TWM517644U - Nanofiber-based affinity chromatographic column - Google Patents

Description

Affinity chromatography column established by nanofiber

The present invention relates to an affinity chromatography column for purifying a substance, in particular to a fixed metal affinity chromatography column for purifying a substance, wherein the polymer nanofiber is used as a static phase carrier film. Substrate.

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⁺ and Co. Metal ions such as 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 scorpion 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. . Because fixed metal affinity chromatography does not use a strong acid base or high salt concentration extraction environment, it is quite mild to protein, it is not easy to denature or inactivate protein, so it is very suitable for mass production with genetic recombination technology. A biologically active protein with high economic value such as an enzyme.

When the affinity chromatography technique is carried out, generally, the target substance to be purified is sufficiently contacted with the static phase carrier material to promote the target substance to be trapped by the static phase carrier material. The static phase carrier material may be filled in the column to contact and adsorb the target substance flowing through the column, or may be placed in a container and mixed with the target substance in a batch manner to facilitate contact and adsorption. Target substance. Many types of commercial affinity chromatography products are commercially available, most of which are made into tubular forms for immediate use. However, these commercial affinity chromatography columns generally use a resin such as agarose, cellulose or dextran as a substrate. 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. For example, the Sartobind ^® IDA 75 affinity chromatography column from Sartorius AG is based on reinforced cellulose with IDA ligand on the surface of the substrate for trapping Histidine labeling. 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.

However, the small affinity of the affinity chromatography column causes the kinetics to be slow, resulting in a relatively time-consuming overall purification process. Although the adsorption amount is large, impurities are easily left. Furthermore, conventional commercial affinity chromatography columns must be kept wet from time to time to avoid loss of binding capacity of the static phase support material, which is often troublesome in operation. In addition, although the resin type substrate has strong binding force, the separation efficiency is not good, and it is usually required to further adjust the buffer and continue the molecular sieve purification process to achieve purification requirements.

Accordingly, there is a great need in the art for an affinity chromatography column that is convenient in use, highly efficient for separation and capture of target species, and that the static phase support material used is less expensive.

In order to meet the needs of the industry, the creator of this case has developed an affinity chromatography column, in which the polymer nanofiber is used to replace the substrate of the conventional static phase carrier material, so that the total amount of the polymer nanofiber can be utilized. The advantages of large surface area, high porosity and fast adsorption speed overcome the above industrial problems and achieve excellent separation and collection efficiency. Moreover, this affinity chromatography column is more cost-effective and suitable for large and fast protein purification processes, and is particularly suitable for disposal after single use.

Accordingly, in accordance with a first aspect of the present disclosure, the present invention relates to an affinity chromatography column for purifying a composition, comprising: a hollow tubular body having a first end opposite to the first a second end of the end, and an internal passage connecting the first end and the second end, the first end forming a feed inlet for receiving the composition to be purified, and the second end forming a flushing port And an adsorbent material assembly filled in the inner passage of the hollow tubular body, comprising a first filter, a second filter, and a first filter and the second filter A static phase carrier film, wherein the static phase carrier film comprises: a polymer nanofiber substrate having a BET specific surface area measured by a specific surface area analyzer of ≧ 5 m 2 /g, The fiber diameter measured by a 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 specific binding The composition.

The characteristics of this creation can be clearly understood by referring to the detailed description of the drawings and the examples.

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.

In a preferred embodiment, the affinity chromatography is fixed metal affinity chromatography (IMAC), also known as metal chelate affinity chromatography (MCAC), which involves multiple The metal ion of the address is 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.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a perspective view of an affinity chromatography column according to a specific example of the present invention, which comprises a hollow tube body 1, and an adsorbent material assembly 2 located inside the hollow tube body 1. The hollow tubular body 1 has a first end 11, a second end 12 opposite the first end 11, and an internal passage 13 connecting the first end 11 and the second end 12. The first end 11 is formed with a feed inlet 15 for receiving the composition to be purified, the washing liquid, and the extract. The second end 12 is formed with a flushing port 16 for discharging material that is not retained in the column. The first end 11 and the second end 12 can also each be configured to be coupled to an upstream or downstream device, such as an external or internal thread for screwing an upstream feed device or a downstream liquid collection device.

In general, the hollow body 1 is made of an inert material that does not physically and chemically react with solid and liquid materials involved in the affinity chromatography process, and is preferably stable to environmental factors such as heat and pressure. In a preferred embodiment, the hollow tubular body 1 is made of medical grade plastic, more preferably made of medical grade plastics that meet biocompatibility requirements, for example, in compliance with the US Food and Drug Administration. Modified plastic material for ISO-10993-1 (FDA-modified ISO-10993-1) or USP CLASS 6 (USP XXV class VI). Types of these medical grade plastics include, but are not limited to, polycarbonate (PC), polyphenylene oxide (PPO), PA6/66 nylon plastic, polycarbonate/acrylonitrile-butadiene-styrene copolymer (PC/ABS) ) Composite plastics, polyethylene terephthalate (PET), polyethyleneimine (PEI), polymethyl methacrylate (PMMA), polyphenylene sulfide (PPS), polyethylene (PE), polypropylene ( Plastic materials such as PP), polystyrene (PS), polyvinyl chloride (PVC), and ethylene/vinyl acetate copolymer (EVA). In another preferred embodiment, the hollow tubular body 1 is made of a medical grade metal material, such as a medical grade stainless steel or titanium metal material.

The manufacturing process of the hollow tubular body 1 is familiar to those of ordinary skill in the related art, and can be adjusted depending on the material. Further, the hollow tubular body 1 can be integrally formed by a single process, or a plurality of components can be separately manufactured by a plurality of processes, and these components can be assembled into a complete hollow tubular body 1. For example, when the hollow tubular body 1 is made of plastic, the processes suitable for the hollow tubular body 1 include, but are not limited to, plastic processing processes such as injection molding, compression molding, and thermoforming. When the hollow tubular body 1 is made of a metal material, it can be produced by a conventional metal working process such as stamping, rolling, compression molding, or forging.

The size of the hollow tubular body 1 can be adjusted depending on the size of the purification, and is not particularly limited. For example, in a mass production type purification process, a tube having a diameter of several centimeters can be used to process a large amount of raw materials, and a capillary body having an inner diameter of less than 1 mm can be used for analysis of a micro sample. When the affinity chromatography column of the present invention is designed in the form of a spin column, the size of the hollow tube 1 must be configured to fit into a centrifuge.

As shown in FIG. 1, the adsorbent material assembly 2 is filled in the internal passage 13 of the hollow tubular body 1. The adsorbent material assembly 2 includes a first filter 21, a second filter 22, and a static phase carrier film 23 sandwiched between the first filter 21 and the second filter 22 so that the feed is fed The fluid entering the inlet 15 may be in contact with the first filter 21, the stationary carrier film 23, and the second filter 22 in sequence. The first filter 21 and the second filter 22 may be made of the same or different porous materials, which have considerable mechanical strength for holding the static phase carrier film 23, and may optionally be used for filtering. The impurity particles in the raw material prevent the impurity particles from contacting the static phase carrier film 23 to enhance the efficiency of the affinity chromatography. Preferably, the first filter 21 and the second filter 22 have a pore diameter greater than or equal to 45 microns, and the material thereof includes, but is not limited to, polyethylene (PE), polyether oxime (PES), nylon, mixed cellulose ester (mixed Cellulose ester; MCE), cellulose acetate (CA), polytetrafluoroethylene (PTFE), and polyvinylidene fluoride (PVDF).

The static phase carrier film 23 comprises a polymer nanofiber substrate and a ligand covalently coupled to the polymer nanofiber for specifically binding the target composition. 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.

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.

The ligand has an appropriate functional group and can be covalently coupled to the surface of the polymer nanofiber by any conventional chemical synthesis process to obtain a static phase carrier film 23. 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:

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 other literature, and N- alkylated by adding carboxy functional reaction conditions can be found, such as a further Zhang, L. et al., reactive & functional Polymers 69 (2009) p.48-54, which The complete disclosure of the literature is incorporated into this case for reference.

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 this creation.

Immersing the static phase carrier film in a metal salt solution, for example, 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 with agitation to allow The chelating group sufficiently adsorbs metal ions. The stationary phase support membrane 23 to which the appropriate metal ions are immobilized is suitable for the affinity chromatography process for purification or detection of the target composition. The ligand of the static phase carrier membrane 23 is particularly suitable for capturing target compositions with histidine, cysteine and/or tryptophan labeling after chelation of metal ions, especially with repeating groups. Aminic acid marker, for example 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.

The static phase carrier film 23 can be combined with the first filter 21 and the second filter 22 by any conventional means to form the absorbent material assembly 2. In a specific example, the diameters of the first filter 21 and the second filter 22 are substantially identical in size to the inner diameter of the hollow tubular body 1, so that the absorbent material assembly 2 can be engaged in the internal passage 13, and The static phase carrier film 23 is filled between the first filter 21 and the second filter 22. In another embodiment, the static phase carrier film 23 is sandwiched between the first filter 21 and the second filter 22, and is hot pressed at 25 to 150 ° C and preferably 80 to 120 ° C. The manners are adhered to each other for 1 to 30 minutes, preferably 10 to 20 minutes, to obtain an absorbent material assembly 2 in the form of a composite film.

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 film of the present invention can be filled in the column to contact and adsorb the target composition flowing through the column, or can be placed in the container in a batch manner and the target composition. Mix to facilitate contact and adsorption of 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 film, thereby obtaining a rinse containing the enriched target composition. Things. 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.

The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

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. 2.

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

Example 3: Preparation of Formula 12 The compound of example that the PAN nanofiber membrane and ethylenediamine (ethylenediamine) are made of a reaction. 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

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

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

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

Example 7: Preparation of Example 23 Compound of Formula repeat of 6, except that a substituted ethylenediamine and hexamethylenediamine so prepared in Example 1 PAN nanofiber membrane outside the reaction. According to this, a compound of the following formula 23 having a nitrogen triacetic acid (NTA) chelating group is obtained. Equation 23

Example 8 : Chelation of metal ions to prepare a nickel sulfate solution containing 2000 ppm of Ni ²⁺ , and 50 ml of each of them was added to an appropriate amount of a compound of the formula 11, the formula 12, the formula 13, the formula 21, the formula 22 and the formula 23 at room temperature. Shake to the next day to make a static phase film. 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. The amount of Ni ²⁺ chelated by the compound of Formula 11, Formula 12, Formula 13, Formula 21, Formula 22 and Formula 23 as a stationary phase support membrane was 59.38 mg/g and 33.97 mg/g, respectively. 26.30 mg/g, 18.71 mg/g, 94.74 mg/g and 8.59 mg/g.

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.

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 film 23, and 1 gram of the static phase carrier film 23 was used. Inserted into a hollow tube body 1 of polypropylene material (volume 25 ml) and filled between two first filter sheets 21 and second filter sheets 22 made of porous polyethylene to make an affinity Chromatographic column.

Example 10: Preparation of analytes Transformation has taken 5 ml Moloney murine leukemia virus reverse transcriptase (moloney murine leukemia virus reverse transcriptase; MMLV RTase) of 6 × His expression plasmid of OverExpress ™ C41 (DE3) pLySs coli 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.

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.

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.

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.

The technical content and technical features of the present invention have been disclosed as above, but those skilled in the art may still make various substitutions and modifications based on the disclosure of the present invention. Therefore, the scope of protection of the present invention is not limited to the embodiments disclosed, but should include various alternatives and modifications, and is covered by the following claims.

Symbol Description
1‧‧‧ hollow body
11‧‧‧ first end
12‧‧‧ second end
13‧‧‧Internal passage
15‧‧‧ Feeding entrance
16‧‧‧
2‧‧‧Adsorption material assembly
21‧‧‧First filter
22‧‧‧Second filter
23‧‧‧Static phase film

1 is a schematic perspective view of an affinity chromatography column according to a specific example of the present invention;

2 is an image of a polyacrylonitrile nanofiber film taken by a scanning electron microscope;

Figures 3A-3C 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‧‧‧ hollow body

11‧‧‧ first end

12‧‧‧ second end

13‧‧‧Internal passage

15‧‧‧ Feeding entrance

16‧‧‧

2‧‧‧Adsorption material assembly

21‧‧‧First filter

22‧‧‧Second filter

23‧‧‧Static phase film

Claims (10)

An affinity chromatography column for purifying a composition, comprising: a hollow tubular body having a first end, a second end opposite the first end, and a first end coupled to the first end An inner passage of the second end, the first end is formed with a feed inlet for receiving the composition to be purified, and the second end is formed with a flushing port; and an adsorbent material assembly is filled in the hollow tube The inner passage of the body comprises a first filter, a second filter, and a static phase carrier membrane sandwiched between the first filter and the second filter, wherein the static phase is carried The body film 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 ± as measured by a scanning electron microscope 50 nm and fiber size concentration is ≧90%; and a ligand covalently coupled to the polymeric nanofiber for specific binding of the composition. The affinity chromatography column of claim 1, wherein the polymer nanofiber is 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 polyacrylonitrile nanofiber membranes. An affinity chromatography column 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 iminodiacetic acid (IDA) ), nitrogen triacetic acid (NTA), tricarboxymethyl ethylene diamine (TED), carboxymethylated aspartic acid (CM-ASP), tetraethylene pentamine (TEPA), O -phosphorusamine A group consisting of acid (OPS), 8-hydroxyquinoline (8-HQ) and tris(2-aminoethyl)amine (TREN). The affinity chromatography column of 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 A group consisting of ³⁺ , Fe ²⁺ , Fe ³⁺ , Al ^{3+ ,} and Ca ²⁺ . An affinity chromatography column according to claim 4, wherein the metal ion is selected from the group consisting of Ni ²⁺ , Co ²⁺ and Zn ²⁺ . The affinity chromatography column of claim 4, 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 moved away from the polymeric nanofiber, wherein the main chain is in the form of a simple -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 individual The ground is an integer selected from 0 to 6. An affinity chromatography column according to claim 6, wherein the static phase carrier material has the formula: (Formula 22). The affinity chromatography column according to claim 7, wherein the diameters of the first filter and the second filter are identical in size to the inner diameter of the hollow tube, so that the adsorbent assembly can be engaged. In the internal passage, the static phase carrier film is filled between the first filter and the second filter. The affinity chromatography column of claim 7, wherein the adsorbent material assembly is in the form of a composite film, wherein the static phase support film is heat-pressed to the first filter and the second filter. between. An affinity chromatography column according to claim 3, wherein the composition is a protein comprising six consecutive histidine residues.