙げられる。該前処理としては、例えば、細胞の分離、硫安沈殿、カプリル酸沈殿、硫酸デキストラン沈殿、ポリビニルピロリドン沈殿、活性炭によるフェノールレッド除去、並びに脱塩およびバッファー交換（例えば、ゲル濾過、透析および限外濾過）が挙げられる。細胞を分離する方法としては、例えば、遠心分離、ろ過、および限外ろ過が挙げられる。 The sample containing the physiologically active protein used in step (1) is a sample that has not been pretreated. Examples of pretreatment of a sample containing a physiologically active protein include pretreatment necessary for purifying a protein by a column method. Examples of the pretreatment include cell separation, ammonium sulfate precipitation, caprylic acid precipitation, dextran sulfate precipitation, polyvinylpyrrolidone precipitation, phenol red removal with activated carbon, and desalting and buffer exchange (eg gel filtration, dialysis and ultrafiltration). ). Examples of the method for separating cells include centrifugation, filtration, and ultrafiltration.
本発明の方法は生理活性タンパク質を含む試料の前処理を経なくてもよいため、生理活性タンパク質の精製に要する時間を短縮することができる。前処理に要する時間は、生理活性タンパク質の種類および量等により異なるが、通常、１５〜３０時間である。例えば、遠心分離により夾雑物である細胞を分離する場合、通常約３０分〜１時間の所用時間である。また、生理活性タンパク質を含む試料が血清の場合、硫安沈殿および脱塩をする必要があるが、例えば、血清の量が１５〜１０００ｍｌの場合、その所要時間は、通常約３０分から１時間である。また、生理活性タンパク質を含む試料が培養上清の場合、フェノールレッド除去を必要に応じてする必要があるが、培養上清が１５〜１０００ｍｌの場合その所要時間は、通常約１時間である。 Since the method of the present invention does not require pretreatment of a sample containing a physiologically active protein, the time required for purification of the physiologically active protein can be shortened. The time required for the pretreatment varies depending on the type and amount of the physiologically active protein, but is usually 15 to 30 hours. For example, when separating cells that are contaminants by centrifugation, the required time is usually about 30 minutes to 1 hour. Further, when the sample containing the physiologically active protein is serum, it is necessary to perform ammonium sulfate precipitation and desalting. For example, when the amount of serum is 15 to 1000 ml, the required time is usually about 30 minutes to 1 hour. . Moreover, when the sample containing a physiologically active protein is a culture supernatant, it is necessary to remove phenol red as necessary. When the culture supernatant is 15 to 1000 ml, the required time is usually about 1 hour.
本発明の方法に用いる磁性微粒子は、平均粒子径（キュムラント解析）が、０．１〜１００μｍであることが好ましく、０．３〜１０μｍであることがより好ましい。磁性微粒子の平均粒子径をこの範囲とすることで、４時間以下の生理活性タンパク質の精製が可能になるからである。平均粒子径（キュムラント解析）は、例えば、レーザーゼータ電位計（大塚電子株式会社製ＥＬＳ−８０００（商品名））を用いて測定することにより算出する。 The magnetic fine particles used in the method of the present invention preferably have an average particle size (cumulant analysis) of 0.1 to 100 μm, and more preferably 0.3 to 10 μm. This is because, by setting the average particle size of the magnetic fine particles within this range, it becomes possible to purify a physiologically active protein in 4 hours or less. The average particle size (cumulant analysis) is calculated by, for example, measuring using a laser zeta electrometer (ELS-8000 (trade name) manufactured by Otsuka Electronics Co., Ltd.).
原料磁性微粒子の素材としては、例えば、マグネタイト、酸化ニッケル、フェライト、コバルト鉄酸化物、バリウムフェライト、炭素鋼、タングステン鋼、ＫＳ鋼、希土類コバルト磁石およびヘマタイトなどの鉄酸化物が挙げられる。 Examples of the raw material magnetic fine particles include iron oxides such as magnetite, nickel oxide, ferrite, cobalt iron oxide, barium ferrite, carbon steel, tungsten steel, KS steel, rare earth cobalt magnet, and hematite.
原料磁性微粒子の調製方法は特に限定はないが、例えば、オレイン酸とドデシルベンゼンスルホン酸ナトリウムを用いて、マグネタイトを二重のミセルとし、水溶液中に分散させる、マグネタイトの調製方法を挙げることができる［バイオカタライシス（Ｂｉｏｃａｔａｌｙｓｉｓ）１９９１年、第５巻、６１〜６９頁］。 The method for preparing the raw magnetic particles is not particularly limited. For example, magnetite can be prepared as a double micelle using oleic acid and sodium dodecylbenzenesulfonate, and dispersed in an aqueous solution. [Biocatalysis 1991, Volume 5, pages 61-69].
原料磁性微粒子は、有機物と鉄酸化物との複合体であってもよい。有機物としては、例えば、多糖および合成高分子が挙げられる。多糖および合成高分子としては、例えば、アガロースおよびポリエチレングリコールの架橋体が好ましく挙げられる。 The raw material magnetic fine particles may be a composite of an organic substance and iron oxide. Examples of organic substances include polysaccharides and synthetic polymers. As the polysaccharide and the synthetic polymer, for example, a cross-linked product of agarose and polyethylene glycol is preferably mentioned.
本発明の方法に用いる磁性微粒子は、その表面がフレキシビリティーを有することが好ましい。磁性微粒子の表面がフレキシビリティを有することで生理活性タンパク質と磁性微粒子に固定化されたリガンドの反応効率が良好になるからである。ここで、「フレキシビリティー」とは、柔軟性、融通性のことを意味する。具体的には、「フレキシビリティーを有する」とは、材料表面に依存する立体障害を受けない状態にすることであり、リガンドと生理活性タンパク質との相互作用（結合定数）を阻害しないことを示す。 The surface of the magnetic fine particles used in the method of the present invention preferably has flexibility. This is because the reaction efficiency of the biologically active protein and the ligand immobilized on the magnetic fine particles is improved because the surface of the magnetic fine particles has flexibility. Here, “flexibility” means flexibility and flexibility. Specifically, “having flexibility” means that the material does not suffer from steric hindrance depending on the surface of the material, and does not inhibit the interaction (binding constant) between the ligand and the bioactive protein. Show.
例えば、原料磁性微粒子と疎水性物質との複合体を形成させ、該複合体の表面を、複数の親水性モノマーからなる共重合体を架橋してなるゲルで被覆することにより、磁性微粒子の表面のフレキシビリティーを増大することができる。ここで「被覆」とは、磁性微粒子の外側を覆って、磁性微粒子の最外にゲル層が形成されてなることを指す。 For example, the surface of the magnetic fine particles is formed by forming a composite of raw magnetic fine particles and a hydrophobic substance and coating the surface of the composite with a gel formed by crosslinking a copolymer composed of a plurality of hydrophilic monomers. The flexibility of the can be increased. Here, “coating” means that the outer side of the magnetic fine particle is covered and a gel layer is formed on the outermost side of the magnetic fine particle.
疎水性物質としては、例えば、酪酸、吉草酸、カプロン酸、エナント酸、カプリル酸、ペラルゴン酸、カプリン酸、ラウリン酸、ミリスチン酸、ペンタデシル酸、パルチミン酸、パルミトイル酸、マルガリン酸、ステアリン酸、オレイン酸、バクセン酸、リノール酸、（９，１２，１５）−リノレン酸、（６，９，１２）−リノレン酸、（６，９，１２）−リノレン酸、ツベルクロステアリン酸、アラキジン酸、アラキドン酸、ベヘン酸およびリグノセリン酸が挙げられる。 Examples of hydrophobic substances include butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, lauric acid, myristic acid, pentadecylic acid, palmitic acid, palmitoyl acid, margaric acid, stearic acid, olein Acid, vaccenic acid, linoleic acid, (9,12,15) -linolenic acid, (6,9,12) -linolenic acid, (6,9,12) -linolenic acid, tuberculostearic acid, arachidic acid, arachidone Examples include acids, behenic acid and lignoceric acid.
親水性モノマーとしては、例えば、アクリル酸、メタクリル酸、アクリルアミド、メタクリルアミド、ヒドロキシメタクリルアミド、グリシジルメタクリレート、ポリエチレングリコールメタクリルエチルメタクリレートとポリエチレングリコールジアクリレートおよびＮ−（３−Ａｍｉｎｏｐｒｏｐｙｌ）ｍｅｔｈａｃｒｙｌａｍｉｄｅ ｈｙｄｒｏｃｈｌｏｒｉｄｅが挙げられる。 Examples of the hydrophilic monomer include acrylic acid, methacrylic acid, acrylamide, methacrylamide, hydroxymethacrylamide, glycidyl methacrylate, polyethylene glycol methacrylethyl methacrylate and polyethylene glycol diacrylate, and N- (3-Aminopropyryl) methacrylate hydrochloride.
また、例えば、架橋剤の添加量や親水性モノマー側鎖の分子量を大きくすることによっても、磁性微粒子の表面のフレキシビリティを増大させることができる。 Further, for example, the surface flexibility of the magnetic fine particles can be increased by increasing the amount of the crosslinking agent added and the molecular weight of the hydrophilic monomer side chain.
生理活性タンパク質に対する親和性物質としては、例えば、プロテインＡ、プロテインＧ、ビオチン、アビジン、グルタチオン、レクチンおよび抗体を磁性微粒子に表面修飾することで、それらに対する特異的吸着作用を有する生理活性タンパク質と特異的に結合できる。該親和性物質がビオチンの場合は、アビジンとの特異的な結合を介してビオチン化された検出対象のタンパク質と、またビオチン化された抗体を用いてそれらの抗原である種々の生理活性タンパク質と更に結合することが可能である。 Examples of affinity substances for physiologically active proteins include protein A, protein G, biotin, avidin, glutathione, lectin, and antibodies that are surface-modified to magnetic microparticles so that they are specific to physiologically active proteins having a specific adsorption action on them. Can be combined. When the affinity substance is biotin, the protein to be detected biotinylated through specific binding to avidin, and various bioactive proteins that are antigens using biotinylated antibodies Further coupling is possible.
本発明の方法では、市販されているアビジンまたはビオチン化タンパク質が利用でき、ビオチン化は、当該技術分野で周知の方法に従えばよい。生理活性タンパク質に対する親和性物質がグルタチオンの場合は、グルタチオン−Ｓ−トランスフェラーゼ（以下、「ＧＳＴ」という。）を含有するタンパク質と特異的に結合できる。このようなＧＳＴ含有タンパク質の調製は当該技術分野で周知の方法に従えばよい。 In the method of the present invention, commercially available avidin or biotinylated protein can be used, and biotinylation may be performed according to a method well known in the art. When the affinity substance for a physiologically active protein is glutathione, it can specifically bind to a protein containing glutathione-S-transferase (hereinafter referred to as “GST”). Such a GST-containing protein may be prepared by a method well known in the art.
磁性微粒子は適当な分散媒に分散させた状態で使用することが好ましい。分散媒としては、例えば、リン酸バッファー、リン酸カリウムバッファー、リン酸ナトリウムバッファー、トリス塩酸バッファー、１，４−ピペラジンジエタンスルフォン酸バッファー（以下、「ＰＩＰＥＳバッファー」という。）、ホウ酸バッファーおよび酢酸バッファーが挙げられる。 The magnetic fine particles are preferably used in a state of being dispersed in an appropriate dispersion medium. Examples of the dispersion medium include a phosphate buffer, a potassium phosphate buffer, a sodium phosphate buffer, a tris hydrochloric acid buffer, a 1,4-piperazine diethane sulfonate buffer (hereinafter referred to as “PIPES buffer”), a borate buffer, and An acetate buffer is mentioned.
磁性微粒子は、生理活性タンパク質を含む試料に、好ましくは１０−１８〜１０−２ｍｏｌ／ｌ、より好ましくは１０−５〜１０−２ｍｏｌ／ｌとなるように加えることが好ましい。この範囲とすることで、４時間以下の生理活性タンパク質の精製が可能になる。 The magnetic fine particles are preferably added to a sample containing a physiologically active protein so that the concentration is preferably 10 −18 to 10 −2 mol / l, more preferably 10 −5 to 10 −2 mol / l. By setting it within this range, it becomes possible to purify a physiologically active protein in 4 hours or less.
精製すべき生理活性タンパク質と磁性微粒子の質量比は１：１〜１：１０００であることが好ましく、１：１０〜１：１００であることがより好ましい。この範囲とすることで、４時間以下の生理活性タンパク質の精製が可能になる。 The mass ratio of the physiologically active protein to be purified and the magnetic fine particles is preferably 1: 1 to 1: 1000, more preferably 1:10 to 1: 100. By setting it within this range, it becomes possible to purify a physiologically active protein in 4 hours or less.
（２）工程（１）で得られた混合液から磁力により磁性微粒子を分離する工程
この工程は、工程（１）で得られた混合液から、生理活性タンパク質の結合した磁性微粒子を磁力により分離する工程である。具体的には、例えば、生理活性タンパク質と磁性微粒子との結合を適当なチューブ内で行った場合、チューブの側壁に磁石を外側から近づけることによって磁性微粒子をチューブの側壁近傍に保持しつつ、チューブ内から上澄み部分となる液体を排出することによって、生理活性タンパク質が結合した磁性微粒子を分離することができる。
(2) Step of separating magnetic fine particles from the mixed solution obtained in step (1) by magnetic force This step separates magnetic fine particles bound with physiologically active protein from the mixed solution obtained in step (1) by magnetic force. It is a process to do. Specifically, for example, when binding of a physiologically active protein and magnetic fine particles is performed in an appropriate tube, the magnetic fine particles are held near the side wall of the tube by bringing a magnet close to the side wall of the tube from the outside. By discharging the liquid that becomes the supernatant portion from the inside, the magnetic fine particles to which the physiologically active protein is bound can be separated.
磁性微粒子の分離に用いる磁石等の磁力は、用いる磁性微粒子の有する磁力の大きさによって異なる。磁力は、目的の磁性微粒子を磁集可能な程度の磁力を適宜使用できる。磁石の素材としては、例えば、上述した磁性微粒子の素材で構成されたものを使用することができる。例えば、ネオジム磁石（マグナ社製）等が利用できる。ネオジム磁石の磁力は、３８００ガウス以上がより好ましい。 The magnetic force of a magnet or the like used for separating the magnetic fine particles varies depending on the magnitude of the magnetic force of the magnetic fine particles used. As the magnetic force, a magnetic force that can collect magnetic particles of interest can be used as appropriate. As a material of the magnet, for example, a material composed of the above-described magnetic fine particle material can be used. For example, a neodymium magnet (manufactured by Magna) can be used. The magnetic force of the neodymium magnet is more preferably 3800 gauss or more.
（３）工程（２）で分離した磁性微粒子から生理活性タンパク質を溶出する工程
この工程は、工程（２）で磁性分離により分離した磁性微粒子から、目的とする生理活性タンパク質を分離し、回収する工程である。生理活性タンパク質の特性に従い、当該技術分野で周知の方法によって磁性微粒子から生理活性タンパク質を分離する。
(3) The step of eluting the bioactive protein from the magnetic fine particles separated in the step (2) In this step, the target bioactive protein is separated and recovered from the magnetic fine particles separated by the magnetic separation in the step (2). It is a process. According to the characteristics of the bioactive protein, the bioactive protein is separated from the magnetic fine particles by a method well known in the art.
具体的には、例えば、チューブ内に適当な溶出液を加えることによって、生理活性タンパク質を磁性微粒子から溶出させる。そして、溶出後、チューブの側壁に磁石を外側から近づけて磁性微粒子をチューブの側壁近傍に保持しつつ、チューブ内から上澄み部分となる液体を採取することによって、磁性微粒子に結合していた生理活性タンパク質を回収することができる。 Specifically, for example, the bioactive protein is eluted from the magnetic fine particles by adding an appropriate eluate into the tube. Then, after elution, the magnet is brought close to the side wall of the tube from outside to hold the magnetic fine particles in the vicinity of the side wall of the tube, and the liquid that becomes the supernatant portion is collected from the inside of the tube, so that the physiological activity bound to the magnetic fine particles Protein can be recovered.
生理活性タンパク質を溶出させる溶出液としては、生理活性タンパク質とリガンドとの親和性を低下させる効果がある液体が好ましい。当該液体としては、例えば、塩酸、イミダゾール、グルタチオンおよびビオチンを含むバッファーが挙げられる。バッファーとしては、例えば、リン酸カリウムバッファー、リン酸ナトリウムバッファー、トリス塩酸塩バッファー、ＰＩＰＥＳバッファー、ホウ酸バッファーおよびグリシン塩酸バッファーが挙げられる。 As the eluate for eluting the bioactive protein, a liquid having an effect of reducing the affinity between the bioactive protein and the ligand is preferable. Examples of the liquid include a buffer containing hydrochloric acid, imidazole, glutathione, and biotin. Examples of the buffer include potassium phosphate buffer, sodium phosphate buffer, Tris hydrochloride buffer, PIPES buffer, borate buffer, and glycine hydrochloride buffer.
尚、磁性微粒子から生理活性タンパク質を溶出させる前に磁性微粒子を洗浄し、夾雑物を除去してもよい。具体的な洗浄方法としては、例えば、０．５質量％のＴｗｅｅｎ２０（ユニヒェマ ヒェミー ベスローテン フエンノートシャップ社の登録商標：Ｐｏｌｙｏｘｙｅｔｈｙｌｅｎｅ Ｓｏｒｂｉｔａｎ Ｍｏｎｏｌａｕｒａｔｅ）を含むリン酸ナトリウムバッファー中に生理活性タンパク質−磁性微粒子複合体を加え、再分散を繰り返すことで疎水性相互作用で結合や巻き込みにより磁性微粒子中に取り込まれていた夾雑物を取り除く方法が挙げられる。 The magnetic fine particles may be washed to remove contaminants before eluting the bioactive protein from the magnetic fine particles. As a specific cleaning method, for example, a physiologically active protein-magnetic fine particle complex is contained in a sodium phosphate buffer containing 0.5% by mass of Tween 20 (registered trademark of Polyoxyethylene Sorbitan Monolaurate). In addition, by repeating redispersion, there is a method of removing contaminants taken in the magnetic fine particles by binding or entrainment by hydrophobic interaction.
上記工程（１）〜（３）を経ることによって、生理活性タンパク質を含む試料から目的の生理活性タンパク質を精製することができる。本発明の方法による、生理活性タンパク質の収率は、６０〜９９．９％であることが好ましく、８０〜９９％であることがより好ましい。 By going through the steps (1) to (3), the target physiologically active protein can be purified from the sample containing the physiologically active protein. The yield of the physiologically active protein according to the method of the present invention is preferably 60 to 99.9%, more preferably 80 to 99%.
本発明の方法において、上記工程（１）〜（３）の所要時間は、４時間以下であることが好ましく、３．５時間以下であることがより好ましい。また、生理活性タンパク質の種類、生理活性タンパク質を含む試料の量および生理活性タンパク質を含む試料中の生理活性タンパク質の濃度などにより適宜調整することができるが、上記工程（１）〜（３）の所要時間は、通常、３分以上であることが好ましく、１０分以上であることがより好ましい。 In the method of the present invention, the time required for the steps (1) to (3) is preferably 4 hours or less, and more preferably 3.5 hours or less. Moreover, although it can adjust suitably by the kind of bioactive protein, the quantity of the sample containing bioactive protein, the density | concentration of the bioactive protein in the sample containing bioactive protein, etc., the said process (1)-(3) The required time is usually preferably 3 minutes or more, and more preferably 10 minutes or more.
以下、本発明を実施例および比較例により説明するが、本発明はこれらの実施例に限定されるものではない。
磁気分離とは、磁性微粒子等を磁石等の磁力によって、液体から収集することをいう。なお、磁気分離には、株式会社二六製作所製ネオジム磁石を使用した。
精製水とは，ミリポア社製純水製造装置「Ｄｉｒｅｃｔ−ＱＴＭ」によって精製された導電率１８ＭΩｃｍの水であり、ＭｉｌｌＱ水と呼ばれることもある。
EXAMPLES Hereinafter, although an Example and a comparative example demonstrate this invention, this invention is not limited to these Examples.
Magnetic separation refers to collecting magnetic fine particles and the like from a liquid by a magnetic force such as a magnet. For magnetic separation, a neodymium magnet manufactured by 26 Manufacturing Co., Ltd. was used.
The purified water is water having a conductivity of 18 MΩcm purified by a pure water production apparatus “Direct-Q ™ ” manufactured by Millipore, Inc., and is sometimes referred to as MillQ water.
実施例１
＜表面にゲル層を持つ磁性微粒子の製造方法＞
２００ｍｌ容のフラスコに、塩化第二鉄・六水和物（１．０ｍｏｌ）及び塩化第一鉄・四水和物（０．５ｍｏｌ）混合水溶液を１００ｍｌ入れ、メカニカルスターラー（ＬＡＢＯＲＡＴＯＲＹ ＨＩＧＨＴ ＰＯＷＥＲ ＭＩＸＥＲ、ＡＳＯＮＥ社製）で攪拌し、この混合溶液を５０℃に昇温した後、２８質量％アンモニア水溶液５．０ｍｌを滴下し、１時間程度攪拌した。この操作で、平均粒子径が約５ｎｍのマグネタイトが得られた。得られたマグネタイトを精製水で３回洗浄し、乾燥した後メタノールにて２０ｍｇ/ｍｌに調製した。マグネタイト分散液１００ｍｌに対し、オレイン酸ナトリウム３ｇを加え８０℃で１２時間還流操作を行った。遠心分離（１００００ｇ、３０分、２５℃）して過剰なオレイン酸ナトリウムを除き、再びメタノールに分散し２０ｍｇ／ｍｌに調製し、マグネタイト−オレイン酸ナトリウム複合体のメタノール溶液を得た。
Example 1
<Method for producing magnetic fine particles having a gel layer on the surface>
A 200 ml flask is charged with 100 ml of a mixed aqueous solution of ferric chloride / hexahydrate (1.0 mol) and ferrous chloride / tetrahydrate (0.5 mol), and a mechanical stirrer (LABORATORY HIGH POWER MIXER, ASONE). The mixture solution was heated to 50 ° C., and 5.0 ml of 28% by mass aqueous ammonia solution was added dropwise and stirred for about 1 hour. By this operation, magnetite having an average particle diameter of about 5 nm was obtained. The obtained magnetite was washed 3 times with purified water, dried and then adjusted to 20 mg / ml with methanol. 3 g of sodium oleate was added to 100 ml of the magnetite dispersion and refluxed at 80 ° C. for 12 hours. Centrifugation (10000 g, 30 minutes, 25 ° C.) removed excess sodium oleate, and the dispersion was again dispersed in methanol to prepare 20 mg / ml to obtain a methanol solution of magnetite-sodium oleate complex.
マグネタイト−オレイン酸ナトリウム複合体（磁性微粒子）のメタノール溶液１００ｍｌにメタクリル酸グリシジル２５ｇ、ポリエチレングリコールメチルエーテルメタクリレート（数平均分子量２０８０）１ｇおよびポリエチレングリコールジアクリレート（数平均分子量５７５）０．１ｇを加え、室温で２時間攪拌した後、２，２’−アゾビスイソブチロニトリルを０．２５ｇ加え、７０℃に加熱し６時間反応させた。得られた反応混合物を室温に戻した後、磁気分離（ネオジム磁石、株式会社二六製作所製）し、メタノールで２回洗浄することで、表面にゲル層を持つ磁性微粒子のメタノール分散液を得た。前記磁性微粒子のメタノール分散液から、磁気分離（ネオジム磁石、株式会社二六製作所製）によりメタノールを除き、精製水に置換し一晩回転混合によりゲル層を膨潤させた。その後、１ｍｌの分散液を分取し、磁気分離を行い上清を除き、水を含有する磁性微粒子の質量を測定し、乾燥（６０℃、６時間、恒温・乾燥器 ＤＫＮ４０２、ヤマト科学社製）させた後、乾燥後の磁性微粒子の質量を測定した。その結果、ゲル層を有する磁性微粒子の含水率は８３質量％であった。 25 ml of glycidyl methacrylate, 1 g of polyethylene glycol methyl ether methacrylate (number average molecular weight 2080) and 0.1 g of polyethylene glycol diacrylate (number average molecular weight 575) are added to 100 ml of methanol solution of magnetite-sodium oleate complex (magnetic fine particles), After stirring at room temperature for 2 hours, 0.25 g of 2,2′-azobisisobutyronitrile was added, heated to 70 ° C., and reacted for 6 hours. After returning the obtained reaction mixture to room temperature, it is magnetically separated (neodymium magnet, manufactured by 26 Co., Ltd.) and washed twice with methanol to obtain a methanol dispersion of magnetic fine particles having a gel layer on the surface. It was. Methanol was removed from the methanol dispersion of the magnetic fine particles by magnetic separation (neodymium magnet, manufactured by 26 Co., Ltd.), replaced with purified water, and the gel layer was swollen overnight by rotary mixing. Thereafter, 1 ml of the dispersion liquid is collected, magnetically separated, the supernatant is removed, the mass of the magnetic fine particles containing water is measured, and dried (60 ° C., 6 hours, constant temperature / dryer DKN402, manufactured by Yamato Scientific Co., Ltd.) The mass of the magnetic fine particles after drying was measured. As a result, the water content of the magnetic fine particles having the gel layer was 83% by mass.
得られた磁性微粒子の平均粒子径をレーザーゼータ電位計（大塚電子株式会社製ＥＬＳ−８０００（商品名））により測定したところ、５２５ｎｍであった。 It was 525 nm when the average particle diameter of the obtained magnetic microparticles | fine-particles was measured with the laser zeta electrometer (Otsuka Electronics Co., Ltd. ELS-8000 (brand name)).
実施例２
＜ストレプトアビジン固定化磁性微粒子によるビオチン化抗体の分離＞
（ストレプトアビジン固定化磁性微粒子の調製）
実施例１と同様に調製したゲル層を持つ磁性微粒子１ｍｌ（２０ｍｇ／ｍｌ）を磁気分離し上清を除いた。そこへ精製水１ｍｌを添加し、磁性微粒子を再分散させた。再び磁気分離後、上清を除き、そこへ２５ｍＭのＭＥＳ ｂｕｆｆｅｒ（ＭＥＳ：２−（Ｎ−Ｍｏｒｐｈｏｌｉｎｏ）ｅｔｈａｎｅ ｓｕｌｆｏｎｉｃ Ａｃｉｄ、ｐＨ４．７５）１ｍｌを加えて再分散させた。ストレプトアビジン（１０ｍｇ／ｍｌ）を２５μｌ添加し、３時間回転混合した。磁気分離を行い、上清を除いた後１ｍｌの１００ｍＭ ＴＢＳ（ＴＢＳ：Ｔｒｉｓ−Ｂｕｆｆｅｒｅｄ Ｓａｌｉｎｅ）を加えて１時間反応させた。磁気分離の後、上清を除去し、１ｍｌの１００ｍＭ ＰＢＳ（ＰＢＳ：Ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒｅｄ ｓａｌｉｎｅ、ｐＨ７．５、０．０１質量％のＢＳＡを含む）を加え、磁性微粒子を再分散させた。同様の操作をもう一度行い、ストレプトアビジン固定化磁性微粒子を得た。
Example 2
<Separation of biotinylated antibody with streptavidin-immobilized magnetic fine particles>
(Preparation of streptavidin-immobilized magnetic fine particles)
1 ml (20 mg / ml) of magnetic fine particles having a gel layer prepared in the same manner as in Example 1 was magnetically separated and the supernatant was removed. 1 ml of purified water was added thereto to redisperse the magnetic fine particles. After magnetic separation again, the supernatant was removed, and 1 ml of 25 mM MES buffer (MES: 2- (N-Morpholino) ethane sulfone Acid, pH 4.75) was added thereto and redispersed. Streptavidin (10 mg / ml) was added in an amount of 25 μl and mixed by rotation for 3 hours. After magnetic separation and removal of the supernatant, 1 ml of 100 mM TBS (TBS: Tris-Buffered Saline) was added and allowed to react for 1 hour. After magnetic separation, the supernatant was removed, and 1 ml of 100 mM PBS (PBS: Phosphate buffered saline, pH 7.5, containing 0.01% by mass of BSA) was added to redisperse the magnetic fine particles. The same operation was performed once again to obtain streptavidin-immobilized magnetic fine particles.
（ストレプトアビジン固定化磁性微粒子によるビオチン化抗体の分離）
ストレプトアビジン固定化磁性微粒子１００μｌに対してビオチン化ＩｇＧ（ＲＯＣＫＬＡＮＤ社製、Ａｎｔｉ−ＩｇＧ（Ｆｃ）、Ｒａｂｂｉｔ、Ｇｏａｔ−Ｐｏｌｙ、Ｂｉｏｔｉｎ）１０μｇを添加し５分間回転混合した。その後１５μｌを分取し、ＳＤＳ−ＰＡＧＥ（ドデシル硫酸ナトリウム−ポリアクリルアミドゲル電気泳動の略。）による電気泳動のＴｏｔａｌサンプルとした。２分間磁気分離し、上清から１５μｌを分取し、ＳＤＳ−ＰＡＧＥによる電気泳動の上清（ｓｕｐ）サンプルとした。上清を除去し、１ｍｌの１００ｍＭ ＰＢＳ（ｐＨ７．５、０．０１質量％のＢＳＡを含む）を加え、磁性微粒子を再分散させた。同様の操作をもう一度行い、上清を除去後１５μｌの１００ｍＭＰＢＳを添加し、ＳＤＳ−ＰＡＧＥによる電気泳動の残渣（ｐｐｔ）サンプルとした。
(Separation of biotinylated antibody using streptavidin-immobilized magnetic microparticles)
10 μg of biotinylated IgG (manufactured by ROCKLAND, Anti-IgG (Fc), Rabbit, Goat-Poly, Biotin) was added to 100 μl of streptavidin-immobilized magnetic fine particles, and mixed by rotation for 5 minutes. Thereafter, 15 μl was taken out and used as a total sample for electrophoresis by SDS-PAGE (abbreviation for sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After magnetic separation for 2 minutes, 15 μl was collected from the supernatant and used as a supernatant sample for electrophoresis by SDS-PAGE. The supernatant was removed, and 1 ml of 100 mM PBS (pH 7.5, containing 0.01% by mass of BSA) was added to redisperse the magnetic fine particles. The same operation was performed once again, and after removing the supernatant, 15 μl of 100 mM PBS was added to obtain a residue (ppt) sample for electrophoresis by SDS-PAGE.
１５μｌの各サンプルをＳＤＳ−ＰＡＧＥによる電気泳動で分析した結果を図１に示す。図１に示したように、Ｔｏｔａｌサンプルに存在するＩｇＧに由来するバンドがｓｕｐサンプルでは消失し、磁性微粒子側（ｐｐｔサンプル）に結合していることがわかった。この結果から、本発明によるストレプトアビジン固定化磁性微粒子により、効率的にビオチン化ＩｇＧを回収できることがわかった。 FIG. 1 shows the result of analyzing 15 μl of each sample by electrophoresis by SDS-PAGE. As shown in FIG. 1, it was found that the band derived from IgG present in the Total sample disappeared in the sup sample and was bound to the magnetic fine particle side (ppt sample). From this result, it was found that biotinylated IgG can be efficiently recovered with the streptavidin-immobilized magnetic fine particles according to the present invention.
実施例３
＜プロテインＡ固定化磁性微粒子による抗体の精製＞
（プロテインＡ固定化磁性微粒子の調製）
実施例１と同様に調製したゲル層を持つ磁性微粒子１ｍｌ（２０ｍｇ／ｍｌ）を磁気分離し、上清を除いた。そこへ精製水１ｍｌを添加し、磁性微粒子を再分散させた。再び磁気分離後、上清を除き、そこへ１ｍｌの２５ｍＭ ＭＥＳ ｂｕｆｆｅｒを加え再分散した。２５μｌのプロテインＡ（１０ｍｇ／ｍｌ）を添加し、３時間回転混合した。磁気分離を行い、上清を除いた後、１ｍｌの１００ｍＭ ＴＢＳを加えて１時間反応させた。磁気分離の後、上清を除去し、１ｍｌの１００ｍＭ ＰＢＳ（ｐＨ７．５）を加え、磁性微粒子を再分散させた。同様の操作をもう一度行い、プロテインＡ固定化磁性微粒子を得た。
Example 3
<Purification of antibodies using protein A-immobilized magnetic particles>
(Preparation of protein A-immobilized magnetic fine particles)
1 ml (20 mg / ml) of magnetic fine particles having a gel layer prepared in the same manner as in Example 1 was magnetically separated, and the supernatant was removed. 1 ml of purified water was added thereto to redisperse the magnetic fine particles. After magnetic separation again, the supernatant was removed, and 1 ml of 25 mM MES buffer was added thereto for redispersion. 25 μl of protein A (10 mg / ml) was added and vortex mixed for 3 hours. After magnetic separation and removal of the supernatant, 1 ml of 100 mM TBS was added and allowed to react for 1 hour. After magnetic separation, the supernatant was removed and 1 ml of 100 mM PBS (pH 7.5) was added to redisperse the magnetic microparticles. The same operation was performed once again to obtain protein A-immobilized magnetic fine particles.
（プロテインＡ固定化磁性微粒子を用いたウサギおよびヒト血清からのＩｇＧの分離）
プロテインＡ固定化磁性微粒子１００μｌに対してウサギおよびヒト血清１０μｌを添加し５分間、ＭＴＲ−１０３（ＡＳ ＯＮＥ社製）によって回転混合させた。上清を除去し、１ｍｌの１００ｍＭ ＰＢＳを加えて再分散させた。再び磁気分離操作を行い、上清を除去し再分散させた。磁気分離の後、上清を除去し、９０μｌの１００ｍＭグリシン塩酸バッファー（ｐＨ３．０）加え３分間振とう混合することでＩｇＧを溶出させた（１回目）。
磁気分離の後、上清を分離し、分離した上清に対して１０μｌの１０×ＰＢＳを添加し、溶出サンプル１とした。
(Separation of IgG from rabbit and human serum using protein A-immobilized magnetic microparticles)
10 μl of rabbit and human serum was added to 100 μl of protein A-immobilized magnetic microparticles, and rotated and mixed with MTR-103 (manufactured by AS ONE) for 5 minutes. The supernatant was removed and redispersed by adding 1 ml of 100 mM PBS. The magnetic separation operation was performed again, and the supernatant was removed and redispersed. After magnetic separation, the supernatant was removed, and 90 μl of 100 mM glycine hydrochloride buffer (pH 3.0) was added and mixed with shaking for 3 minutes to elute IgG (first time).
After the magnetic separation, the supernatant was separated, and 10 μl of 10 × PBS was added to the separated supernatant to obtain an eluted sample 1.
再び９０μｌの１００ｍＭグリシン塩酸バッファー（ｐＨ３．０）加え、３分間振とう混合して、ＩｇＧを溶出させた（２回目）。分離した上清に対して１０μｌの１０×ＰＢＳを添加し、溶出サンプル１とした。磁気分離の後、上清を分離し、上清に対して１０μｌの１０×ＰＢＳを添加し、溶出サンプル２とした。 Again 90 μl of 100 mM glycine hydrochloride buffer (pH 3.0) was added and mixed with shaking for 3 minutes to elute IgG (second time). To the separated supernatant, 10 μl of 10 × PBS was added to obtain eluted sample 1. After magnetic separation, the supernatant was separated, and 10 μl of 10 × PBS was added to the supernatant to obtain Elution Sample 2.
１５μｌの各サンプルをＳＤＳ−ＰＡＧＥによる電気泳動で分析した結果を図２に示す。図２に示すように、本発明によるプロテインＡ固定化磁性微粒子を用いて、ウサギ血清およびヒト血清のいずれからでもＩｇＧを分離・溶出できることがわかった。 FIG. 2 shows the results of analyzing 15 μl of each sample by electrophoresis by SDS-PAGE. As shown in FIG. 2, it was found that IgG can be separated and eluted from either rabbit serum or human serum using protein A-immobilized magnetic microparticles according to the present invention.
実施例４
＜プロテインＡ固定化磁性微粒子及びプロテインＧ固定化磁性微粒子と従来製品との比較＞
（ＩｇＧの分離）
実施例３と同様に、プロテインＡ固定化磁性微粒子及びプロテインＧ固定化磁性微粒子を調製した。各３ｍｇのプロテインＡ固定化磁性微粒子及プロテインＧ固定化磁性微粒子を２００μｇのＩｇＧ Ｆｒａｃｔｉｏｎ ｏｆ Ａｎｔｉ−Ｓｔｒｅｐｔａｖｉｄｉｎ Ｒａｂｂｉｔ（ＲＯＣＫＬＡＮＤ社製）を含む１００μｌの１００ｍＭ ＰＢＳ溶液に添加し２５℃で１５分間、ＭＴＲ−１０３（ＡＳ ＯＮＥ社製）によって回転混合させた。２分間磁気分離の後、上清を除去して２００μｌの１００ｍＭ ＰＢＳ−Ｔ（０．０５質量％のＴｗｅｅｎ２０を含むＰＢＳ溶液）を加え、磁性微粒子を再分散させた。再び磁気分離操作を行い、上清を除去し再分散させた。さらに２回洗浄操作を行い、上清を除去した。４５μｌの１００ｍＭ グリシン塩酸バッファー（ｐＨ２．７）を加え、ピペットにより混合した。ボルテックスを５分間行い、磁気分離の後に上清を回収した。さらに再度抽出操作を行い、得られた２回分の上清を混合し、８μｌの１Ｎ ＮａＯＨを加え中和した。中和した各上清をＳＤＳ−ＰＡＧＥによる電気泳動を行った。
Example 4
<Comparison of protein A immobilized magnetic particles and protein G immobilized magnetic fine particles with conventional products>
(Separation of IgG)
In the same manner as in Example 3, protein A-immobilized magnetic fine particles and protein G-immobilized magnetic fine particles were prepared. 3 mg each of protein A-immobilized magnetic microparticles and protein G-immobilized magnetic microparticles were added to 100 μl of 100 mM PBS solution containing 200 μg of IgG Fraction of Anti-Streptavidin Rabbit (manufactured by ROCKLAND), and MTR-103 at 25 ° C. for 15 minutes. (Made by AS ONE). After magnetic separation for 2 minutes, the supernatant was removed and 200 μl of 100 mM PBS-T (PBS solution containing 0.05% by mass of Tween 20) was added to redisperse the magnetic microparticles. The magnetic separation operation was performed again, and the supernatant was removed and redispersed. The washing operation was further performed twice to remove the supernatant. 45 μl of 100 mM glycine hydrochloride buffer (pH 2.7) was added and mixed with a pipette. Vortexed for 5 minutes and the supernatant was collected after magnetic separation. Further, the extraction operation was performed again, and the resulting supernatants were mixed, and neutralized by adding 8 μl of 1N NaOH. Each neutralized supernatant was subjected to electrophoresis by SDS-PAGE.
電気泳動の結果はＣＳ Ａｎａｌｙｚｅｒ（ＡＴＴＯ社製）により解析した。なお、検量線は、ＩｇＧ Ｆｒａｃｔｉｏｎ ｏｆ Ａｎｔｉ−Ｓｔｒｅｐｔａｖｉｄｉｎ Ｒａｂｂｉｔを用いて作成した。また、Ｄｙｎａｂｅａｄｓ ＰｒｏｔｅｉｎＡおよびＧ（Ｉｎｖｉｔｏｒｏｇｅｎ社製）についても同様の操作を行った。 The result of electrophoresis was analyzed by CS Analyzer (manufactured by ATTO). The calibration curve was prepared using IgG Fraction of Anti-Streptavidin Rabbit. The same operation was performed for Dynabeads Protein A and G (Invitrogen).
電気泳動の結果、プロテインＡ固定化磁性微粒子は、１５８．４μｇのＩｇＧを分離し、Ｄｙｎａｂｅａｄｓ ＰｒｏｔｅｉｎＡは、３６．８μｇのＩｇＧを分離した。また、プロテインＧ固定化磁性微粒子は、９７．４μｇのＩｇＧを分離し、Ｄｙｎａｂｅａｄｓ ＰｒｏｔｅｉｎＧは、２７．３μｇのＩｇＧを分離した。 As a result of electrophoresis, protein A-immobilized magnetic microparticles separated 158.4 μg of IgG, and Dynabeads Protein A separated 36.8 μg of IgG. Protein G-immobilized magnetic fine particles separated 97.4 μg of IgG, and Dynabeads Protein G separated 27.3 μg of IgG.
上記の結果から、Ｄｙｎａｂｅａｄｓ ＰｒｏｔｅｉｎＧを用いた場合に対して、本発明によるプロテインＡ固定化磁性微粒子では、最大４．３倍、プロテインＧ固定化磁性微粒子では、３．６倍のＩｇＧを分離できることが分かった。 From the above results, it is possible to separate IgG up to 4.3 times with protein A-immobilized magnetic microparticles according to the present invention and 3.6 times with protein G-immobilized magnetic microparticles when Dynabeads Protein G is used. I understood.
（希薄溶液からのＩｇＧの分離）
プロテインＡ固定化磁性微粒子を実施例３と同様に調製した。１ｍｇのプロテインＡ固定化磁性微粒子を５０μｇのＩｇＧ Ｆｒａｃｔｉｏｎ ｏf Ａｎｔｉ−Ｓｔｒｅｐｔａｖｉｄｉｎ Ｒａｂｂｉｔ（ＲＯＣＫＬＡＮＤ社製）を含む５０ｍｌの希薄溶液に添加し３０分間回転混合させた。磁気分離の後、上清を除去し１ｍｌの１００ｍＭ ＰＢＳを加えて磁性微粒子を再分散させた。再び磁気分離操作を行い、上清を除去し、磁性微粒子を再分散させた。磁気分離の後、上清を除去し、磁性微粒子をＳＤＳ−ＰＡＧＥによる電気泳動で分析した。
(Separation of IgG from dilute solution)
Protein A-immobilized magnetic fine particles were prepared in the same manner as in Example 3. 1 mg of protein A-immobilized magnetic fine particles were added to 50 ml of a dilute solution containing 50 μg of IgG Fraction of Anti-Streptavidin Rabbit (manufactured by ROCKLAND), and mixed by rotation for 30 minutes. After magnetic separation, the supernatant was removed and 1 ml of 100 mM PBS was added to redisperse the magnetic microparticles. The magnetic separation operation was performed again, the supernatant was removed, and the magnetic fine particles were redispersed. After magnetic separation, the supernatant was removed, and the magnetic microparticles were analyzed by electrophoresis with SDS-PAGE.
電気泳動の結果をＣＳ Ａｎａｌｙｚｅｒ（ＡＴＴＯ社製）により解析した。なお、検量線の作成はＩｇＧ Ｆｒａｃｔｉｏｎ ｏｆ Ａｎｔｉ−Ｓｔｒｅｐｔａｖｉｄｉｎ Ｒａｂｂｉｔを用いて行った。また、Ｄｙｎａｂｅａｄｓ ＰｒｏｔｅｉｎＡ（Ｉｎｖｉｔｏｒｏｇｅｎ社製）についても同様の操作を行った。 The result of electrophoresis was analyzed by CS Analyzer (manufactured by ATTO). The calibration curve was created using IgG Fraction of Anti-Streptavidin Rabbit. The same operation was performed for Dynabeads Protein A (Invitrogen).
その結果、プロテインＡ固定化磁性微粒子により２５μｇのＩｇＧが分離できた。一方、Ｄｙｎａｂｅａｄｓ ＰｒｏｔｅｉｎＡにより６μｇのＩｇＧが分離できた。この結果から、本発明によるプロテインＡ固定化磁性微粒子はＤｙｎａｂｅａｄｓ ＰｒｏｔｅｉｎＡと比較して、希薄溶液から約４倍のＩｇＧを分離できることがわかった。 As a result, 25 μg of IgG could be separated by protein A-immobilized magnetic fine particles. On the other hand, 6 μg of IgG could be separated by Dynabeads Protein A. From this result, it was found that the protein A-immobilized magnetic fine particles according to the present invention can separate about 4 times as much IgG from a dilute solution as compared with Dynabeads Protein A.
（磁性微粒子とＩｇＧの反応速度の比較）
プロテインＡ固定化磁性微粒子を実施例３と同様に調製した。１０ｍｇのプロテインＡ固定化磁性微粒子を２５μｇのＩｇＧ Ｆｒａｃｔｉｏｎ ｏｆ Ａｎｔｉ−Ｓｔｒｅｐｔａｖｉｄｉｎ Ｒａｂｂｉｔ（ＲＯＣＫＬＡＮＤ社）を含む５００μｌのＰＢＳ溶液に添加し、０．５、１、５、１０、２０、３０分の反応の後に磁気分離を行い、上清を１５μｌ分取し、電気泳動を行った。
(Comparison of reaction rate between magnetic fine particles and IgG)
Protein A-immobilized magnetic fine particles were prepared in the same manner as in Example 3. 10 mg of Protein A-immobilized magnetic microparticles are added to 500 μl of PBS solution containing 25 μg of IgG Fraction of Anti-Streptavidin Rabbit (ROCKLAND), and after reaction for 0.5, 1, 5, 10, 20, and 30 minutes Magnetic separation was performed, and 15 μl of the supernatant was collected and subjected to electrophoresis.
電気泳動の結果は、ＣＳ Ａｎａｌｙｚｅｒ（ＡＴＴＯ社製）により解析した。また、Ｄｙｎａｂｅａｄｓ ＰｒｏｔｅｉｎＡ（Ｉｎｖｉｔｏｒｏｇｅｎ社製）についても同様の操作を行った。プロテインＧ固定化磁性微粒子についても同様に比較を行った。その結果を図３に示す。 The result of electrophoresis was analyzed by CS Analyzer (manufactured by ATTO). The same operation was performed for Dynabeads Protein A (Invitrogen). The same comparison was made for protein G-immobilized magnetic fine particles. The result is shown in FIG.
図３の上図に示すように、プロテインＡ固定化磁性微粒子は、約０．５分でほぼ１００％のＩｇＧを分離した。一方、Ｄｙｎａｂｅａｄｓ ＰｒｏｔｅｉｎＡは、３０分で８５％のＩｇＧを分離した。また、図３の下図に示すように、プロテインＧ固定化微粒子は、０．５分でほぼ１００％のＩｇＧを分離した。一方、Ｄｙｎａｂｅａｄｓ ＰｒｏｔｅｉｎＧは、３０分で２７％のＩｇＧを分離した。これらの結果から、本発明によるプロテインＡ固定化磁性微粒子およびプロテインＧ固定化磁性微粒子は、Ｄｙｎａｂｅａｄｓ ＰｒｏｔｅｉｎＡおよびＤｙｎａｂｅａｄｓ ＰｒｏｔｅｉｎＧと比較して、ＩｇＧとの反応速度が速いことがわかった。 As shown in the upper diagram of FIG. 3, the protein A-immobilized magnetic fine particles separated almost 100% IgG in about 0.5 minutes. On the other hand, Dynabeads Protein A separated 85% IgG in 30 minutes. Further, as shown in the lower diagram of FIG. 3, the protein G-immobilized microparticles separated almost 100% IgG in 0.5 minutes. On the other hand, Dynabeads Protein G separated 27% IgG in 30 minutes. From these results, it was found that the protein A-immobilized magnetic microparticles and protein G-immobilized magnetic microparticles according to the present invention have a faster reaction rate with IgG than Dynabeads Protein A and Dynabeads Protein G.
実施例５
＜ハイブリドーマ培養上清５０ｍｌからのプロテインＡ固定化磁性微粒子によるＩｇＧ２ａの分離＞
（処理１）
５０ｍｌのハイブリドーマ培養上清を３００ｍｌ容積の三角フラスコに分注し、１０質量％ Ｔｗｅｅｎ２０を２５０μｌ加えた（最終濃度：０．０５質量％）。そこへ、実施例３と同様に調製した４ｍｌのプロテインＡ固定化磁性微粒子(１０ｍｇ／ｍｌ)を加え、室温で１時間、泡立たぬ程度に振とう機（９０回／分、ＭＳ−１ Ｍｉｎｉｓｈａｋｅｒ、ＩＫＡ社製）によりインキュベートした。
Example 5
<Separation of IgG2a from Protein A Immobilized Magnetic Fine Particles from 50 ml of Hybridoma Culture Supernatant>
(Process 1)
50 ml of the hybridoma culture supernatant was dispensed into a 300 ml Erlenmeyer flask, and 250 μl of 10 mass% Tween 20 was added (final concentration: 0.05 mass%). Thereto, 4 ml of protein A-immobilized magnetic fine particles (10 mg / ml) prepared in the same manner as in Example 3 were added, and shaken at a room temperature for 1 hour without foaming (90 times / minute, MS-1 Minishaker, Incubated by IKA).
（処理２）
５０ｍｌ遠心チューブ２本に均等に移し、磁気分離を室温にて１０分間行った。上清を三角フラスコに戻し、再度４ｍｌのプロテインＡ固定化磁性微粒子を加え、１時間同様にインキュベートした。
(Process 2)
The sample was evenly transferred to two 50 ml centrifuge tubes, and magnetic separation was performed at room temperature for 10 minutes. The supernatant was returned to the Erlenmeyer flask, and 4 ml of protein A-immobilized magnetic microparticles were added again and incubated in the same manner for 1 hour.
（洗浄操作）
処理１で磁気分離した磁性微粒子に３ｍｌのＰＢＳ−Ｔ（Ｔｗｅｅｎ２０を０．０５質量％含むＰＢＳ）を加え、十分に分散させた後、２ｍｌのマイクロチューブに２本に移した。空になった遠心チューブに再度１ｍｌのＰＢＳ−Ｔを加え、壁面を洗い、上記の２ｍｌマイクロチューブへ０．５ｍｌのＰＢＳ−Ｔを加えた。磁気分離装置（マグナスタント−８、マグナビート株式会社製）にセットし、氷水中で５〜１０分間静置し磁気分離した。上清を除去し、再びＰＢＳ−Ｔを添加して磁性微粒子を分散させ、５〜１０分間磁気分離を行い、上清を除去した。洗浄操作を再び行い、上清を除去した。
(Washing operation)
3 ml of PBS-T (PBS containing 0.05% by weight of Tween 20) was added to the magnetic fine particles magnetically separated in the treatment 1, and the mixture was sufficiently dispersed, and then transferred to two in a 2 ml microtube. 1 ml of PBS-T was added again to the emptied centrifuge tube, the wall surface was washed, and 0.5 ml of PBS-T was added to the 2 ml microtube. The magnetic separation device (Magnastant-8, manufactured by Magna Beat Co., Ltd.) was set and left in ice water for 5 to 10 minutes for magnetic separation. The supernatant was removed, PBS-T was added again to disperse the magnetic fine particles, magnetic separation was performed for 5 to 10 minutes, and the supernatant was removed. The washing operation was performed again and the supernatant was removed.
（抽出操作）
各チューブに４５０μｌの１００ｍＭ グリシン塩酸バッファー(ｐＨ２．７)加え、十分に混合させた。マクロチューブをフロートへ指し、氷水中に装着し、ソニケーター（Ｓ３０Ｈ Ｅｌｍａｓｏｎｉｃ、Ｅｌｍａ社製）に浮かべ、超音波洗浄を行った。超音波洗浄は、１０秒間行った後、１０秒間休止し、これを洗浄操作のサイクルとし、これを５分間繰り返した。磁気分離操作の後、上清を分離し、新しいマイクロチューブに移し、８０μｌの１Ｍ ＮａＯＨを加え中和し、精製ＩｇＧ２ａを得た。抽出操作をさらにもう一度行った。再度（処理２）プロテインＡ固定化磁性微粒子を加え分離操作を行ったものは、同様に洗浄・抽出操作を行った。
(Extraction operation)
450 μl of 100 mM glycine hydrochloride buffer (pH 2.7) was added to each tube and mixed well. The macrotube was pointed to the float, mounted in ice water, floated on a sonicator (S30H Elmasonic, manufactured by Elma), and subjected to ultrasonic cleaning. The ultrasonic cleaning was performed for 10 seconds and then paused for 10 seconds. This was used as a cleaning operation cycle, which was repeated for 5 minutes. After the magnetic separation operation, the supernatant was separated, transferred to a new microtube, and neutralized by adding 80 μl of 1M NaOH to obtain purified IgG2a. The extraction operation was performed once more. Again (treatment 2) Protein A-immobilized magnetic fine particles were added and subjected to separation operation, and washing and extraction operations were performed in the same manner.
１５μｌの各サンプルをＳＤＳ−ＰＡＧＥによる電気泳動で分析した結果を図４に示す。
図４に示したように、上清の洗浄液には、ＩｇＧに由来するバンドはなく溶出液にのみＩｇＧに由来するバンドが見られた。この結果から、本発明による磁性微粒子を用いた操作により、ＩｇＧを特異的に精製することができることがわかった。また、回収率は９３％であり、所要時間は約４時間であった。
FIG. 4 shows the results of analyzing 15 μl of each sample by electrophoresis by SDS-PAGE.
As shown in FIG. 4, in the supernatant washing solution, there was no band derived from IgG, and only a band derived from IgG was seen in the eluate. From this result, it was found that IgG can be specifically purified by the operation using the magnetic fine particles according to the present invention. The recovery rate was 93%, and the required time was about 4 hours.
実施例６
＜ＡＢ−ＮＴＡ固定化磁性微粒子によるＨｉｓ−プロテインＡの回収＞
（ＡＢ−ＮＴＡの固定化）
実施例１と同様に調製したゲル層を持つ磁性微粒子１０ｍｌ（２０ｍｇ／ｍｌ）を磁気分離し上清を除いた。そこへ１０ｍｌの精製水を添加し、磁性微粒子を再分散させた。再び磁気分離後、上清を除き、そこへ１０ｍｌの１００ｍＭ ホウ酸バッファー（ｐＨ８．５）を加えて磁性微粒子を再分散させた。１００ｍｇのＡＢ−ＮＴＡ ｆｒｅｅ ａｃｉｄ（ＤＯＪＩＤＯ社製）を添加し、１２時間回転混合した。磁気分離の後、上清を除去し、１０ｍｌの精製水を加えて磁性微粒子を再分散させた。同様の操作をさらに３回行い、ＡＢ−ＮＴＡ固定化磁性微粒子を得た。
Example 6
<Recovery of His-protein A by AB-NTA-immobilized magnetic fine particles>
(Immobilization of AB-NTA)
10 ml (20 mg / ml) of magnetic fine particles having a gel layer prepared in the same manner as in Example 1 were magnetically separated and the supernatant was removed. 10 ml of purified water was added thereto to redisperse the magnetic fine particles. After magnetic separation again, the supernatant was removed, and 10 ml of 100 mM borate buffer (pH 8.5) was added thereto to redisperse the magnetic fine particles. 100 mg of AB-NTA free acid (manufactured by DOJIDO) was added and mixed by rotation for 12 hours. After magnetic separation, the supernatant was removed, and 10 ml of purified water was added to redisperse the magnetic fine particles. The same operation was further performed three times to obtain AB-NTA-immobilized magnetic fine particles.
（ニッケルとの複合体の形成）
１０ｍｌのＡＢ−ＮＴＡ固定化磁性微粒子を磁気分離の後、上清を除去し、１０ｍｌの１Ｍ濃度硫酸ニッケル溶液に懸濁し、６時間撹拌した。その後、磁気分離を行い、上清を除去し、１０ｍｌの精製水を加えて磁性微粒子を再分散させた。同様の操作をさらに３回行い、ＰＢＳ（ｐＨ７．５）中に再懸濁しＮｉ−ＮＴＡ磁性微粒子を保存した。
(Formation of a complex with nickel)
After magnetic separation of 10 ml of AB-NTA-immobilized magnetic fine particles, the supernatant was removed, suspended in 10 ml of 1M nickel sulfate solution, and stirred for 6 hours. Thereafter, magnetic separation was performed, the supernatant was removed, and 10 ml of purified water was added to redisperse the magnetic fine particles. The same operation was further performed three times, and the suspension was resuspended in PBS (pH 7.5) to store Ni-NTA magnetic fine particles.
（Ｈｉｓ−プロテインＡの回収）
Ｈｉｓ−ｔａｇをＮ−末端に有するＲｅｃｏｍｂｉｎａｎｔ−ＰｒｏｔｅｉｎＡ（フナコシ社製）１００μｇを含む１０μｌのＧｏａｔ血清に、１ｍｇのＮｉ−ＮＴＡ磁性微粒子を添加した。５分間の反応の後、上清を除去した。そこへＴｗｅｅｎ２０を０.１質量％含むＰＢＳを１ｍｌ添加して、Ｎｉ−ＮＴＡ磁性微粒子を再分散させた。同様にＴｗｅｅｎ２０を０．１質量％含むＰＢＳ１ｍｌで５回洗浄操作を行った。磁気分離の後、上清を除去し、５０μｌの５００ｍＭイミダゾールＰＢＳ溶液を添加して溶出操作を行った。１５μｌの上清を分取しＳＤＳ−ＰＡＧＥによる電気泳動で分析した結果を図５に示す。
(Recovery of His-Protein A)
1 mg of Ni-NTA magnetic fine particles was added to 10 μl of Goat serum containing 100 μg of Recombinant-Protein A (manufactured by Funakoshi) having His-tag at the N-terminus. After 5 minutes reaction, the supernatant was removed. 1 ml of PBS containing 0.1% by mass of Tween 20 was added thereto to redisperse the Ni-NTA magnetic fine particles. Similarly, the washing operation was performed 5 times with 1 ml of PBS containing 0.1% by mass of Tween20. After magnetic separation, the supernatant was removed, and 50 μl of a 500 mM imidazole PBS solution was added for elution. FIG. 5 shows the result of separating 15 μl of the supernatant and analyzing it by electrophoresis by SDS-PAGE.
図５に示したように、溶出液にＲｅｃｏｍｂｉｎａｎｔ−ＰｒｏｔｅｉｎＡに由来するバンドがみられた。この結果から、本発明によるＮｉ−ＮＴＡ磁性微粒子により、Ｈｉｓタグを有するタンパク質を特異的に分離できることがわかった。 As shown in FIG. 5, a band derived from Recombinant-Protein A was observed in the eluate. From this result, it was found that Ni-NTA magnetic fine particles according to the present invention can specifically separate proteins having His tags.
Claims (9)
（１）生理活性タンパク質を１０−１８〜１０−２ｍｏｌ／ｌの濃度で含み、かつ前処理していない試料と、生理活性タンパク質に対する親和性物質が結合した、平均粒子径が、０．１〜１００μｍである、表面に、メタクリル酸グリシジルとポリエチレングリコールメチルエーテルメタクリレートとからなる共重合体をポリエチレングリコールジアクリレートにより架橋して得られるゲル層を有する磁性微粒子を混合し、混合液とする工程
（２）工程（１）で得られた混合液から磁力により磁性微粒子を分離する工程
（３）工程（２）で分離した磁性微粒子から生理活性タンパク質を溶出させる工程 A method for purifying a physiologically active protein comprising at least the following steps (1) to (3), wherein the time required for steps (1) to (3) is 4 hours or less.
(1) An average particle diameter of 0.1 to 10 with a concentration of 10 −18 to 10 −2 mol / l of a physiologically active protein and a non-pretreated sample and an affinity substance for the physiologically active protein bound thereto A step of mixing magnetic fine particles having a gel layer obtained by crosslinking a copolymer of glycidyl methacrylate and polyethylene glycol methyl ether methacrylate with polyethylene glycol diacrylate on the surface, which is ˜100 μm to form a mixed solution ( 2) Step of separating magnetic fine particles by magnetic force from the mixture obtained in step (1) (3) Step of eluting physiologically active protein from the magnetic fine particles separated in step (2)
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