TW201709961A

TW201709961A - Nanofiber-based affinity chromatographic process

Info

Publication number: TW201709961A
Application number: TW104129272A
Authority: TW
Inventors: 陳子智
Original assignee: 益生生技開發股份有限公司
Priority date: 2015-09-04
Filing date: 2015-09-04
Publication date: 2017-03-16

Abstract

The present invention primarily relates to an affinity chromatographic process for purifying a target substance using a stationary phase support material, wherein a polymeric nanofiber is used as the matrix of the stationary phase support material. Preferably, the polymeric nanofiber is made from polyacrylonitrile by electro-spinning. Taking advantage of its large specific surface area and high porosity, the stationary phase support material exhibits an excellent ability to capture the target substance.

Description

以奈米纖維建立之親和性層析法Affinity chromatography established by nanofibers

本發明關於一種運用靜相擔體材料來純化物質的親和性層析法，特別是一種運用所述靜相擔體材料來純化物質的固定金屬親和層析法，其中高分子奈米纖維被使用做為靜相擔體材料中的基材。The present invention relates to an affinity chromatography method for purifying a substance using a static phase carrier material, in particular, a fixed metal affinity chromatography method using the static phase carrier material to purify a substance, wherein the polymer nanofiber is used. As a substrate in a static phase carrier material.

親和性層析技術是一種利用分子間的專一性結合差異來進行分離和純化的技術，其中固定金屬親和層析法（immobilized metal affinity chromatography；IMAC）中最常見的一種乃是將Ni²⁺ 、Co²⁺ 、Zn²⁺ 等金屬離子固定在靜相擔體材料上，另運用遺傳工程技術在欲分離純化的目標蛋白的N端或C端綴加一個聚組胺酸標記（poly-histidine tag）而形成融合蛋白，利用組胺酸上的氮原子作為路易士鹼，與做為路易士酸的金屬離子形成配位鍵結。當含有融合蛋白的萃出物通過所述靜相擔體材料時，金屬離子將會與聚組胺酸標記產生專一性結合使融合蛋白滯留在靜相擔體材料上，雜質成份則因為不會與聚組胺酸標記產生專一性結合而被洗除，後續再運用高濃度的咪唑溶液與聚組胺酸標記競爭金屬離子而使融合蛋白脫離靜相擔體材料，從而達成分離純化蛋白的目的。由於固定金屬親和層析法不會使用強酸鹼值或高鹽濃度的沖提環境，對蛋白質而言相當溫和，不容易使蛋白質發生變性或失去活性，所以非常適合於搭配基因重組技術來大量生產如酵素等具有高經濟價值的生物活性蛋白質。Affinity chromatography is a technique for separation and purification using specific binding differences between molecules. The most common one of immobilized metal affinity chromatography (IMAC) is Ni ²⁺ , Metal ions such as Co ²⁺ and Zn ²⁺ are immobilized on the stationary phase support material, and a poly-histidine tag is added to the N-terminus or C-terminus of the target protein to be separated and purified by genetic engineering. The fusion protein is formed by using a nitrogen atom on histidine as a Lewis base to form a coordination bond with a metal raft as a Lewis acid. When the extract containing the fusion protein passes through the static phase carrier material, the metal scorpion will specifically bind to the polyhistidine label to cause the fusion protein to remain on the static phase carrier material, and the impurity component will not It is washed out with the specificity of the polyhistidine labeling, and then the high concentration of the imidazole solution and the polyhistidine acid labeling the competing metal ions to decouple the static phase carrier material, thereby achieving the purpose of separating and purifying the protein. . Since immobilized metal affinity chromatography does not use a strong acid base or a high salt concentration, it is quite mild to proteins, and it is not easy to denature or inactivate proteins. Therefore, it is very suitable for genetic recombination. Production of biologically active proteins with high economic value such as enzymes.

上述固定金屬親和層析法一般使用瓊脂糖（agarose）、纖維素或葡聚醣（dextran）等樹脂做為基材。舉例而言，常用的商品化基材包括由法瑪西亞公司（Pharmacia Inc., Piscataway, NJ, USA）所出品的Sepharose®4B和Sepharose®6B。藉由在基材表面進行官能基團改質，使基材表面接枝有亞胺基二乙酸（iminodiacetic acid；IDA）、氮基三乙酸（nitrilotriacetic acid；NTA）或是三羧甲基乙二胺（N,N,N-tris(carboxymethyl) ethylene diamine；TED）等多牙螯合化合物，以供螯合金屬離子。另外也有使用多孔性材料做為基材，再以塗佈或接枝的方式在其表面上進行改質，以製成薄膜材質的靜相擔體材料。The above fixed metal affinity chromatography generally uses a resin such as agarose, cellulose or dextran as a substrate. For example, commonly used commercial substrates include Sepharose® 4B and Sepharose® 6B from Pharmacia Inc., Piscataway, NJ, USA. The surface of the substrate is grafted with iminodiacetic acid (IDA), nitrilotriacetic acid (NTA) or tricarboxymethylethylene by modifying the functional group on the surface of the substrate. A multidentate chelate compound such as an amine (N, N, N-tris (carboxymethyl) ethylene diamine; TED) for sequestering metal rafts. In addition, a porous material is used as a substrate, and then modified on the surface by coating or grafting to form a static phase carrier material of a film material.

然而，上述樹脂型基材的缺點在於吸附速度慢，孔洞大使得動力學變慢，在工業上長期使用時容易受微生物污染或分解，而且通常保存在含有醇類的保存液中，所以在使用前必須事先進行繁複的前處理程序以除去保存液，避免醇類影響目標蛋白的品質。薄膜型基材則是比表面積（specific surface area）太小，造成目標蛋白的分離捕集效率不高。再者，商品化的靜相擔體材料一般而言價格相當高昂，而且被預設成供操作者重覆使用。但是，靜相擔體材料的使用次數增加無可避免地會造成雜質污染，阻礙金屬離子的結合位址，導致對於目標物的分離捕集效率降低。However, the above-mentioned resin-type substrate has disadvantages in that the adsorption speed is slow, the pores are large, the kinetics are slow, and it is easily contaminated or decomposed by microorganisms when used industrially for a long period of time, and is usually stored in a preservation liquid containing an alcohol, so that it is used. Beforehand, a complicated pre-treatment procedure must be performed to remove the preservation solution to prevent the alcohol from affecting the quality of the target protein. The film-type substrate has a specific surface area that is too small, resulting in low separation and collection efficiency of the target protein. Furthermore, commercial static phase carrier materials are generally quite expensive and are preset for repeated use by the operator. However, an increase in the number of times the static phase carrier material is used inevitably causes impurity contamination, hinders the binding site of the metal ions, and causes a decrease in separation and collection efficiency for the target.

因此，親和性層析相關技術領域中亟需一種施行方便、對於目標物質的分離捕集具有高效率，而且所使用的靜相擔體材料成本較低的純化方法。Therefore, in the art of affinity chromatography, there is a need for a purification method which is convenient to carry out, has high efficiency in separation and collection of a target substance, and has a low cost of a stationary phase carrier material.

本案發明人已進行廣泛的研究，希望能滿足上述產業需求。現在，本案發明人發現，使用高分子奈米纖維來取代習用樹脂型和薄膜型基材做為施行親和性層析時所使用之靜相擔體材料的基材，可以憑藉著高分子奈米纖維的總表面積大、孔隙度高和吸附速度快等優點，克服上述產業問題，並獲致極為優異的分離捕集效率。而且，這種新的靜相擔體材料亦具有成本低廉的優勢，適合大量及快速之蛋白質純化製程使用，特別適合於一次性使用後即丟棄的情況。The inventor of the present case has conducted extensive research and hopes to meet the above industrial needs. Now, the inventors of the present invention have found that the use of a polymer nanofiber to replace a conventional resin type and a film type substrate as a substrate for a static phase carrier material used for affinity chromatography can be achieved by means of a polymer nanometer. The fiber has the advantages of large total surface area, high porosity and fast adsorption speed, which overcomes the above industrial problems and achieves excellent separation and collection efficiency. Moreover, this new static phase carrier material also has the advantage of low cost, suitable for large and fast protein purification processes, and is particularly suitable for disposal after disposable use.

依據本案所揭第一態樣，本發明關於一種親和性層析法，其包含使一所欲純化的組成物與一靜相擔體材料相接觸；其中該靜相擔體材料包含：高分子奈米纖維基材，其藉由比表面積分析儀所測得的BET比表面積為≧5平方米/克，藉由掃瞄式電子顯微鏡所測得的纖維直徑為300±50奈米和纖維尺寸集中度為≧90%；以及配體，其被共價地耦合於該高分子奈米纖維，供專一性結合該組成物。According to a first aspect of the present invention, the present invention relates to an affinity chromatography comprising contacting a composition to be purified with a static phase support material; wherein the static phase support material comprises: a polymer A nanofiber substrate having a BET specific surface area measured by a specific surface area analyzer of ≧5 m 2 /g, a fiber diameter of 300 ± 50 nm measured by a scanning electron microscope, and a fiber size concentration The degree is ≧90%; and a ligand covalently coupled to the polymeric nanofiber for specific binding of the composition.

依據本案所揭第二態樣，本發明關於一種用於親和性層析的靜相擔體材料，其包含：高分子奈米纖維基材，其選自於由聚丙烯腈之均聚物、無規共聚物和嵌段共聚物或彼等之組合所製成的聚丙烯腈奈米纖維膜，且其藉由比表面積分析儀所測得的BET比表面積為≧5平方米/克，藉由掃瞄式電子顯微鏡所測得的纖維直徑為300±50奈米和纖維尺寸集中度為≧90%；以及配體，其被共價地耦合於該高分子奈米纖維，供專一性結合一組成物，其中該配體包含固定有金屬離子的多牙螯合基團，且該多牙螯合基團選自於由亞胺基二乙酸（IDA）、氮基三乙酸（NTA）、三羧甲基乙二胺（TED）、羧甲基化天冬胺酸（CM-ASP）、四亞乙基五胺（TEPA）、O -磷絲胺酸（OPS）、8- 羥基喹啉（8-HQ）和三(2-胺基乙基)胺（TREN）所組成的群組，而該金屬離子選自於由Ni²⁺ 、Co²⁺ 和Zn²⁺ 所組成的群組。According to a second aspect of the present invention, the present invention relates to a static phase carrier material for affinity chromatography, comprising: a polymer nanofiber substrate selected from the group consisting of a homopolymer of polyacrylonitrile, a polyacrylonitrile nanofiber film made of a random copolymer and a block copolymer or a combination thereof, and having a BET specific surface area measured by a specific surface area analyzer of ≧5 m 2 /g, by The fiber diameter measured by the scanning electron microscope is 300±50 nm and the fiber size concentration is ≧90%; and the ligand is covalently coupled to the polymer nanofiber for the specificity to combine a composition, wherein the ligand comprises a polydentate chelating group immobilized with a metal ion, and the polydentate chelating group is selected from the group consisting of iminodiacetic acid (IDA), nitrogen triacetic acid (NTA), and three Carboxymethylethylenediamine (TED), carboxymethylated aspartic acid (CM-ASP), tetraethylenepentamine (TEPA), O -phosphoric acid (OPS), 8-hydroxyquinoline ( a group consisting of 8-HQ) and tris(2-aminoethyl)amine (TREN), and the metal ion is selected from the group consisting of Ni ²⁺ , Co ²⁺ and Zn ²⁺ Group.

除非另行說明，否則本案專利說明書和各請求項中所使用的下列用語具有下文給予的定義。請注意，本案說明書和各請求項中所使用的單數形用語「一」意欲涵蓋在一個以及一個以上的所載事項，例如至少一個、至少二個或至少三個，而非意味著僅僅具有單一個所載事項。此外，本案各請求項中使用的「包含」、「具有」等開放式連接詞是表示請求項中所記載的元件或成分之組合中，不排除請求項未載明的其他元件或成分。Unless otherwise stated, the following terms used in the patent specification and claims of this case have the definitions given below. Please note that the singular expression "a" used in the description and the claims is intended to cover one or more items, such as at least one, at least two, or at least three, A matter of inclusion. In addition, the open connection words such as "including" and "having" used in the claims of the present invention are combinations of elements or components described in the claims, and do not exclude other elements or components not specified in the claims.

本案說明書中所使用的「親和性層析」此用語是指藉由靜相擔體材料上所搭載之配體（ligand）與目標組成物之間的專一性結合來純化或檢測目標組成物的層析方法。一般而言，目標組成物具有特殊的化學結構或生物活性，藉此與所述配體形成氫鍵、疏水性鍵結及/或偶極-偶極交互作用。親和性分子配對的具例範例包括抗原-抗體、酵素-受質、荷爾蒙-受器、酵素-抑制因子、mRNA-寡聚去氧胸苷、金屬離子-特定標記等，例如使用榖胱甘肽做為配體來捕集帶有榖胱甘肽轉移酶（GST）標記的重組蛋白質。如相關技術領域中具有通常知識者所熟悉，上述專一性結合可以經由從外部加入競爭性配體，抑或是改變酸鹼度、極性或離子強度等條件，從而回收而獲得目標組成物。The term "affinity chromatography" as used in the specification refers to the purification or detection of a target composition by a specific combination between a ligand carried on a stationary phase carrier and a target composition. Chromatography method. In general, the target composition has a particular chemical structure or biological activity whereby hydrogen bonds, hydrophobic bonds, and/or dipole-dipole interactions are formed with the ligand. Examples of affinity molecule pairings include antigen-antibody, enzyme-substrate, hormone-receiver, enzyme-inhibitor, mRNA-oligodeoxythymidine, metal scorpion-specific label, etc., for example, using glutathione As a ligand, a recombinant protein with a glutathione transferase (GST) tag is captured. As is well known to those of ordinary skill in the relevant art, the above specific binding can be achieved by adding a competitive ligand from the outside or by changing conditions such as pH, polarity or ionic strength to recover the target composition.

在一較佳具體例中，所述親和性層析是固定金屬親和層析法（IMAC），亦被稱為金屬螯合親和層析法（metal chelate affinity chromatography；MCAC），其涉及將具有多個配位址的金屬離子固定在靜相擔體材料上，以捕集帶有組胺酸、半胱胺酸及/或色胺酸殘基等電子提供者的目標組成物。所述金屬離子的範例包括但不限於Ni²⁺ 、Cu²⁺ 、Zn²⁺ 、Co²⁺ 、Co³⁺ 、Fe²⁺ 、Fe³⁺ 、Al³⁺ 和Ca²⁺ ，而以Ni²⁺ 、Co²⁺ 和Zn²⁺ 尤佳。這些金屬離子是被配體上的多牙（multidentate）螯合基團所固定。一般而言，所選定的金屬離子對於螯合基團的親和力應該要大於對目標組成物的親和力，以免在沖提過程中排放出過多的金屬離子。適用於固定金屬離子的螯合基團種類為相關技術領域所熟悉，具體範例包括但不限於亞胺基二乙酸（IDA）、氮基三乙酸（NTA）、三羧甲基乙二胺（TED）、羧甲基化天冬胺酸（carboxymethylated aspartic acid；CM-ASP）、四亞乙基五胺（tetraethylene pantamine；TEPA）、O -磷絲胺酸（O -phosphoserine；OPS）、8- 羥基喹啉（8-hydroxyquinoline；8-HQ）和三(2-胺基乙基)胺（tris(2-aminoethyl)amine；TREN），較佳者為IDA、NTA和TED。適用於本發明的其他螯合基團可見於美國專利第6,623,655號中，該件專利的完整揭露內容納入於本案，以供參照。In a preferred embodiment, the affinity chromatography is fixed metal affinity chromatography (IMAC), also known as metal chelate affinity chromatography (MCAC), which relates to The metal ions of the assigned sites are immobilized on the stationary phase support material to capture the target composition of the electron donor with histidine, cysteine, and/or tryptophan residues. Examples of the metal ion include, but are not limited to, Ni ²⁺ , Cu ²⁺ , Zn ²⁺ , Co ²⁺ , Co ³⁺ , Fe ²⁺ , Fe ³⁺ , Al ^{3+ ,} and Ca ²⁺ , and Ni ^{2 +} , Co ²⁺ and Zn ^{2+ are} especially preferred. These metal ions are immobilized by a multidentate chelating group on the ligand. In general, the affinity of the selected metal ion for the chelating group should be greater than the affinity for the target composition to avoid escaping excessive metal ions during the stripping process. The types of chelating groups suitable for immobilizing metal ions are well known in the relevant art, and specific examples include, but are not limited to, iminodiacetic acid (IDA), nitrogen triacetic acid (NTA), tricarboxymethylethylenediamine (TED). ), carboxymethylated aspartate (carboxymethylated aspartic acid; CM-ASP ), tetraethylene pentamine (tetraethylene pantamine; TEPA), O - P serine (O -phosphoserine; OPS), 8- hydroxy Quinoline (8-hydroxyquinoline; 8-HQ) and tris(2-aminoethyl)amine; TREN, preferably IDA, NTA and TED. Other chelating groups suitable for use in the present invention can be found in U.S. Patent No. 6,623,655, the entire disclosure of which is incorporated herein by reference.

本案說明書中所使用的「高分子奈米纖維」此用語是指纖維直徑位在數十至數百奈米之間的化學纖維，與直徑通常在約50微米左右的傳統化纖相比較，在相同的體積下，高分子奈米纖維具有表面積大、孔隙度高以及高孔洞均勻度的優勢，適合做為各種濾材。靜電紡絲技術（electro-spinning）是目前唯一能夠直接、有效率且可連續生產高分子奈米纖維的方法，其大致上是將高分子溶液輸送至針頭形成液滴，當施加高壓電後，隨著高分子電荷相互排斥並克服溶液表面張力後，從液滴表面噴射出帶電液柱，並在電場作用下，以每小時約三百英里的速度以螺旋狀向下進行甩動延伸，使其比一般微米噴嘴所製造出來的纖維細一千倍，在過程中液柱溶液中的溶劑迅速揮發，最後可在接地的收集板上取得纖維直徑為奈米等級的纖維膜。藉由此工序製成的高分子奈米纖維所具有的比表面積為0.1至1000平方米/克且較佳為1至100平方米/克，纖維尺寸集中度為10至99.5%且較佳為80至99.5%，以及孔洞尺寸為0.01至10微米且較佳為0.1至0.8微米。也可進一步藉由改變靜電紡絲製程中的電紡距離、電紡電壓以及接收器轉速，而獲得所希望的纖維直徑尺寸。在較佳的具體例中，高分子奈米纖維具有BET比表面積為≧5平方米/克，以及纖維直徑為300±50奈米和纖維尺寸集中度為≧90%的特性。上述比表面積是藉由比表面積分析儀測得，而纖維直徑則藉由掃瞄式電子顯微鏡測得，獲得纖維直徑的平均值，並算出纖維直徑的標準差，再藉由（１- (標準差／平均纖維直徑)）× 100%的算式，求出纖維尺寸集中度，以做為纖維直徑均勻性的度量。靜電紡絲製程的詳細條件和設備參數可見於中華民國專利公告號I414345，該件專利的完整揭露內容納入於本案，以供參照。The term "polymer nanofiber" as used in the specification refers to a chemical fiber having a fiber diameter of between several tens and hundreds of nanometers, which is the same as a conventional chemical fiber having a diameter of usually about 50 μm. Under the volume, the polymer nanofiber has the advantages of large surface area, high porosity and high pore uniformity, and is suitable as various filter materials. Electro-spinning is currently the only method that can directly and efficiently produce high-molecular-weight nanofibers. It is generally used to transport a polymer solution to a needle to form droplets. After the polymer charges repel each other and overcome the surface tension of the solution, the charged liquid column is ejected from the surface of the droplet, and under the action of the electric field, the helix is downwardly spiraled downward at a speed of about three hundred miles per hour. It is one thousand times thinner than the fiber produced by the general micro nozzle. During the process, the solvent in the liquid column solution is quickly volatilized, and finally the fiber membrane with the fiber diameter of nanometer grade can be obtained on the grounded collecting plate. The polymer nanofiber produced by this process has a specific surface area of 0.1 to 1000 m 2 /g and preferably 1 to 100 m 2 /g, and a fiber size concentration of 10 to 99.5% and preferably 80 to 99.5%, and the pore size is 0.01 to 10 μm and preferably 0.1 to 0.8 μm. It is also possible to further obtain a desired fiber diameter size by changing the electrospinning distance, the electrospinning voltage, and the receiver rotation speed in the electrospinning process. In a preferred embodiment, the polymer nanofibers have a BET specific surface area of ≧5 m 2 /g, a fiber diameter of 300 ± 50 nm, and a fiber size concentration of ≧90%. The above specific surface area is measured by a specific surface area analyzer, and the fiber diameter is measured by a scanning electron microscope to obtain an average value of the fiber diameter, and the standard deviation of the fiber diameter is calculated, and (1- (standard deviation) / Average fiber diameter)) × 100% of the formula, the fiber size concentration is determined as a measure of the fiber diameter uniformity. The detailed conditions and equipment parameters of the electrospinning process can be found in the Republic of China Patent Publication No. I414345, the entire disclosure of which is incorporated herein by reference.

所述高分子奈米纖維可由任何水溶性、水不溶性或膠態高分子所製成，只要其帶有適當官能基或可經由化學改質而具有適當官能基，能夠經由共價鍵結與配體相耦合即可。適合靜電紡絲成為奈米纖維的高分子材料包括但不限於聚丙烯腈、聚甲基丙烯酸、聚甲基丙烯酸甲酯、聚氯乙烯、聚對苯二甲酸酯、聚丙烯醯胺、聚苯乙烯、聚乙烯、聚丙烯、聚醯胺、聚碸、聚酯、彼等之均聚物（homopolymer）、無規共聚物（random copolymer）和嵌段共聚物（block copolymer），以及彼等之組合。在一較佳具體例中，所述高分子奈米纖維為選自於由聚丙烯腈（PAN）之均聚物、無規共聚物和嵌段共聚物或彼等之組合所製成的PAN奈米纖維膜，例如由丙烯腈單體和甲基丙烯酸單體所構成之嵌段共聚物。The polymeric nanofibers can be made of any water-soluble, water-insoluble or colloidal polymer, as long as they carry appropriate functional groups or can have appropriate functional groups via chemical modification, and can be bonded and coordinated via covalent bonding. The body can be coupled. Polymer materials suitable for electrospinning into nanofibers include, but are not limited to, polyacrylonitrile, polymethacrylic acid, polymethyl methacrylate, polyvinyl chloride, polyethylene terephthalate, polypropylene decylamine, poly Styrene, polyethylene, polypropylene, polyamide, polyfluorene, polyester, homopolymers, random copolymers, and block copolymers, and their The combination. In a preferred embodiment, the polymeric nanofiber is a PAN selected from the group consisting of a polyacrylonitrile (PAN) homopolymer, a random copolymer, and a block copolymer or a combination thereof. A nanofiber membrane such as a block copolymer composed of an acrylonitrile monomer and a methacrylic monomer.

配體帶有適當的官能基，可以藉由任何習用的化學合成製程而被共價地耦合於高分子奈米纖維表面，以得到本案靜相擔體材料。習用的耦合手段包括但不限於：(1)以碳化二亞胺（carbodiimide）使胺基與酸基脫水而生成醯胺鍵；(2)以CNBr活化醇基，用以接合胺基；(3)以N -羥基琥珀醯亞胺（N -hydroxysuccinimide）活化，供與胺基進行反應；以及(4)以戊二醛（glutaraldehyde）為中間架橋物，供連接兩個胺基。在使用PAN奈米纖維膜做為高分子奈米纖維的較佳具體例中，使纖維表面上的氰基官能性與多元胺其中一末端的胺基發生反應，使該多元胺經由醯胺鍵的生成而共價地耦合於PAN奈米纖維表面，從而致使PAN奈米纖維帶有胺基官能性。此反應如下式所示： The ligand has an appropriate functional group and can be covalently coupled to the surface of the polymeric nanofiber by any conventional chemical synthesis process to obtain the static phase support material of the present invention. Conventional coupling means include, but are not limited to, (1) dehydrating an amine group and an acid group with a carbodiimide to form a guanamine bond; (2) activating an alcohol group with CNBr for bonding an amine group; ) from N - hydroxysuccinimide (PEI) (N -hydroxysuccinimide) activated for reaction with amine groups; and (4) using glutaraldehyde (glutaraldehyde) was intermediate bridge for connecting two amine groups. In a preferred embodiment in which a PAN nanofiber membrane is used as a polymer nanofiber, the cyano functionality on the surface of the fiber is reacted with an amine group at one end of the polyamine to cause the polyamine to pass through a guanamine bond. The formation is covalently coupled to the surface of the PAN nanofibers, thereby causing the PAN nanofibers to carry amine functionalities. This reaction is shown in the following equation:

在某些具體例中，該胺基官能性進一步經由與氯乙酸（CAA）等烷基化試劑發生N-烷基化反應，加上羧基官能性，從而形成配體的螯合基團。PAN奈米纖維的胺基官能化反應條件可進一步參見諸如 Denget al .,Langmuir , 24(19), (2008) p.10961-67；以及Denget al .,Water Research 38, (2008) p.2431-37等文獻，而藉由N-烷基化加上羧基官能性的反應條件可進一步參見諸如 Zhang, L.et al .,Reactive & Functional Polymers 69 (2009) p.48–54，這些文獻的完整揭露內容納入於本案，以供參照。In certain embodiments, the amine functionality is further N-alkylated with an alkylating agent such as chloroacetic acid (CAA), plus carboxyl functionality to form a chelating group for the ligand. The amine functionalization conditions of PAN nanofibers can be further described, for example, in Deng et al ., Langmuir , 24 (19), (2008) p. 10961-67; and Deng et al ., Water Research 38, (2008) p .2431-37 and the like, and the reaction conditions by N-alkylation plus carboxyl functionality can be further described, for example, in Zhang, L. et al ., Reactive & Functional Polymers 69 (2009) p. 48-54. The complete disclosure of the literature is incorporated into this case for reference.

在其他具體例中，PAN奈米纖維上的胺基官能性可以經由在鹼性環境下與如表氯醇、表溴醇等表鹵醇類（epihalohydrins）、縮水甘油醚類、縮水甘油酯類等環氧化物進行反應，使其活化成為帶有氧口元（oxirane）的高反應性基團，以便在接合如離胺酸等另一多元胺，隨後再進一步經由N-烷基化反應加入羧基官能性，從而形成配體的螯合基團。在此例中，經由共價鍵結數個有機分子，致使螯合基團和高分子奈米纖維此二者間相隔有一懸臂結構。懸臂結構的構築有助於使螯合基團遠離高分子奈米纖維，避免因為高分子奈米纖維構成立體障礙而阻擋了配體和目標組成物之間的交互作用，導致分離捕集效率下降。所述懸臂結構通常是一個飽和或不飽和、經取代或未經取代、線性或環狀、直鏈或分支的原子鏈結，其主鏈由不超過25個原子所構成，更佳為由不超過15個原子所構成。主鏈的構成原子較佳為碳、氧、氮、硫、矽、硒和磷，以碳、氧、硫和氮尤佳。舉例來說，該主鏈可以簡式-CO-NH-(CH₂ )_m X(CH₂ )_n -或-CO-NH-(CH₂ )_m X₁ (CH₂ )_n X₂ (CH₂ )_p -來表示，其中X、X₁ 及X₂ 個別地選自於O、S、NH及共價鍵；而m、n及p則個別地為一個選自於0至6中之整數。懸臂結構的構築反應條件可進一步參見諸如S¸enkal, BFet al .,Reactive & Functional Polymers 49, (2001) p.151–157；以及Chang TCet al .,Polymer Degradation and Stability 87, (2005) p.87-94，這些文獻的完整揭露內容納入於本案，以供參照。其他類型的懸臂結構及其構築方法亦為本發明所屬技術領域所熟知，也可適用於本發明。In other specific examples, the amine functionality on the PAN nanofibers can be via an alkaline environment with epihalohydrins such as epichlorohydrin, epibromohydrin, glycidyl ethers, glycidyl esters. The epoxide is reacted to activate it into a highly reactive group with an oxirane to join another polyamine such as an amide acid, which is then further added via an N-alkylation reaction. Carboxyl functionality, thereby forming a chelating group for the ligand. In this case, by covalently bonding a plurality of organic molecules, the chelating group and the polymeric nanofiber are separated by a cantilever structure. The structure of the cantilever structure helps to keep the chelating group away from the polymer nanofiber, avoiding the interaction between the ligand and the target composition due to the steric hindrance of the polymer nanofiber, resulting in a decrease in separation and collection efficiency. . The cantilever structure is usually a saturated or unsaturated, substituted or unsubstituted, linear or cyclic, linear or branched atomic chain, the main chain of which is composed of no more than 25 atoms, more preferably no It consists of more than 15 atoms. The constituent atoms of the main chain are preferably carbon, oxygen, nitrogen, sulfur, antimony, selenium and phosphorus, and particularly preferably carbon, oxygen, sulfur and nitrogen. For example, the backbone can be a simple formula -CO-NH-(CH ₂ ) _m X(CH ₂ ) _n - or -CO-NH-(CH ₂ ) _m X ₁ (CH ₂ ) _n X ₂ (CH ₂ And _p - to represent wherein X, X ₁ and X _{2 are} individually selected from O, S, NH and a covalent bond; and m, n and p are each an integer selected from 0 to 6. The conditions for the construction of the cantilever structure can be further described, for example, in S ̧enkal, BF et al ., Reactive & Functional Polymers 49, (2001) p. 151–157; and Chang TC et al ., Polymer Degradation and Stability 87, (2005). P.87-94, the complete disclosure of these documents is incorporated herein by reference. Other types of cantilever structures and methods of construction thereof are also well known in the art to which the present invention pertains, and are also applicable to the present invention.

使本案靜相擔體材料浸潤於金屬鹽溶液中，例如浸潤於稀硫酸鎳或稀硫酸銅水溶液中，歷時1至12小時，例如歷時3至8小時，任擇地施以攪動，以容許螯合基團充分吸附金屬離子。固定有適當金屬離子的靜相擔體材料適用於親和性層析法，以供純化或檢測目標組成物。本案所揭靜相擔體材料的配體在螯合金屬離子後特別適用於捕集帶有組胺酸、半胱胺酸及/或色胺酸標記的目標組成物，尤其是帶有重覆組胺酸標記者，例如帶有6個連續組胺酸標記（6×His）者。實際操作時，可以將欲純化之特定蛋白質的對應核苷酸序列選殖進入一個帶有6×His基因序列的表現載體中，例如Novagen, Inc.所出品的pET-15b或pET-20b蛋白質表現系統，經過在宿主細胞中大量表現帶有6×His標記的融合蛋白後，即可藉由習用的均質化、親和層析和沖提手段來收集所欲純化的蛋白。The static phase carrier material of the present invention is infiltrated into the metal salt solution, for example, by infiltrating in a dilute nickel sulfate or a dilute copper sulfate aqueous solution for 1 to 12 hours, for example, for 3 to 8 hours, optionally subjected to agitation to allow the chelate. The chelating group sufficiently adsorbs metal ions. A stationary phase support material to which a suitable metal ion is immobilized is suitable for affinity chromatography for purification or detection of a target composition. The ligands of the static phase support materials disclosed in the present invention are particularly suitable for capturing target compositions with histidine, cysteine and/or tryptophan labeled after chelation of metal ions, especially with repeated A histidine marker, such as one with 6 consecutive histidine labels (6 x His). In practice, the corresponding nucleotide sequence of the specific protein to be purified can be cloned into a expression vector with a 6×His gene sequence, such as pET-15b or pET-20b protein produced by Novagen, Inc. Systematic, after a large number of fusion proteins with a 6xHis tag are displayed in the host cell, the desired protein can be collected by conventional homogenization, affinity chromatography and extraction means.

因此，本發明之另一態樣係關於一種利用所揭靜相擔體材料來純化一組成物的方法，其中該組成物對於靜相擔體材料的配體具有親和性，該方法包含使該組成物與本案所揭靜相擔體材料相接觸的步驟。此處所稱「純化」意指致使目標組成物呈現出相較於該組成物在原先所存在的環境中更為豐富的狀態。值得注意的是，本案所稱「組成物」不僅意指具有連續組胺酸、半胱胺酸及/或色胺酸殘基的天然蛋白以及藉由基因重組技術而融合有組胺酸、半胱胺酸及/或色胺酸標記的蛋白，尤其是融合有組胺酸標記的蛋白，更意欲涵蓋所有對於被螯合的金屬離子具有親和性以及能夠透過化學、酵素、基因重組方法而直接或間接綴合有組胺酸標記的所有分子，包括蛋白、磷蛋白、寡肽、DNA、RNA、寡核苷酸，以及合成和天然產物。此處所使用「具有親和性」此用語意指配體和目標組成物可以經由距離的接近（亦即接觸）而產生彼此結合的交互作用，而且這個結合的強度遠高於非專一性結合，致使兩者的解離常數介於10^-4 至10^-8 之間。據此，本案靜相擔體材料可以被填充在管柱中以接觸並吸附流過管柱的目標組成物，也可在置放在容器中以批次方式（batch manner）與目標組成物混合以利接觸並吸附目標組成物。其後，藉由加入如咪唑等競爭配體或改變酸鹼值、鹽濃度等習用沖提手段，使目標組成物脫離靜相擔體材料，即可以獲得含有豐富化目標組成物的沖提物。經過純化後，可以運用各種習用手段對於目標組成物進行定性或定量檢測，抑或是使此沖提物接受透析脫鹽、濃縮等製程回收獲取高純度的目標組成物。Accordingly, another aspect of the present invention is directed to a method of purifying a composition using the disclosed static phase support material, wherein the composition has an affinity for a ligand of the static phase support material, the method comprising The step of contacting the composition with the static phase carrier material disclosed in the present application. By "purified" herein is meant a state in which the target composition exhibits a richer state than the composition in the environment in which it was originally present. It is worth noting that the term "composition" in this case refers not only to natural proteins with continuous histidine, cysteine and/or tryptophan residues, but also to histidine by genetic recombination techniques. Cystamine and/or tryptophan-labeled proteins, especially those fused with histidine-tagged proteins, are intended to cover all of the affinity for chelated metal ions and are directly permeable to chemical, enzymatic, and genetic recombination methods. Or indirectly conjugated to all molecules labeled with histidine, including proteins, phosphoproteins, oligopeptides, DNA, RNA, oligonucleotides, and synthetic and natural products. As used herein, the term "having affinity" means that the ligand and the target composition can interact with each other via proximity (ie, contact) of the distance, and the strength of the combination is much higher than the non-specific combination, resulting in The dissociation constant of the two is between 10 ^-4 and 10 ^-8 . Accordingly, the static phase carrier material of the present invention can be filled in the column to contact and adsorb the target composition flowing through the column, or can be mixed with the target composition in a batch manner in the container. In order to contact and adsorb the target composition. Thereafter, by adding a competitive ligand such as imidazole or changing the acid-base value, salt concentration, and the like, the target composition is removed from the static phase carrier material, and the extract containing the enriched target composition can be obtained. . After purification, various target methods can be used for qualitative or quantitative detection of the target composition, or the extract can be subjected to dialysis desalination, concentration and other processes to obtain a high-purity target composition.

下列實例僅供用於例示本發明，而非意欲限制本發明之範圍。The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention.

實例 1 ：高分子奈米纖維之製備本案各實例中所使用的奈米纖維是由財團法人紡織產業綜合研究所運用中華民國專利公告號I414345中所揭露的靜電紡絲製程所生產出的聚丙烯腈奈米纖維。簡言之，將由93.5重量%丙烯腈單體和6.5重量%甲基丙烯酸單體所構成的聚丙烯腈共聚物原料（購自於福海工業股份有限公司）溶於二甲基甲醯胺（購自於于成股份有限公司），製成14 w/v%之聚合物溶液。隨後將聚合物溶液輸送至利用靜電紡絲機（紡織產業綜合研究所）。在紡絲過程中，將收集器設定為360度旋轉、使用雙組份噴嘴以及噴嘴以左右移動之方式操作，且進一步設定20公分的幅寬、20kV的電壓、10公分/秒的收集器轉速、20公分的電紡距離、1毫升/小時的進料流速以及12次/分鐘的左右移動次數，最後可在接地的收集板上取得纖維直徑為奈米等級的纖維膜，從而獲得具有高孔隙度、高孔洞均勻度之聚丙烯腈（PAN）奈米纖維薄膜。在本實例中，利用靜電紡絲技術所製得PAN奈米纖維膜，其藉由比表面積分析儀（Micromeritics, ASAP 2000）所測得的比表面積為13平方米/克，藉由掃瞄式電子顯微鏡所測得的纖維直徑約為 220-270 奈米，纖維尺寸集中度為91%。電子顯微鏡所觀測到之影像如圖1所示。 Example 1 : Preparation of polymeric nanofibers The nanofibers used in the examples of the present invention are polypropylene produced by the electrospinning process disclosed in the Republic of China Textile Industry Research Institute using the Republic of China Patent Publication No. I414345. Nitrile nanofibers. Briefly, a polyacrylonitrile copolymer raw material (purchased from Fuhai Industrial Co., Ltd.) composed of 93.5 wt% of acrylonitrile monomer and 6.5% by weight of methacrylic acid monomer was dissolved in dimethylformamide (purchased) From Yucheng Co., Ltd.), a 14 w/v% polymer solution was prepared. The polymer solution is then transferred to an electrospinning machine (Industrial Industry Research Institute). During the spinning process, the collector was set to rotate 360 degrees, the two-component nozzle was used, and the nozzle was operated to move left and right, and the width of 20 cm, the voltage of 20 kV, and the collector speed of 10 cm/sec were further set. , 20 cm electrospinning distance, 1 ml / hour feed flow rate and 12 times / minute of the number of left and right movements, and finally a fiber membrane with a fiber diameter of nanometer grade can be obtained on the grounded collecting plate, thereby obtaining high porosity Polyacrylonitrile (PAN) nanofiber film with uniformity and high hole uniformity. In the present example, a PAN nanofiber membrane produced by an electrospinning technique having a specific surface area measured by a specific surface area analyzer (Micromeritics, ASAP 2000) of 13 m 2 /g, by scanning electrons The fiber diameter measured by the microscope was about 220-270 nm, and the fiber size concentration was 91%. The image observed by the electron microscope is shown in Fig. 1.

實例 2 ：式 11 化合物之製備將2克在實例1中所製成的PAN奈米纖維膜與50毫升二乙烯三胺（diethylenetriamine；DETA）在120^o C下施以加熱回流以進行反應，歷時50分鐘，以使得奈米纖維膜具有胺官能性。隨後，使胺官能化的聚丙烯腈奈米纖維膜在70^o C下與氯乙酸（chloroacetic acid；CAA）進行反應，歷時3小時，以獲得帶有三羧甲基乙二胺（TED）螯合基團的下式11化合物。式11 Example 2: Preparation of Compound of Formula 11 2 g of PAN nanofiber membrane prepared in Example 1 and 50 ml of diethylene triamine (diethylenetriamine; DETA) subjected heated to reflux at 120 ^o C for reaction, which lasted 50 minutes to give the nanofiber membrane an amine functionality. Subsequently, the amine-functionalized polyacrylonitrile nano fiber membrane with chloroacetic acid (chloroacetic acid; CAA) at 70 ^o C for reaction for 3 hours to obtain (the TED) chelate with trimethylol ethylenediamine A compound of the formula 11 below. Equation 11

實例 3 ：式 12 化合物之製備使實例1中所製成的PAN奈米纖維膜與乙二胺（ethylenediamine）進行反應。隨後，使經過改質的聚丙烯腈奈米纖維膜與10%甲基丙烯酸縮水甘油酯（glycidyl methacrylate；GMA）進行反應，歷時2小時。接著，使所得化合物在70^o C下與氯乙酸（CAA）進行反應，歷時3小時。獲得帶有三羧甲基乙二胺（TED）螯合基團的下式12化合物。式12 Example 3 : Preparation of compound of formula 12 The PAN nanofiber membrane produced in Example 1 was reacted with ethylenediamine. Subsequently, the modified polyacrylonitrile nanofiber membrane was reacted with 10% glycidyl methacrylate (GMA) for 2 hours. Then, the resultant compound is reacted with chloroacetic acid (CAA) at 70 ^o C, for 3 hours. A compound of the following formula 12 is obtained with a tricarboxymethylethylenediamine (TED) chelating group. Equation 12

實例 4 ：式 13 化合物之製備重覆實例3，除了使二乙烯三胺（DETA）取代乙二胺與實例1中所製成的PAN奈米纖維膜進行反應以外。據此獲得帶有三羧甲基乙二胺（TED）螯合基團的下式13化合物。式13 Example 4 : Preparation of compound of formula 13 Example 3 was repeated except that the diethylenetriamine (DETA) substituted ethylenediamine was reacted with the PAN nanofiber membrane produced in Example 1. According to this, a compound of the following formula 13 having a tricarboxymethylethylenediamine (TED) chelating group is obtained. Equation 13

實例 5 ：式 21 化合物之製備使實例1中所製成的PAN奈米纖維膜與離胺酸進行反應。隨後，使所得化合物在70^o C下與氯乙酸（CAA）進行反應，歷時3小時。獲得帶有氮基三乙酸（NTA）螯合基團的下式21化合物。式21 Example 5 : Preparation of compound of formula 21 The PAN nanofiber membrane produced in Example 1 was reacted with an amide acid. Subsequently, the resulting compound is reacted with chloroacetic acid (CAA) at 70 ^o C, for 3 hours. A compound of the following formula 21 having a nitrogen triacetic acid (NTA) chelating group is obtained. Equation 21

實例 6 ：式 22 化合物之製備使實例1中所製成的PAN奈米纖維膜與乙二胺（ethylenediamine）進行反應。隨後，使經過改質的聚丙烯腈奈米纖維膜與表氯醇（epichlorohydrin）進行反應，歷時2小時。使所得化合物與離胺酸進行反應。隨後，使所得化合物在70^o C下與氯乙酸（CAA）進行反應，歷時3小時。獲得帶有氮基三乙酸（NTA）螯合基團的下式22化合物。式22 Example 6 : Preparation of compound of formula 22 The PAN nanofiber membrane produced in Example 1 was reacted with ethylenediamine. Subsequently, the modified polyacrylonitrile nanofiber membrane was reacted with epichlorohydrin for 2 hours. The resulting compound is reacted with an amine acid. Subsequently, the resulting compound is reacted with chloroacetic acid (CAA) at 70 ^o C, for 3 hours. A compound of the following formula 22 having a nitrogen triacetic acid (NTA) chelating group is obtained. Equation 22

實例 7 ：式 23 化合物之製備重覆實例6，除了使己二胺取代乙二胺與使實例1中所製成的PAN奈米纖維膜進行反應以外。據此獲得帶有氮基三乙酸（NTA）螯合基團的下式23化合物。式23 Example 7 : Preparation of compound of formula 23 Example 6 was repeated except that hexamethylenediamine substituted ethylenediamine was reacted with the PAN nanofiber membrane produced in Example 1. According to this, a compound of the following formula 23 having a nitrogen triacetic acid (NTA) chelating group is obtained. Equation 23

圖2顯示式21、式22和式23化合物的簡要合成流程。Figure 2 shows a brief synthetic scheme for the compounds of Formula 21, Formula 22 and Formula 23.

實例 8 ：金屬離子之螯合配製含有2000 ppm Ni²⁺ 的硫酸鎳溶液，取50ml 分別加入適量的式11、式12、式13、式21、式22和式23化合物中，在室溫下搖晃至隔日，以製成靜相擔體材料。各化合物的Ni²⁺ 吸附量Q （毫克Ni²⁺ /克化合物）係藉由下列等式(1)換算而得：Q ＝(c₀ －c_t ) /G ×V …………(1) 其中c₀ 代表初始金屬離子濃度（毫克/升），c_t 代表吸附後的金屬離子濃度，V 代表溶液體積，而G 則表示化合物乾重。經過換算，式11、式12、式13、式21、式22和式23化合物做為靜相擔體材料時所螯合的Ni²⁺ 數量分別為59.38毫克/克、33.97毫克/克、26.30毫克/克、18.71毫克/克、94.74毫克/克和8.59毫克/克。 Example 8 : Chelation of metal ions A nickel sulfate solution containing 2000 ppm of Ni ²⁺ was prepared, and 50 ml of each was added to a suitable amount of the compound of Formula 11, Formula 12, Formula 13, Formula 21, Formula 22 and Formula 23 at room temperature. Shake to the next day to make a static phase carrier material. The Ni ²⁺ adsorption amount Q (mg Ni ²⁺ / gram compound) of each compound is obtained by the following equation (1): Q = ( c ₀ - c _t ) / G × V ...... (1 Wherein c ₀ represents the initial metal ion concentration (mg/L), c _t represents the metal ion concentration after adsorption, V represents the solution volume, and G represents the dry weight of the compound. After conversion, the compounds of Formula 11, Formula 12, Formula 13, Formula 21, Formula 22 and Formula 23 were chelated as the stationary phase carrier, and the amount of Ni ²⁺ sequestered was 59.38 mg/g, 33.97 mg/g, 26.30, respectively. Mg/g, 18.71 mg/g, 94.74 mg/g and 8.59 mg/g.

採用式22化合物做為範例，以每次100毫升的去離子水洗滌吸附Ni²⁺ 後的式22化合物，總共洗滌 5次。取第六次水樣品，檢測出 Ni²⁺ 濃度為0ppm。隨後，再以咪唑溶液清洗吸附Ni²⁺ 後的式22化合物，總共5次。取第六次水樣，檢測出 Ni²⁺ 濃度為37.5 ppm，約等於吸附量的3.6%。本實例的測試結果顯示出，本案所揭露的化合物對於金屬離子具有極為優異的螯合能力。Using the compound of formula 22 as an example, the compound of formula 22 after adsorption of Ni ²⁺ was washed with 100 ml of deionized water each time for a total of 5 washes. The sixth water sample was taken and the concentration of Ni ^{2+ was} detected to be 0 ppm. Subsequently, the compound of formula 22 after adsorption of Ni ²⁺ was washed with an imidazole solution for a total of 5 times. Taking the sixth water sample, the concentration of Ni ^{2+ was} detected to be 37.5 ppm, which was approximately equal to 3.6% of the adsorption amount. The test results of this example show that the compounds disclosed in the present invention have extremely excellent chelating ability for metal ions.

實例 9 ：親和性層析管柱之製備將實例8中所製備出之螯合有Ni²⁺ 的式22化合物做為靜相擔體材料1，將1克的靜相擔體材料1置入層析用塑膠管柱3內（容積25毫升），並填充於二個多孔聚乙烯膜片2之間，以製成一個親和性層析管柱，如圖3所示。 Example 9 : Preparation of affinity chromatography column The compound of the formula 22 chelated with Ni ²⁺ prepared in Example 8 was used as the static phase carrier material 1, and 1 gram of the static phase carrier material 1 was placed. The chromatography was carried out in a plastic column 3 (volume 25 ml) and filled between two porous polyethylene membranes 2 to form an affinity chromatography column, as shown in FIG.

實例 10 ：分析物之製備取5毫升轉形有莫洛尼鼠白血病病毒反轉錄酶（moloney murine leukemia virus reverse transcriptase；MMLV RTase）的6×His表現質體的OverExpress™ C41(DE3)pLySs大腸桿菌轉形株（購自於美國威斯康辛州Middleton市Lucigen Corp.）菌液，加入0.25公升LB培養基內，於37℃下以轉速150rpm搖瓶培養至OD 600nm下之光學密度（O.D.）值為0.6-0.8。再加入IPTG至最終濃度為0.1至1mM，而將菌液置於30至37℃下，以轉速150rpm搖瓶培養3小時。藉此於大腸桿菌宿主細胞中表現出N端帶有6個組胺酸標記之RTase融合蛋白，其分子量預計約為75kDa。 Example 10 : Preparation of Analyte: 5 ml of 6×His plastid-expressing OverExpressTM C41(DE3) pLySs Escherichia coli with moloney murine leukemia virus reverse transcriptase (MMLV RTase) The transgenic strain (purchased from Lucigen Corp., Middleton, Wisconsin, USA) was added to 0.25 liter LB medium and cultured at 37 ° C in a shake flask at 150 rpm to an optical density (OD) of 0.6 at OD 600 nm. 0.8. Further, IPTG was added to a final concentration of 0.1 to 1 mM, and the bacterial solution was placed at 30 to 37 ° C, and cultured in a shake flask at 150 rpm for 3 hours. This results in a 6-histidine-tagged RTase fusion protein at the N-terminus in E. coli host cells, which is expected to have a molecular weight of approximately 75 kDa.

在12,000rpm下將菌液予以離心5分鐘後，得到1.4克的轉形菌株沈澱物。將20毫升裂解緩衝液（50-300mM Tris-HCl, pH 5-9；100-450mM氯化鈉），在4℃下施以超音波震盪破菌，而得到轉型株之破菌液。在13,000rpm下將破菌液予以離心20分鐘，並收集上清液，總體積約19毫升。取10微升上清液樣品，標註為餾分S（Fraction S）備用。After the bacterial solution was centrifuged at 12,000 rpm for 5 minutes, 1.4 g of the transformed strain precipitate was obtained. 20 ml of lysis buffer (50-300 mM Tris-HCl, pH 5-9; 100-450 mM sodium chloride) was subjected to ultrasonic shock sterilization at 4 ° C to obtain a bacterial strain of the transformed strain. The bacterial suspension was centrifuged at 13,000 rpm for 20 minutes, and the supernatant was collected for a total volume of about 19 ml. Ten microliters of the supernatant sample was taken and labeled as Fraction S.

實例 11 ：親和性層析第一階段將實例10中所製得之上清液（標註為S）饋入實例9所製得之親和性層析管柱內，並以每4秒一滴的流出速率收集流出液（1毫升/餾分），各餾分取10微升樣品，標註為流出液餾分1~10（FF1~FF10）備用。第二階段以20毫升裂解緩衝液洗滌管柱，收集洗出液（1.5毫升/餾分），各餾分取10微升樣品，標註為洗出液餾分1~9（W1~W9）備用。將10毫升沖提緩衝液（50-300mM Tris-HCl, pH 5-9；100-450mM氯化鈉；100-500mM咪唑）。收集沖提物（1毫升/餾分），各餾分取10微升樣品，標註為沖提物餾分1~9（E1~E9）備用。 Example 11 : Affinity Chromatography First Stage The supernatant (labeled S) prepared in Example 10 was fed into the affinity chromatography column prepared in Example 9 and flowed out every 4 seconds. The effluent was collected at a rate (1 ml/fraction), and 10 μl of each sample was taken and labeled as effluent fraction 1~10 (FF1~FF10) for use. In the second stage, the column was washed with 20 ml of lysis buffer, and the eluate (1.5 ml/fraction) was collected. Ten microliters of each fraction was taken and labeled as the eluate fraction 1~9 (W1~W9) for use. 10 ml of buffering buffer (50-300 mM Tris-HCl, pH 5-9; 100-450 mM sodium chloride; 100-500 mM imidazole) was added. The extract (1 ml/fraction) was collected, and 10 μl of each sample was taken and labeled as the extract fraction 1~9 (E1~E9) for use.

實例 12 ： SDS-PAGE 分析將實例11中所收集到各餾分樣品饋入含有12%十二烷基硫酸鈉的聚丙烯醯胺膠體中，在NE1067型電泳槽中（美國加州舊金山市Hoefer, Inc.）以120V持續30分鐘再以140V持續2小時的條件進行SDS-PAGE電泳分析。隨後以考馬西氏藍（Coomassie blue）將膠體加以染色，其結果示於圖4A至4C。RTase融合蛋白的分子量約為75kDa。電泳分析的結果顯示，N端帶有6個組胺酸標記之RTase蛋白被充分地吸附於親和性層析管柱中，以致於留存於流出液餾分中的RTase蛋白非常有限。另一方面，洗滌步驟進行至W4後雜蛋白幾乎已被完全清洗乾淨，而以咪唑進行競爭性沖提也可極有效率地沖提出高濃度的RTase融合蛋白。 Example 12 : SDS-PAGE analysis Samples collected in Example 11 were fed into a polypropylene guanamine colloid containing 12% sodium lauryl sulfate in a NE1067 electrophoresis tank (Hoefer, Inc., San Francisco, CA, USA) .) SDS-PAGE electrophoresis analysis was carried out at 120 V for 30 minutes and then at 140 V for 2 hours. The colloid was subsequently dyed with Coomassie blue, and the results are shown in Figures 4A to 4C. The RTase fusion protein has a molecular weight of approximately 75 kDa. The results of electrophoretic analysis showed that the 6 histidine-tagged RTase protein at the N-terminus was sufficiently adsorbed in the affinity chromatography column, so that the RTase protein retained in the effluent fraction was very limited. On the other hand, after the washing step is carried out until W4, the heteroprotein is almost completely cleaned, and the competitive elution with imidazole can also efficiently extract a high concentration of the RTase fusion protein.

雖然本發明已被詳細描述於說明書中，但在本發明精義和範圍內的各種修改和變化對於本領域具有通常知識者而言屬於明顯。基於前述的揭露內容，相關技術中的知識以及在發明背景和發明的詳細敘述中所討論到的參考文獻的揭露內容完全地被納入於此作為參考。While the invention has been described in detail hereinabove, it will be apparent to those of ordinary skill in the art. The disclosure of the related art and the disclosure of the references discussed in the Detailed Description of the Invention and the Detailed Description of the Invention are hereby incorporated by reference.

符號說明
1‧‧‧靜相擔體材料
2‧‧‧膜片
3‧‧‧塑膠管柱Symbol Description
1‧‧‧Static phase carrier material
2‧‧‧ diaphragm
3‧‧‧Plastic column

圖1為掃瞄式電子顯微鏡所攝得之聚丙烯腈奈米纖維薄膜影像；Figure 1 is a film of a polyacrylonitrile nanofiber film taken by a scanning electron microscope;

圖2為有機合成流程圖，顯示本案式21、式22和式23化合物的製造；Figure 2 is a flow chart of organic synthesis showing the production of the compounds of the formula 21, formula 22 and formula 23;

圖3為一示意圖，顯示填充有式22化合物的親和性層析管柱；以及Figure 3 is a schematic diagram showing an affinity chromatography column packed with a compound of formula 22;

圖4A-4C為SDS-PAGE分析圖譜，顯示利用圖3所示親和性層析管柱對於6×His-反轉錄酶融合蛋白的捕集效率，其中S代表上清液， FF1~FF10代表流出液餾分， W1~W9代表洗出液餾分，以及E1~E9代表沖提物餾分。Figures 4A-4C are SDS-PAGE analysis maps showing the capture efficiency of the 6xHis-reverse transcriptase fusion protein using the affinity chromatography column shown in Figure 3, where S represents the supernatant and FF1~FF10 represents the efflux. The liquid fraction, W1~W9 represents the eluate fraction, and E1~E9 represents the extract fraction.

1‧‧‧靜相擔體材料 1‧‧‧Static phase carrier material

2‧‧‧膜片 2‧‧‧ diaphragm

3‧‧‧塑膠管柱 3‧‧‧Plastic column

Claims

一種親和性層析法，其包含使一所欲純化的組成物與一靜相擔體材料相接觸，而該組成物對於該靜相擔體材料具有親和性，其中該靜相擔體材料包含：高分子奈米纖維基材，其藉由比表面積分析儀所測得的BET比表面積為≧5平方米/克，藉由掃瞄式電子顯微鏡所測得的纖維直徑為300±50奈米和纖維尺寸集中度為≧90%；以及配體，其被共價地耦合於該高分子奈米纖維，供專一性結合該組成物。An affinity chromatography comprising contacting a composition to be purified with a static phase support material, the composition having an affinity for the static phase support material, wherein the static phase support material comprises : a polymer nanofiber substrate having a BET specific surface area measured by a specific surface area analyzer of ≧5 m 2 /g, and a fiber diameter of 300 ± 50 nm as measured by a scanning electron microscope The fiber size concentration is ≧90%; and a ligand covalently coupled to the polymeric nanofiber for specific binding of the composition.

如請求項1之親和性層析法，其中該高分子奈米纖維為選自於由聚丙烯腈之均聚物、無規共聚物和嵌段共聚物和彼等之組合所製成的聚丙烯腈奈米纖維膜所組成的群組。The affinity chromatography method of claim 1, wherein the polymer nanofiber is a polymer selected from the group consisting of a homopolymer of a polyacrylonitrile, a random copolymer, and a block copolymer, and a combination thereof. A group consisting of acrylonitrile nanofiber membranes.

如請求項1或2之親和性層析法，其中該配體具有多牙螯合基團用於固定金屬離子，而該多牙螯合基團選自於由亞胺基二乙酸（IDA）、氮基三乙酸（NTA）、三羧甲基乙二胺（TED）、羧甲基化天冬胺酸（CM-ASP）、四亞乙基五胺（TEPA）、O -磷絲胺酸（OPS）、8- 羥基喹啉（8-HQ）和三(2-胺基乙基)胺（TREN）所組成的群組。An affinity chromatography according to claim 1 or 2, wherein the ligand has a polydentate chelating group for immobilizing a metal ion, and the multidentate chelating group is selected from the group consisting of imidodiacetic acid (IDA) , nitrogen triacetic acid (NTA), tricarboxymethyl ethylene diamine (TED), carboxymethylated aspartic acid (CM-ASP), tetraethylene pentamine (TEPA), O -phosphoric acid (OPS), a group consisting of 8-hydroxyquinoline (8-HQ) and tris(2-aminoethyl)amine (TREN).

如請求項3之親和性層析法，其中該多牙螯合基團選自於由亞胺基二乙酸（IDA）、氮基三乙酸（NTA）、三羧甲基乙二胺（TED）所組成的群組。An affinity chromatography according to claim 3, wherein the polydentate chelating group is selected from the group consisting of iminodiacetic acid (IDA), nitrogen triacetic acid (NTA), and tricarboxymethylethylenediamine (TED). The group formed.

如請求項3之親和性層析法，其中該多牙螯合基團固定有金屬離子，而該金屬離子選自於由Ni²⁺ 、Cu²⁺ 、Zn²⁺ 、Co²⁺ 、Co³⁺ 、Fe²⁺ 、Fe³⁺ 、Al³⁺ 和Ca²⁺ 所組成的群組。An affinity chromatography according to claim 3, wherein the polydentate chelating group is immobilized with a metal ion selected from the group consisting of Ni ²⁺ , Cu ²⁺ , Zn ²⁺ , Co ²⁺ , Co ³ a group consisting of ⁺ , Fe ²⁺ , Fe ³⁺ , Al ^{3+ ,} and Ca ²⁺ .

如請求項5之親和性層析法，其中該金屬離子選自於由Ni²⁺ 、Co²⁺ 和Zn²⁺ 所組成的群組。An affinity chromatography as claimed in claim 5, wherein the metal ion is selected from the group consisting of Ni ²⁺ , Co ²⁺ and Zn ²⁺ .

如請求項6之親和性層析法，其中該金屬離子是Ni²⁺ 。An affinity chromatography as claimed in claim 6, wherein the metal ion is Ni ²⁺ .

如請求項3之親和性層析法，其中該多牙螯合基團和該高分子奈米纖維之間以一懸臂結構連接，該懸臂結構具有不超過25個原子所構成的主鏈，使該多牙螯合基團遠離該高分子奈米纖維，其中該主鏈以簡式-CO-NH-(CH₂ )_m X(CH₂ )_n -或-CO-NH-(CH₂ )_m X₁ (CH₂ )_n X₂ (CH₂ )_p -來表示，其中X、X₁ 及X₂ 個別地選自於O、S、NH及共價鍵；而m、n及p則個別地為一個選自於0至6中之整數。An affinity chromatography method according to claim 3, wherein the polydentate chelating group and the polymeric nanofiber are connected by a cantilever structure having a main chain of not more than 25 atoms, The polydentate chelating group is remote from the polymeric nanofiber, wherein the main chain is a simple form -CO-NH-(CH ₂ ) _m X(CH ₂ ) _n - or -CO-NH-(CH ₂ ) _m X ₁ (CH ₂ ) _n X ₂ (CH ₂ ) _p - represents wherein X, X ₁ and X _{2 are} individually selected from O, S, NH and a covalent bond; and m, n and p are individually Is an integer selected from 0 to 6.

如請求項8之親和性層析法，其中該靜相擔體材料具有下式：（式22）。An affinity chromatography as claimed in claim 8, wherein the static phase support material has the formula: (Formula 22).

如請求項3之親和性層析法，其中該組成物為一含有連續6個組胺酸殘基的蛋白。An affinity chromatography as claimed in claim 3, wherein the composition is a protein comprising six consecutive histidine residues.