PS-peg resin, and mRNA separation and purification by column chromatography using same

Abstract

The present invention relates to mRNA separation and purification by PS-PEG resin column chromatography, which a core made of a polymer resin is used, thereby providing higher durability compared to conventional polymer supports, and an activation group is attached to the outer surface so that a desired biomaterial can be separated with high efficiency. The present invention provides a polymer support having a core-shell structure, the polymer support comprising: a non-reactive core layer made of a polymer resin; a first shell layer crosslinked with the core; and a second shell layer formed on the outer periphery of the first shell layer and including an activation group on the outer side thereof, wherein the polymer support is packed into a chromatography column.

Description

PS-PEG resin and its purification and separation of mRNA by column chromatography

[0001] The present invention relates to a PS-PEG resin and to mRNA separation and purification by column chromatography using the same. More specifically, the present invention relates to a core-shell type (cs) PS-PEG resin, which utilizes a core made of polystyrene (PS) polymer resin and thus has higher durability than conventional polymer supports, and has an activator attached to the outside thereof, enabling the high-efficiency separation of a desired biomaterial, and to mRNA separation and purification by column chromatography using the same.

[0002] Chromatography is a technique that uses appropriate stationary phases and mobile phases to separate each component from a mixture containing the analyte substances by utilizing the difference in migration speed. Liquid chromatography is a chromatographic separation method that uses a liquid as the mobile phase. In this case, porous particles are mainly used as the stationary phase support of liquid chromatography, and the materials used include silica (SiO2), alumina (Al2O3), and zirconia (ZrO2).

[0003] However, these conventionally used supports have a disadvantage in that the reaction area is small compared to the volume of the filled support because the reaction occurs only on the surface of the particle.

[0004] Meanwhile, the effectiveness of solid-phase synthesis or sequence analysis of nucleic acids and peptides is affected by the solid or polymeric support that immobilizes the reactive sites. For example, polystyrene gels or porous glass have been used as solid or polymeric supports for peptide sequencing. The solvents used in these processes can alter the volume of polystyrene particles and potentially cause blockage and backpressure in the reaction column.

[0005] In particular, when such blockages occur in chromatography columns, the polymer support within the column must be replaced, as no further fluid can enter under normal pressure. Furthermore, porous glass has the disadvantage of requiring periodic replacement of the entire column, as separation is difficult after adsorption with the target substance.

[0006] To address this issue, technologies have been developed that utilize spherically shaped cellulose, agar, or agarose as biopolymer supports. These supports, once expanded beyond a certain size, do not expand further, minimizing column clogging and facilitating disposal or regeneration.

[0007] However, in the case of the support as described above, durability is low, and when the speed or pressure of the mobile phase (fluid) in the chromatography column is increased, the structure shape is not maintained and it collapses, which is a disadvantage.

[0008] Therefore, a new type of polymer support is needed to improve the shortcomings of existing polymer supports.

[0009] In order to solve the above-mentioned problem, the present invention provides a method for separating and purifying mRNA by PS-PEG resin column chromatography, which uses a core manufactured from polystyrene (PS) polymer resin and has higher durability than existing polymer supports, and has an activator attached to the outside so that a desired biomaterial can be separated with high efficiency.

[0010] In order to solve the above-described problem, the present invention provides a polymer support having a core-shell structure, which comprises a non-reactive core layer made of a polymer resin; a first shell layer crosslinked with the core; and a second shell layer formed on the outer surface of the first shell layer and including an activating group on the outer surface, wherein the polymer support is filled inside a chromatography column.

[0011] In one embodiment, the core layer and the first shell layer can be crosslinked via an internal linker molecule.

[0012] In one embodiment, the internal linker molecule may be a molecule having two or more functional groups.

[0013] In one embodiment, the functional group is a halogen group, a hydroxyl group, a thiol group, or an amine group, and the internal linker molecule may comprise a cyclic structure.

[0014] In one embodiment, the internal linker molecule can be cyanuric chloride, 1,3,5-tri(halomethyl) benzene, or 1,3,5-triformyl benzene.

[0015] In one embodiment, the polymer support may have a swelling ratio (ml / g) in a solvent of 1 to 5, as measured by the following equation 1.

[0016] [Formula 1]

[0017] Swelling = Swelling volume of polymer support / Dry weight of polymer support

[0018] In one embodiment, the polymer support may be applied to a column having a pressure of 1 to 20 psi.

[0019] In one embodiment, the core layer may be polystyrene or a derivative thereof having an amine group, and the first shell layer and the second shell layer may be polyethylene glycol or a derivative thereof.

[0020] In one embodiment, the polymer support may be used for separation or purification of proteins or nucleic acids.

[0021] In one embodiment, the nucleic acid may be used for isolation or purification of a nucleic acid comprising at least one selected from mRNA, tRNA, rRNA, miRNA, single-stranded RNA, double-stranded RNA, or siRNA.

[0022] In one embodiment, the separation or purification of the protein or nucleic acid can be performed in a solution containing 10 to 30 volume % ethanol and 1 to 500 mM NaCl.

[0023] The present invention also provides a resin for purifying a biomaterial, comprising: a non-reactive core layer made of a polymer resin; a first shell layer crosslinked with the core; a second shell layer formed on the outer surface of the first shell layer and including an activator on the outer surface; and a first spacer attached to the outer surface of the shell layer.

[0024] In one embodiment, the first spacer may include a structure represented by the following chemical formulas 1 to 4.

[0025] [Chemical Formula 1]

[0026] -[CO(CH2) n NH] m -

[0027] (In the above chemical formula 1, n and m are integers from 1 to 10)

[0028] [Chemical Formula 2]

[0029] -CO(CH2) n O-

[0030] (In the above chemical formula 2, n is an integer from 1 to 10)

[0031] [Chemical Formula 3]

[0032]

[0033] [Chemical Formula 4]

[0034] -[NH-CO(CH2) n CO] m -

[0035] (In the above chemical formula 4, n and m are integers from 1 to 10)

[0036] In one embodiment, the terminal of the first spacer further includes an external linker, and the external linker may include a structure represented by the following chemical formulas 3 and 4.

[0037] [Chemical Formula 3]

[0038]

[0039] [Chemical Formula 4]

[0040] -[NH-CO(CH2) n CO] m -

[0041] (In the above chemical formula 4, n and m are integers from 1 to 10)

[0042] The present invention also provides a polymer support having a core-shell structure, which comprises a non-reactive core layer made of a polymer resin; a first shell layer crosslinked with the core; and a second shell layer formed on the outer surface of the first shell layer and including an activating group on the outer surface, wherein the polymer support is used in a spindown process.

[0043] In one embodiment, the spin down may be performed at 10,000 rpm or more.

[0044] The present invention also provides a resin for purifying a biomaterial, comprising: a non-reactive core layer made of a polymer resin; a first shell layer crosslinked with the core; a second shell layer formed on the outer surface of the first shell layer and including an activating group on the outer surface; a first spacer attached to the outer surface of the shell layer; and a second spacer, a ligand or an antibody attached to the first spacer.

[0045] In one embodiment, the biomaterial purification resin may be used in a spin-down process or a chromatography process.

[0046] In one embodiment, the first and second spacers may include structures represented by the following chemical formulas 1 and 2.

[0047] [Chemical Formula 1]

[0048] -CO(CH2) n NH-

[0049] (In the above chemical formula 1, n is an integer from 1 to 10)

[0050]

[0051] [Chemical Formula 2]

[0052] -CO(CH2) n O-

[0053] (In the above chemical formula 2, n is an integer from 1 to 10)

[0054]

[0055] In one embodiment, the ligand may be graphene oxide, a nucleic acid, a nucleic acid derivative, a peptide, or a peptoid.

[0056] In one embodiment, the antibody may bind to mRNA, tRNA, rRNA, miRNA, dsRNA or siRNA.

[0057] In one embodiment, the antibody may be a J2 antibody.

[0058] Since the polymer support according to the present invention has higher pressure resistance than existing polymer supports, it is possible to prevent the disadvantages of existing polymer supports, such as a decrease in flow rate and shape collapse at high pressure.

[0059] In addition, since the polymer support according to the present invention can be used under high pressure conditions, separation and purification of nucleic acids are possible at a faster rate than conventional chromatography for nucleic acid purification.

[0060] In addition, since the polymer support according to the present invention has higher pressure resistance than existing polymer supports, it can be used without damage to the support even when high rpm is applied in a spin-down or column chromatography process.

[0061] In addition, since the polymer support according to the present invention can be used under high rpm conditions, spin-down or column chromatography processes can be performed at a faster speed than conventional supports.

[0062] In addition, the polymer support according to the present invention can be used not only in a spin-down process but also in a continuous flow centrifugation process operated at high rpm.

[0063] Figure 1 shows the results of single-stranded RNA isolation using a support according to one embodiment of the present invention.

[0064] Figure 2 shows the results of single-stranded RNA isolation using a support according to one embodiment of the present invention.

[0065] Figure 3 shows the pressure change according to the flow rate according to one embodiment of the present invention.

[0066] Figure 4 shows the results of single-stranded RNA separation using a column including a support according to one embodiment of the present invention.

[0067] Figure 5 illustrates a process of binding an antibody during resin production according to one embodiment of the present invention.

[0068] Figure 6 illustrates a process of combining graphene oxide in the manufacture of resin according to one embodiment of the present invention.

[0069] Figure 7 shows an SEM photograph of a resin bonded with graphene oxide according to one embodiment of the present invention.

[0070] Figure 8 shows the experimental results of a resin combined with graphene oxide according to one embodiment of the present invention.

[0071] Figure 9 shows the results of RNA isolation using an Oligomer His ligand resin according to one embodiment of the present invention.

[0072] Figure 10 shows the results of RNA isolation using an Oligomer RGG ligand resin according to one embodiment of the present invention.

[0073] Hereinafter, preferred embodiments of the present invention will be described in detail. In describing the present invention, if a detailed description of related known technology is judged to obscure the gist of the present invention, the detailed description thereof will be omitted. Throughout the specification, singular expressions should be understood to include plural expressions unless the context clearly indicates otherwise, and terms such as “comprise” or “have” should be understood to indicate the presence of described features, numbers, steps, operations, components, parts, or combinations thereof, but not to exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof. In addition, in performing a method or a manufacturing method, each step constituting the method may occur in a different order from the stated order, unless the context clearly indicates a specific order. That is, each step may occur in the same order as the stated order, may be performed substantially simultaneously, or may be performed in the reverse order.

[0074] The technology disclosed in this specification is not limited to the implementation examples described herein and may be embodied in other forms. However, the implementation examples introduced herein are provided to ensure that the disclosed content is thorough and complete and to ensure that the technical spirit of the present technology can be sufficiently conveyed to those skilled in the art. In the drawings, the dimensions of each device component, such as width and thickness, are somewhat enlarged to clearly represent the components. The drawings are described from the perspective of an observer, and when an element is mentioned as being positioned above another element, this includes the meaning that the element is positioned directly above the other element or that additional elements may be interposed between them. Furthermore, those skilled in the art will be able to implement the spirit of the present invention in various other forms without departing from the technical spirit of the present invention. In addition, the same reference numerals in multiple drawings indicate substantially the same elements.

[0075] As used herein, the term "and / or" includes any combination of multiple listed items or any one of multiple listed items. As used herein, "A or B" may include "A," "B," or "both A and B."

[0076] The present invention relates to a polymer support having a core-shell structure, which comprises a non-reactive core layer made of a polymer resin; a first shell layer crosslinked with the core; and a second shell layer formed on the outer surface of the first shell layer and including an activating group on the outer surface, wherein the polymer support can be filled into the interior of a chromatography column.

[0077] The core layer is made of a polymer resin and may have a specific degree of cross-linking. Agarose, a polymer support widely used in conventional chromatography, exhibits a tendency to collapse and lose its original shape when exposed to high pressure or increased flow rates within the column. Furthermore, agarose is susceptible to decay by external microorganisms during long-term storage.

[0078] However, in the case of the present invention, by using the crosslinked polymer as described above in the core, the shape can be maintained even under high-flow or high-pressure conditions as described above, and long-term storage is possible because spoilage by microorganisms does not occur.

[0079] At this time, the cross-linking degree is not directly measured, but can be indirectly measured by the swelling degree described later.

[0080] That is, in the case of the present invention, the polymer support may have a swelling degree (ml / g) in a solvent of 1 to 5, as measured by the following equation 1.

[0081] [Formula 1]

[0082] Swelling = Swelling volume of polymer support / Dry weight of polymer support

[0083] When the swelling degree is low, it means that the degree of crosslinking of the polymer support is high and swelling due to the solvent is less likely to occur, and when the swelling degree is high, it means that the degree of crosslinking is low.

[0084] In particular, when the swelling degree is 1, proper swelling may not occur, which may reduce the efficiency of the organic reaction and separation reaction of the resin in the column, and when the swelling degree exceeds 5, the degree of crosslinking may decrease, which may lower the pressure resistance of the polymer support.

[0085] In addition, the polymer support can be used in various solvents, but dimethylformamide (DMF) can be used to specify the swelling degree. Separately, when used in the column, it can be swollen using a solvent used for the separation or purification of proteins or nucleic acids, such as water or a mixed solution of DMSO, acetonitrile, ethanol, or water.

[0086] The core layer may be polystyrene or a derivative thereof having an amine group or a hydroxyl group. The amine group is a portion that is bonded to an internal linker molecule as described below, and thus the core portion may be bonded to the first shell layer via the internal linker molecule.

[0087] In the polymer support of the present invention, a shell layer can be formed around the core as described above. Typically, polymer supports used in chromatography are manufactured in a spherical shape to increase surface area and ensure stability. These spherical polymer supports are typically manufactured to be porous, and reactions or separations occur on the surface and within the pores of the polymer support.

[0088] However, in the case of conventional spherical polymer supports, despite the formation of pores, reaction and separation occur mainly only in the outer portion of a certain thickness, which is known to be a portion corresponding to less than 25% of the total volume. In other words, a portion corresponding to more than 75% of the total volume of the polymer support constitutes the polymer support, but is not involved in reaction and separation.

[0089] Conventional single-component polymer supports are easy to manufacture because they contain identical components from the periphery to the interior. However, their porous nature limits their durability. In particular, as discussed above, agarose, widely used as a polymer support for chromatography, has low pressure resistance. When performed under high-pressure conditions, it may not maintain its shape or disintegrate into small, irregularly shaped pieces.

[0090] To improve this, beads made of glass or ceramic are used as supports, but in the case of these glass or ceramics, the reaction or separation occurs only on the surface, which is less than 1% of the total volume, so the efficiency is known to be greatly reduced.

[0091] In the case of the present invention, the polymer support of the present invention is manufactured to have a structure of a core and a shell by utilizing the fact that the selective adsorption and desorption process as described above is mainly performed on the surface layer of the polymer support, and the core portion is manufactured with a non-reactive polymer resin to ensure high durability, and a large number of activating groups are present on the outside of the shell to ensure high reactivity.

[0092] At this time, the core is made of a non-reactive polymer and may have the degree of crosslinking as described above. In the present invention, the “non-reactive core” means a material that does not react with the separation target or the reaction target, and as described below, a crosslinking reaction may be performed with part or all of the shell, and further, as described above, a crosslinking reaction may be performed inside to form a polymer. In addition, as described above, the core may also swell to a certain extent due to a solvent and increase in volume.

[0093] A first shell layer may be formed on the outside of the non-reactive core, which is bonded or cross-linked with the shell layer. In this case, the first shell layer and the core may be cross-linked via an internal linker molecule.

[0094] At this time, the internal linker molecule may be a molecule having two or more functional groups.

[0095] As described above, the internal linker molecule can connect the core and the first shell layer. To this end, the linker molecule preferably has two or more functional groups, preferably three or more, and more preferably three functional groups.

[0096] The functional group is a portion that binds to a constituent molecule of the core or the first shell during a crosslinking reaction by the internal linker molecule. The functional group as described above readily reacts through a condensation substitution reaction, polymerization reaction, dehydration reaction, or hydrolysis reaction, and a molecule of the core or the first shell layer can bind to the portion where the functional group reacted. At this time, the functional group is preferably a halogen group, a hydroxyl group, a thiol group, or an amine group, more preferably a halogen group, and most preferably chlorine.

[0097] In addition, in the case of the first shell layer and the core, although they do not directly perform chemical bonding, a crosslinking reaction is performed by the internal linker molecule, so that they can be chemically bonded to form a core-shell structure. In addition, due to the crosslinking reaction as described above, the polymer support can have high pressure resistance when used.

[0098] The above linker molecule preferably has a structure in which two or more functional groups as described above can be bonded, and more preferably may include an aromatic ring structure. The above ring structure is preferably formed by carbon or nitrogen, and the reactivity can be controlled by the functional group being bonded at a symmetrical position with respect to the aromatic ring. If the functional group is bonded at an asymmetrical position, the bonding positions of the core and the first shell may become close during a crosslinking reaction by the internal linker molecule, thereby reducing the reactivity.

[0099] Therefore, it is preferable that the internal linker molecule has a benzene ring structure or a cyanuric structure, more preferably cyanuric chloride, 1,3,5-tri(halomethyl) benzene or 1,3,5-triformyl benzene, and most preferably cyanuric chloride.

[0100] The second shell layer is a layer formed on the outside of the first shell layer and includes an activator on the outside. At this time, the second shell layer is preferably formed of a material that is compatible with the first shell layer, and more preferably, it is formed of the same component as the first shell layer. At this time, the first shell layer and the second shell layer may be polyethylene glycol or a derivative thereof.

[0101] In the present invention, the first shell layer and the second shell layer are parts separated by function, and when manufactured from the same material as described above, they may not be optically or chemically distinguished. That is, as described above, the part that is bonded to the core through the internal linker molecule may be referred to as the first shell layer, and the part where an activation group is formed on the outside of the first shell layer, as described below, and which is bonded to a ligand and an antibody may be referred to as the second shell layer.

[0102] The polymer support may be used by being filled inside a chromatography column, and at this time, the polymer support may be applied to a column having a pressure of 11 to 10 psi, preferably 1.5 to 5 psi.

[0103] As described above, the polymer support of the present invention has a core-shell structure, and the core is manufactured using a non-reactive polymer resin. Furthermore, the core and the first shell layer are chemically cross-linked via internal linker molecules. Accordingly, the polymer support of the present invention can exhibit high durability, and in particular, can exhibit higher pressure resistance than existing polymer supports.

[0104] Conventional polymer supports are composed of a single component, and agarose, a widely used polymer support, is known to have very poor pressure resistance, although it can be porous. Therefore, such agarose-based polymer supports cannot be used at high flow rates or pressures, and thus act as a factor reducing the fluid velocity within the column. However, the present invention can have high pressure resistance due to its partially cross-linked core-shell structure as described above, and thus can be applied to columns with high flow rates and pressures.

[0105] The flow rate inside the above column can be changed according to the specifications of the packed column. When using a 12.8x60 mm packed column used for purifying biological substances including proteins or nucleic acids, the flow rate inside the column can be increased to 30 ml / min, and this flow rate inside the column can be applied differently depending on the length and diameter of the column. In addition, by controlling the flow rate inside the column, the pressure inside the column can be maintained at 1 to 20 psi, preferably 1.5 to 10 psi, as described above.

[0106] The present invention also provides a polymer support having a core-shell structure, which comprises a non-reactive core layer made of a polymer resin; a first shell layer crosslinked with the core; and a second shell layer formed on the outer surface of the first shell layer and including an activating group on the outer surface, wherein the polymer support is used in a spin-down process or column chromatography.

[0107] In the polymer support used in the above spin-down process, the non-reactive core layer, the first shell layer, and the second shell layer are the same as described above, so their description will be omitted.

[0108] The polymer support may be used in a spin-down process or column chromatography. In this case, the spin-down process may be performed at 10,000 rpm or higher. Conventional spin-down processes are performed at 4,000 to 10,000 rpm. In particular, as described in the background art, when used to separate cells prone to hemolysis or when using a support with low pressure resistance, hemolysis and support collapse can be prevented by rotating at a low speed as described above.

[0109] In the case of the polymer support widely used in the existing spin-down process, it is composed of a single component, and in particular, agarose, which is a widely used polymer support, is known to have very low pressure resistance although it can have porosity. Therefore, in the case of such agarose-based polymer support, it cannot be used at high rpm, and thus acts as a factor reducing the speed of the spin-down process. However, in the case of the present invention, since it has a core-shell structure that is cross-linked to a certain extent as described above, it can have high pressure resistance, and thus can be applied to the spin-down process performed at high rpm. In addition, since this high rpm spin-down process is the same process as high-speed centrifugation, the support of the present invention can also be used in the centrifugation process.

[0110] The polymer support may be used for the separation or purification of proteins or nucleic acids. The polymer support may be used for the separation or purification of various molecules using chromatography, but is preferably used for the separation or purification of proteins or nucleic acids. In this case, when used for the separation of proteins or nucleic acids, the polymer support is manufactured in the form of beads as described below and binds to proteins or nucleic acids, thereby allowing the proteins or nucleic acids to be separated from the elution solution passing through the column. In addition, when used for the purification of proteins or nucleic acids, the polymer support is manufactured in the form of beads and does not bind to impurities in the solution containing proteins or nucleic acids, thereby allowing the proteins or nucleic acids to be purified from the liquid passing through the column.

[0111] At this time, the nucleic acid may include at least one selected from mRNA, tRNA, rRNA, miRNA, ssRNA, double-stranded RNA, or siRNA. The polymer support of the present invention can be used to isolate such nucleic acids, but preferably, it can be used to isolate or purify RNA, more preferably, mRNA, tRNA, rRNA, miRNA, ssRNA, double-stranded RNA, or siRNA, and most preferably, ssRNA or double-stranded RNA.

[0112] The present invention also relates to a resin for purifying a biological material including a protein or nucleic acid, comprising: a non-reactive core layer made of a polymer resin; a first shell layer crosslinked with the core; a second shell layer formed on the outer surface of the first shell layer and including an activator on the outer surface; and a first spacer attached to the outer surface of the shell layer.

[0113] The present invention also relates to a resin for purifying a biomaterial, comprising: a non-reactive core layer made of a polymer resin; a first shell layer crosslinked with the core; a second shell layer formed on the outer surface of the first shell layer and including an activating group on the outer surface; a first spacer attached to the outer surface of the shell layer; and a second spacer, ligand or antibody attached to the first spacer.

[0114] The above-mentioned resin for purifying biomaterials refers to a filler used by attaching functional groups and antibodies, etc. to the surface layer of the above-mentioned polymer support, and generally fills the interior of a column or reactor to expand the surface area while simultaneously enabling selective separation. In the case of the present invention, a resin for purifying biomaterials capable of separating or purifying proteins or nucleic acids can be provided by attaching spacers, external linkers, ligands, antibodies, etc. to the surface of a polymer support manufactured by the above-mentioned method.

[0115] Since the non-reactive core layer, first shell layer, and second shell layer have the same composition as the polymer support, their description will be omitted.

[0116] The spacer is attached to the surface layer of the polymer support and is used to secure a certain distance between the ligand or antibody, which will be described later, and the polymer support. Looking at this in detail, when the ligand and antibody are directly attached to the second shell layer, the ligand or antibody may be bound in a form that is in close contact with the second shell layer. In this case, when the nucleic acid is separated or purified, sufficient binding with the second shell layer may be difficult due to the large size of the nucleic acid molecule, and even when bound to the second shell layer, adjacent ligands or antibodies may be covered, resulting in a reduced number of ligands or antibodies.

[0117] Therefore, in order to increase the reactivity with these macromolecules, such as nucleic acids, the spacer can be used to bind the ligand or antibody at a certain distance from the second shell layer.

[0118] The above first spacer may include a structure represented by the following chemical formulas 1 to 4.

[0119] [Chemical Formula 1]

[0120] -[CO(CH2) n NH] m -

[0121] (In the above chemical formula 1, n and m are integers from 1 to 10)

[0122]

[0123] [Chemical Formula 2]

[0124] -CO(CH2) n O-

[0125] (In the above chemical formula 2, n is an integer from 1 to 10)

[0126] [Chemical Formula 3]

[0127]

[0128] [Chemical Formula 4]

[0129] -[NH-CO(CH2) n CO] m -

[0130] (In the above chemical formula 4, n and m are integers from 1 to 10)

[0131] By changing n and m in the structures of the above chemical formulae 1, 2, and 4, the length of the CH2 portion and the number of repeating amide bonds can be controlled, thereby controlling the length of the spacer. In this case, if n or m is less than 1, the effect as the spacer is not great, so the activation degree of the resin of the present invention may decrease, and if it exceeds 10, the hydrophobicity of the spacer may increase, so that its role may be limited.

[0132] The first spacer may further include an external linker at the end. The external linker is a portion that is attached to the end of the first spacer and binds to the second spacer, ligand, or antibody, which will be described later. This external linker can separate the ligand using an acid in the step described later, confirm the structure of the ligand, and because it is bulky, it can create a certain space between PEGs. Therefore, the external linker may be Rink linker, Suberol, Sieber, or N-methyl-p-hydroxybenzamide (MHBA), and most preferably, Rink Amide Linker can be used. In particular, since the Rink linker has an amine group in the molecule, when the ligand or antibody is attached, it is possible to easily separate the ligand or antibody from the resin and analyze it through hydrolysis, etc.

[0133] The above external linker may include a structure represented by the following chemical formulas 3 and 4.

[0134] [Chemical Formula 3]

[0135]

[0136] [Chemical Formula 4]

[0137] -(NH-CO(CH2) n CO) m -

[0138] (In the above chemical formula 4, n and m are integers from 1 to 10)

[0139] That is, in the case of the above chemical formulas 3 and 4, they can be used not only as spacers but also as external linkers themselves as described above. In this case, if the external linker has the structure of chemical formula 4, a second spacer may be additionally combined as described above.

[0140] In addition, when introducing succinic anhydride or glutaric anhydride at the N-terminus and then introducing an enzyme or the like through an activation step, an external linker having the structure of the chemical formula 4 can be used, and at this time, it is preferable that n is 2 or 3.

[0141] The ligand may be graphene oxide, a nucleic acid, a nucleic acid derivative, a peptide, or a peptoid. In the present invention, a resin is provided that is filled inside a column and used for the separation or purification of proteins or nucleic acids. At this time, the ligand as described above can be used for the separation or purification of the protein or nucleic acid. In particular, since the nucleic acid binds to a nucleic acid having a complementary base sequence, it is possible to separate the nucleic acid using this. In addition, since the nucleic acid can bind to a complementary peptide nucleic acid, peptoid, or similar nucleic acid, the separation of the nucleic acid is possible by binding this to the linker.

[0142] In particular, when using the above graphene oxide, the resin can selectively bind to double-stranded RNA. Therefore, when using graphene oxide as a ligand, selective separation or purification of double-stranded RNA is possible.

[0143] Additionally, it is also possible to use an antibody that binds to the nucleic acid, and in this case, an antibody that can cause an antigen-antibody reaction with the desired nucleic acid can be used. The antibody used may preferably be an antibody that binds to mRNA, tRNA, rRNA, miRNA, ssRNA, dsRNA, or siRNA, and most preferably, a J2 antibody can be used.

[0144] The above nucleic acid separation or purification can be performed in a solution containing 10 to 30% by volume of ethanol and 1 to 500 mM of NaCl. Within the above range, appropriate nucleic acid separation can be performed, but outside the above range, the nucleic acid may be denatured or appropriate separation may not be performed.

[0145]

[0146] Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings so that those skilled in the art can easily implement them. Furthermore, when describing the present invention, detailed descriptions of related, known functions or configurations will be omitted if they are deemed to unnecessarily obscure the gist of the present invention. Furthermore, certain features presented in the drawings may be enlarged, reduced, or simplified for ease of explanation, and the drawings and their components are not necessarily drawn to scale. However, those skilled in the art will readily understand these details.

[0147]

[0148] Example 1

[0149] 60 g (123 mmol, 9.6 mmol / g) of aminomethyl polystyrene resin (Aminomethyl PS resin, 2.05 mmol / g, 1% DVB crosslinked) and 3500 mL of CHCl were added to a 1 L reactor, and the mixture was allowed to swell at room temperature for 30 minutes and then filtered.

[0150] A mixed solution of 11.3 g (61.5 mmol, 0.5 eq) of cyanuric chloride as an internal linker and 3800 mL of CHCl was added to the reactor after filtering.

[0151] 7.95 g (61.5 mmol, 0.5 eq) of diisopopylethylamine was added and stirred at 25°C for 5 hours. Afterwards, the temperature was raised to 50°C, stirred for 4 hours, and filtered.

[0152] 700 mL of tetrahydrofuran (THF) and 7.95 g (61.5 mmol, 0.5 eq) of diisopropylethyleneamine were added to the filtrate, heated to 65°C, stirred for 20 hours, and then filtered.

[0153] The filtrate was washed with dichloromethane, isopropanol, and toluene in that order and filtered.

[0154] 369 g (350 mL) (615 mmol, 5 eq) of polyethylene glycol (Jeffamine ED-600) and 400 mL of toluene were added to the filtrate, stirred for 10 minutes, then 72.7 g (615 mmol: 5 eq) of diisopropylethyleneamine was added, heated to 90°C, stirred for 24 hours, and filtered.

[0155] The filtrate was washed sequentially with dimethylformamide (DMF), isopropyl alcohol (IPA), dichloromethane (DCM), and acetonitrile (ACN) and filtered.

[0156] The filtrate was placed in a tray and dried under reduced pressure in a vacuum oven for 24 hours to produce a polymer support.

[0157] The polymer support manufactured at this time had a PEG content of 33% according to weight gain. Swelling volume: H2O 2.0 ml / g, MeOH 2.4 ml / g, Toluene 2.8 ml / g, ACN 2.6 ml / g, DMF 3.6 ml / g, DCM 4.0 ml / g.

[0158]

[0159] Example 2

[0160] The same procedure as in Example 1 was performed except that 1,3,5-tri(halomethyl)benzene was used as an internal linker.

[0161]

[0162] Example 3

[0163] The same procedure as in Example 1 was performed except that 1,3,5-triformyl benzene was used as an internal linker.

[0164]

[0165] Comparative Example 1

[0166] The same procedure as in Example 1 was performed except that no internal linker was used.

[0167]

[0168] Comparative Example 2

[0169] The same procedure as in Example 1 was performed except that 1,4-diformylbenzene was used as an internal linker.

[0170]

[0171] Experimental Example 1

[0172] Pressure resistance tests were conducted for the above Examples 1 to 3 and Comparative Examples 1 to 2.

[0173] The polymer supports manufactured in Examples 1 to 3 and Comparative Examples 1 to 2 were filled into a column used for HPLC, and then the inside was pressurized for 60 minutes at the pressure shown in Table 1 below to allow a solvent to pass through. The solvent used at this time was water, and in order to prevent damage to the column during pressurization, the column was installed in a chamber, and then the outside of the column was pressurized to maintain the pressure difference between the inside and outside of the column at less than 1 psi.

[0174] After each pressurization, the supports were recovered and the percentage of supports that were broken or did not maintain their shape was investigated.

[0175] Pressure (psi) 11.5 2 3 5 7 10 Example 10.0 10.20.5 0.9 1.14.3 10.4 Example 20.0 20.20.4 0.8 1.14.4 11.5 Example 30.0 10.30.6 1.11.0 4.2 10.9 Comparative Example 10.0 30.6 4.38.4 11.116.5 21.5 Comparative Example 20.0 40.4 3.8 6.5 8.4 11.5 18.4

[0176] As shown in Table 1, in the cases of Examples 1 to 3 of the present invention, it was confirmed that damage to the beads was minimized even when operating under pressure of up to 5 psi. However, in the case of Comparative Example 1, which did not use an internal linker, it was confirmed that damage became severe when operating at a pressure of 1.5 psi or higher. However, in the case of Comparative Example 1, it was confirmed that many supports showed damage to the shell portion rather than damage to the core portion. In addition, in the case of Comparative Example 2, which used diformylbenzene having two functional groups as an internal linker molecule, it was shown to have an improved effect compared to Comparative Example 1.

[0177]

[0178] Comparative Example 3

[0179] The same procedure was followed as in Example 1 above, except that an equivalent amount of agarose was used instead of polystyrene as the non-reactive core layer.

[0180]

[0181] Comparative Example 4

[0182] In the above Example 1, a commercially available agarose carrier (Sigma-Aldrich) was used instead of the polymer support.

[0183]

[0184] Experimental Example 2

[0185] Pressure resistance tests were conducted according to the rotation speed for the above examples 1 to 3 and comparative examples 1 to 4.

[0186] A commercially available centrifuge (Benchmark Scientific C1612) was used to determine the extent of support damage at each rotational speed (rpm). Each experiment was performed for 10 minutes at the rpm shown in Table 1 below, and water was used as the solvent.

[0187] After spin-down was completed under these conditions, the supports were recovered and the percentage (%) of supports that were broken or did not maintain their shape was investigated.

[0188] Rotational speed (rpm) 4000 6000 8000 10000 12000 15000 Example 10.0 10.0 30.0 9 0.5 1.12 0 Example 20.0 20.0 30.0 7 0.6 1.2 2.4 Example 30.0 20.0 40.10 4 1.12 1 Comparative Example 10.0 5 0.12 2.5 6 13.5 22.4 Comparative Example 20.0 40.0 9 1.8 3.5 6.9 11.5 Comparative Example 30.11 5 3.0 8.4 22.5 38.9 Comparative Example 40.21 9 5.2 16.8 35.7 65.4

[0189] As shown in Table 2, in the cases of Examples 1 to 3 of the present invention, it was confirmed that damage to the support was minimized even when the rotation speed was increased to 15,000 rpm. However, in the case of Comparative Example 1, which did not use an internal linker, it was confirmed that damage became severe when operated at a pressure of 8,000 rpm or higher. However, in the case of Comparative Example 1, it was confirmed that many supports showed damage to the shell part rather than damage to the core part. In addition, in the case of Comparative Example 2, which used diformylbenzene having two functional groups as an internal linker molecule, it was shown to have an improved effect compared to Comparative Example 1.

[0190] In the case of Comparative Examples 3 and 4 using existing agarose, it was found that the support was difficult to use due to severe damage to the support under conditions of 10,000 rpm or higher, and in particular, unlike Comparative Example 1, it was confirmed that the core part was damaged and the support was split.

[0191] As shown in Table 2 above, it was found that the existing support (Comparative Example 4) could be used in the existing spin-down process (4000 to 8000 rpm), but in the case of the high rpm conditions suggested in the present invention, the existing support could not be used, and it was confirmed that only Examples 1 to 3 of the present invention could be used.

[0192]

[0193] The following examples were performed using the support prepared in Example 1 above.

[0194]

[0195] Experimental Example 3

[0196] The most effective ethanol and NaCl composition for IVT RNA isolation and purification was selected. Loading buffer containing 50 μg of IVT RNA (996 nt) was used in Pierce TM The resin of Example 1 was mixed with a spin column (Thermo Fisher Scientific) at room temperature for 10 minutes, and then centrifuged (3000×g) for 10 seconds to separate the resin and the liquid phase. When a loading buffer (HEPES (pH 7.2)) with a composition of 20% ethanol and 200–300 mM NaCl was used, the IVT RNA flow-through containing a small amount of double-stranded RNA could be recovered.

[0197] Looking at the above experimental method in detail, as shown in Fig. 1, 10 mM HEPES (pH 7.2) containing 50 μg of IVT RNA (996 nt) and a loading buffer of ethanol and NaCl of various compositions were mixed with the resin packed in a Pierce™ spin column (Thermo Fisher Scientific) at room temperature for 10 minutes, and then centrifuged (3000Xg) for 10 seconds to separate the resin and the liquid phase. Afterwards, the RNA present in the flowthrough recovered through centrifugation was confirmed by agarose gel electrophoresis, and the presence of double-stranded RNA present in the flowthrough was confirmed through J2 antibody dot blotting assay. In order to desorb the IVT RNA remaining in the resin, the resin and 8 M UREA were mixed at room temperature for 10 minutes, and then centrifuged (3000Xg) for 10 seconds to separate the resin and the liquid phase. Afterwards, the RNA present in the eluant recovered through centrifugation was confirmed by agarose gel electrophoresis, and the presence of double-stranded RNA present in the eluant was confirmed through J2 antibody dot blotting assay.

[0198]

[0199] Experimental Example 4

[0200] Pierce 300 mM NaCl loading buffer containing 50 μg of IVT RNA (996 nt) TM After mixing the resin packed in a spin column (Thermo Fisher Scientific) at room temperature for 10 minutes, centrifugation (3000Xg) for 10 seconds separated the resin and liquid phase. At this time, the amount of RNA contained in the flow through was 44.1% of the total RNA amount, and it was confirmed that double-stranded RNA was reduced by 90.8%.

[0201] Afterwards, the IVT RNA remaining in the resin was eluted in a step-gradient manner by lowering the concentration of NaCl by 50 mM each time, and it was confirmed that a large amount of double-stranded RNA was included when eluted with 50 mM and 0 mM NaCl elution buffers.

[0202] The above experimental method is described in detail as follows. As shown in Fig. 2(A), a loading buffer of 10 mM HEPES (pH 7.2), 20% (v / v) ethanol, and 300 mM NaCl containing 50 μg of IVT RNA (996 nt) was mixed with the resin packed in a Pierce™ spin column (Thermo Fisher Scientific) at room temperature for 10 minutes, and then centrifuged (3000×g) for 10 seconds to separate the resin and the liquid phase. The RNA present in the flowthrough recovered through centrifugation was confirmed by agarose gel electrophoresis, and the presence of double-stranded RNA in the flowthrough was confirmed through a J2 antibody dot blotting assay. To detach the IVT RNA remaining on the resin, an elution buffer of 10 mM HEPES (pH 7.2), 20% (v / v) ethanol, and 250 mM NaCl was mixed with the resin again at room temperature for 10 minutes, and then centrifuged (3000Xg) for 10 seconds to separate the resin and the liquid phase. In the same manner, a total of 6 elution steps were performed using elution buffers of 200 mM, 150 mM, 100 mM, 50 mM, and 0 mM NaCl. Afterwards, the RNA present in the eluant recovered through centrifugation was confirmed by agarose gel electrophoresis, and the presence or absence of double-stranded RNA in the eluant was confirmed through a J2 antibody dot blotting assay.

[0203] In addition, the agarose gel and J2 antibody dot blotting assay results were quantified as shown in Fig. 2(B). The intensity of the signal appearing in the agarose gel was quantified as an image to obtain the total recovered RNA amount, and then the amount of RNA recovered at each step was expressed as a percentage. In addition, the signal appearing in the J2 antibody dot blotting assay results was quantified as an image to obtain the total recovered double-stranded RNA amount, and then the amount of double-stranded RNA included at each step was expressed as a percentage.

[0204] Through the above experimental examples 2 and 3, it was confirmed that the resin manufactured by the method of the present invention can be used for the separation and recovery of single-stranded RNA.

[0205]

[0206] Example 4

[0207] An empty cartridge for chromatography (ID12.8 x H60 mm) was filled with core-shell type PS-PEG resin (4 g), connected to an HPLC (High Performance Liquid Chromatography) system, and the pressure change was observed when water was flowed at various flow rates. Starting with a flow rate of 5 ml / min and increasing to 30 mL / min, it was confirmed that the pressure gradient according to the flow rate was linear up to 7 bar (Fig. 3).

[0208]

[0209] Experimental Example 5

[0210] To demonstrate that the resin manufactured by the method of the present invention can isolate single-stranded RNA, the following experiment was conducted. Developing solvents A and B for column chromatography were prepared as follows. Solution A: 300 mM NaCl, 20% (v / v) ethanol in HEPES buffer (pH 7.2); Solution B: 20% (v / v) ethanol in HEPES buffer (pH 7.2). 4 g of PS-PEG resin (Example 1) was packed into an 8 mL column, and then Solution A containing RNA (encoding eGFP, 500 μg) was flowed at a rate of 1 mL / min for 20 minutes, followed by flowing the same solution for 108 minutes. The concentration of NaCl was continuously changed from Solution A to Solution B for 64 minutes, and the eluate was collected in 8 mL portions. The amount of RNA present in each fraction was analyzed using the Ribogreen assay, and the amount of double-stranded RNA present in each fraction was analyzed using the Dot blot assay, resulting in a chromatogram as shown in Figure 4. It was confirmed that most ssRNA was eluted when the NaCl salt concentration was between 300 and 50 mM, and that double-stranded RNA was eluted in the absence of NaCl (see Figure 4).

[0211]

[0212] Example 5

[0213] Resin synthesis with J2 Antibody (Anti-dsRNA) attached

[0214] 100 mg of the resin of Example 1, into which several spacers (-CO-RINK-NH-COCH2CH2NH-CO(CH2)5-, -CO-CH2CH2-, -COCH2CH2NH-CO(CH2)5-) of different functions and lengths were introduced, was mixed with 2 ml of dimethylformamide (DMF), and then 5 equivalents of succinic anhydride and diisopropylethylamine relative to the amount of amine groups of the resin were added and reacted at 25°C for 2 hours. After completion of the reaction, the mixture was washed using DMF, dichloromethane, and methanol, and dried in a vacuum desiccator to prepare a succinylated resin.

[0215] 50 mg of the succinylated resin thus obtained was added to 1 ml of a mixed solution of H2O:DMF (1:5 to 5:1) and mixed, and then 1-Ethyl-3-(3'-dimethylaminopropyl)-ethylcarbodiimide (EDC, Sigma-Aldrich) and N-Hydroxysuccinimide (NHS, Sigma-Aldrich) were added in excess amounts (10 mg and 12 mg, respectively) and reacted at 25°C for 2 hours. Afterwards, the resin was washed three times each with distilled water and PBS (pH 7.4) solution, and the activated ester form of the resin thus obtained was mixed with 50 μl of J2 Antibody (Anti-dsRNA antibody, Sigma-Aldrich) dissolved in PBS (pH 7.4) solution (1.5 ml). After reacting at 25°C for 2 hours, the filtrate was removed, washed several times with PBS (pH 7.4) solution, and stored in the refrigerator as a 50 mg / ml of PBS (pH 7.4) solution of Resin (see Fig. 5).

[0216]

[0217] Cleaning of resin bound to J2 antibody

[0218] The resin conjugated with J2 antibody was mixed with PBS (pH 7.4) at a ratio of 0.2 g / ml to obtain a resin slurry solution. Then, the resin slurry solution containing 4 mg of the resin was transferred to a microcentrifuge spin column and centrifuged (3000 X g) for 10 seconds to leave only the resin in the spin column. The washing solution was added to the microcentrifuge spin column and stirred for 5 minutes to mix the resin and the washing solution, and then centrifuged (3000 X g) for 10 seconds to recover only the resin in the spin column. The above process was repeated 2-3 times. Washing was performed according to the composition of the chromatography buffer, which is the condition for binding dsRNA to the resin conjugated with J2 antibody.

[0219]

[0220] Example 6

[0221] After swelling 100 mg of the resin of Example 5 in 1 ml of DMF (Dimethylformaldehyde), 3 equivalents of DIC (N,N′-Diisopropylcarbodiimide), HOBt (Hydroxybenzotriazole), and DIEA were each added based on the amount of resin amine groups, and 1 ml of graphene oxide (GO) aqueous solution (GO 10 mg / ml H2O) was added and reacted at 25°C for 12 to 24 hours (see Fig. 6). After the reaction was completed, the normally synthesized resin could be recovered by washing with DMF and water.

[0222]

[0223] Example 8

[0224] After mixing 100 mg of PS-PEG resin with 1 ml of H2O, 3 equivalents of EDC and sulfo-NHS (Hydroxy-2,5-dioxopyrrolidine-3-sulfonicacid sodium salt) were added based on the amount of resin amine groups, and 1 ml of GO aqueous solution (10 mg / ml H2O, Ultra-highly concentrated GO aqueous solution) was added, and the mixture was reacted at 25°C for 12–24 hours (see Fig. 6). After the reaction was completed, the resin with GO bound to it was recovered normally (see Fig. 7).

[0225]

[0226] Example 9

[0227] 100 mg of PS-PEG resin was mixed with 1 ml of H2O, and 1 ml of GO aqueous solution (GO 10 mg / ml H2O) was added and reacted at room temperature for 12 to 24 hours (see Fig. 6). As a result of performing the above, GO binding reaction did not occur, and thus the desired resin could not be recovered.

[0228]

[0229] Experimental Example 5

[0230] In Example 7, the surface-modified graphene oxide prepared using PS-PEG resin (PS-PEG-GO) was used to purify IVT m1Ψ-mRNA (Fig. 8.A). The degree of double-stranded RNA removal after IVT was compared with a conventional double-stranded RNA removal system using cellulose chromatography. IVT m1Ψ-mRNA purification using PS-PEG-GO resin chromatography showed an mRNA recovery rate of approximately 80-90%, and it was confirmed that double-stranded RNA was removed by approximately 90% regardless of LiCl precipitation. In addition, it was shown that the components present in the IVT reaction solution did not affect the mRNA purification efficiency using PS-PEG-GO resin chromatography, suggesting that this purification method can be effectively applied even without a prior LiCl salt precipitation step. Moreover, when IVT m1Ψ-mRNA was purified through LiCl-precipitation, it was confirmed that the double-stranded RNA removal efficiency using PS-PEG-GO resin chromatography was similar to that of chromatography using cellulose (Fig. 8.B).

[0231] To further verify whether PS-PEG-GO resin chromatography removed IVT reaction residues, the presence of residual nucleotides and enzymes in each chromatography fraction after purification was examined. After applying the purified m1Ψ-mRNA without LiCl precipitation to PS-PEG-GO resin chromatography, the presence of nucleotides in the flowthrough fraction (F) and eluant fraction (E) was analyzed using non-denaturing polyacrylamide gel electrophoresis (Fig. 8C). The analysis results showed that nucleotides were mainly detected in the flowthrough fraction, and the amount was similar to that before purification. In contrast, no nucleotides were detected in the eluant fraction, suggesting that most nucleotides were not bound to the PS-PEG-GO resin.

[0232] In addition, the fractionation of enzymes used in the IVT reaction was evaluated using SDS-gel electrophoresis (Fig. 8D). As a result, enzymes were mostly detected in the flowthrough fraction and not in the eluant fraction. These results suggest that PS-PEG-GO resin chromatography can effectively remove enzymes remaining after the IVT reaction without a prior salt precipitation process. Therefore, the nucleotides and enzymes used in the IVT reaction were eluted without binding to the PS-PEG-GO resin, allowing for the effective separation of IVT mRNA from the IVT reaction mixture.

[0233]

[0234] Example 10

[0235] b-Ala-PS-PEG resin synthesis

[0236] 25 g (0.43 mmol / g: 10.75 mmol) of PS-PEG resin and 250 mL of THF were added to a 500 mL reactor, and the mixture was swelled at 20 to 25 degrees for 30 minutes and then filtered. A solution of 8.76 g (21.5 mmol: 2 eq) of Fmoc-B-Ala-OSu and 4.17 g (32.25 mmol: 3 eq) of diisopropylethylamine (DIEA) dissolved in 250 mL of THF was added and stirred at 20 to 25 degrees for 3 hours. The mixture was filtered, and the filtrate was washed twice with 250 mL of THF, twice with 250 mL of DCM, and twice with 250 mL of MeOH, placed in a vacuum oven, and dried under reduced pressure at 30 degrees for 14 hours to recover 26.57 g of Fmoc-b-Ala-PS-PEG resin.

[0237] To remove Fmoc from the Fmoc-b-Ala-PS-PEG resin, 250 mL of a 20% piperidine DMF solution was added and stirred for 25 minutes. The residue was washed sequentially with 500 mL of DMF four times and 500 mL of DCM twice, placed on a tray, placed in a vacuum oven, and dried under reduced pressure at 30°C for 14 hours to recover 22.3 g (0.42 mmol / g) of b-Ala-PS-PEG.

[0238] Synthesis of maleimide-b-Ala-PS-PEG resin

[0239] 10 g (0.42 mmol / g, 4.2 mmol) of the above resin was taken, 100 mL of DMF was added, swelling was performed, and then filtered. A complete solution of 1.77 g (6.3 mmol, 1.5 eq) of Maleimide-NHS, 0.81 g (6.3 mmol: 1.5 eq) of DIEA, and 80 mL of DMF was added, stirred at 20-25°C for 16 hours, and then filtered. The filtrate was washed sequentially with 500 mL of DMF four times and 500 mL of DCM twice, placed on a tray, placed in a vacuum oven, and dried under reduced pressure at 30°C for 14 hours to recover 10.2 g of Maleimide-B-Ala-PS-PEG resin.

[0240] PS-PEG resin with peptide ligand introduced

[0241] The above resin (Maleimide-b-Ala-HiCore) 1 g (theoretical substitution rate: 0.39 mmol / g, 0.39 mmol) was swollen in DMF for 30 minutes and filtered. 0.23 g (0.38 mmol, 0.97 eq) of Fmoc-RGGC-NH2 was dissolved in 10 mL of DMF and added, and stirred at 20-25 degrees for 16 hours. After filtering, the filtrate was washed sequentially with 500 mL of DMF four times and 500 mL of DCM twice, placed on a tray, placed in a vacuum oven, and dried under reduced pressure at 30 degrees for 14 hours to recover the Fmoc-RGGC-Maleimide-B-Ala-PS-PEG resin. Fmoc was removed by the above method, washed, and dried to recover the RGGC-Maleimide-B-Ala-PS-PEG resin.

[0242] At this time, the loading value was measured by quantifying the Fmoc group from the UV value of the solution from which Fmoc was removed. Using the same method, Fmoc-RGG-RGGC-NH2, Fmoc-RGG-RGG-RGGC-NH2, RGG-RGGC-Maleimide-B-Ala-PS-PEG, and RGG-RGG-RGGC-Maleimide-B-Ala-PS-PEG resins were manufactured.

[0243] In the same way, H HHHHHC(H6C)-Maleimide-b-Ala-PS-PEG and HHHHH HHHHH-C(H10C)-Maleimide-b-Ala-PS-PEG resins were prepared, respectively.

[0244] Ligand conjugated-PS-PEG resinLoading (mmol / g)RGGC-Maleimide-b-Ala-PS-PEG resin0.1589RGG-RGGC-Maleimide-b-Ala-PS-PEG resin0.0857RGG-RGG-RGGC-Maleimide-b-Ala-PS-PEG resin0.0329H6C-Maleimide-b-Ala-PS-PEG resin0.0353H10C-Maleimide-b-Ala-PS-PEG resin0.002

[0245] Results of using Oligomer His ligand

[0246] 30 mg of the above resin coupled with His oligomer (6x or 10x) with or without the N-terminal Fmoc group removed (H6C; H10C) or not (Fmoc-H6C; Fmoc-H10C) was loaded onto a spin column and sufficiently swelled with different buffer solutions depending on the pH [20% (v / v) EtOH, 300 mM NaCl, phosphate (pH 6.5) or HEPES (pH 7.5) or Tris (pH 8.5)]. eGFP RNA (25 mg in the same buffer solution) was loaded onto the column and eluted with 10 mM buffer solution containing no NaCl in 20% (v / v) EtOH [phosphate (pH 6.5) or HEPES (pH 7.5), or Tris (pH 8.5)]. 10 μL of the elution solution was applied to a 1% GelRED pre-stained agarose gel, electrophoresed, and the RNA band was quantified to confirm the recovered RNA, and dsRNA was quantified using a dot blotting assay.

[0247]

[0248] As shown in Fig. 9, in the case of His oligomer resin (H6C) with the N-terminal Fmoc group removed, when the pH increased from 6.5 to 7.5, the RNA recovery rate increased from 64.3% to 85.0%, but the double-stranded RNA was eluted as RNA without removal (dsRNA content ratio; 99%), so dsRNA was not removed.

[0249] His oligomer resin (H10C) with the N-terminal Fmoc group removed showed 87% mRNA recovery at pH 7.5, despite a ligand loading amount 1 / 18 less than that of His6. Compared to His6, His10 showed an increased efficiency in removing double-stranded RNA, and the dsRNA content in the eluted RNA decreased from 99% to 82.4%.

[0250]

[0251] Results of using Oligomer RGG ligand resin

[0252] 30 mg of resins in which peptide ligands with 1, 2, and 3 Arg-Gly-Gly (RGG) motifs with the N-terminal Fmoc group removed were introduced (named RGGC-Maleimide-βAl, RGG RGGC-Maleimide-βAla, and RGG RGG RGGC-Maleimide-βAla, respectively) were added to a spin column and mixed with three different buffer solutions with different ethanol contents and salt solution cations of Na or K; [Buffer 1]: 20% (v / v) EtOH, 300 mM NaCl in 10 mM HEPES (pH 7.2); [Buffer 2]: 100 mM NaCl in 10 mM HEPES (pH 7.2); [Buffer 3]: The resin was sufficiently swollen with 100 mM KCl in 10 mM HEPES (pH 7.2). eGFP RNA (25 mg in the same buffer) was loaded and eluted with the same buffer (Flowthrough, F). The RNA remaining on the resin was then eluted with 8 M urea solution (Elution, E). 10 μL each of the F and U fractions of the elution solution were loaded onto a 1% GelRED pre-stained agarose gel and subjected to electrophoresis to quantify the RNA bands to confirm the recovered RNA. dsRNA was quantified using a dot blotting assay.

[0253]

[0254] As shown in Fig. 10, as the RGG motif repeat sequence increased from 1 to 3, the amount of mRNA bound to the resin increased, and the average yield of RNA recovered after resin binding increased from 30% (RGG) to 52% (RGG-RGG-RGG). In addition, when using a resin introduced with an RGG ligand, a buffer solution containing potassium salt (K) instead of sodium salt (Na) at neutral pH could be used as an RNA resin binding buffer without using ethanol. For example, in the case of a resin introduced with RGG-RGG-RGGG, the RNA recovery rate increased from 38% to 63% when using [Buffer Solution 3] compared to when using [Buffer Solution 2].

[0255] The removal efficiency of double-stranded RNA was superior to that of the His peptide-introduced resin. When oligo His was introduced, most of the dsRNA (more than 90%) eluted unbound to the resin when RNA was loaded and eluted with the same buffer (flowthrough, F). When the resin-bound RNA was subsequently eluted with an 8 M urea solution, less than 10% of the dsRNA was included.

[0256] When RNA was attached to the resin introduced with RGG and the double-stranded RNA contained in the RNA eluted with 8M urea, the resin introduced with RGG-RGG-RGG showed a high dsRNA separation ability with a residual relative ratio of 58% when using [buffer solution 3].

[0257] When mRNA was bound to a resin using a resin in which an oligo RGG motif was introduced, it was confirmed that as the number of RGG repeat sequences introduced into the resin increased, the RNA binding ability increased and the double-stranded RNA removal efficiency was maintained at a high level when a buffer solution containing potassium salt (K) instead of sodium salt (Na) was used at neutral pH without using ethanol.

[0258]

[0259] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A non-reactive core layer made of a polymer resin; a first shell layer cross-linked with the core; and A second shell layer formed on the outer surface of the first shell layer and including an activator on the outer surface; In a polymer support including: The above polymer support is a polymer support having a core-shell structure that is filled inside a chromatography column.

2. In paragraph 1, A polymer support having a core-shell structure in which the core layer and the first shell layer are cross-linked through internal linker molecules.

3. In paragraph 2, A polymer scaffold having a core-shell structure, wherein the internal linker molecule is a molecule having two or more functional groups.

4. In paragraph 3, The above functional group is a halogen group, a hydroxyl group, a thiol group or an amine group, The above internal linker molecule is a polymer scaffold having a core-shell structure including a cyclic structure.

5. In paragraph 4, A polymer support having a core-shell structure, wherein the internal linker molecule is cyanuric chloride, 1,3,5-tri(halomethyl) benzene or 1,3,5-triformyl benzene.

6. In paragraph 1, The above polymer support is a polymer support having a core-shell structure with a swelling ratio (ml / g) in a solvent of 1 to 5, as measured by the following equation 1. [Formula 1] Swelling = Swelling volume of polymer support / Dry weight of polymer support 7. In paragraph 1, The above polymer support is a polymer support having a core-shell structure that is applied to a column having a pressure of 1 to 20 psi.

8. In paragraph 1, A polymer support having a core-shell structure, wherein the core layer is polystyrene or a derivative thereof having an amine group, and the first shell layer and the second shell layer are polyethylene glycol or a derivative thereof.

9. In paragraph 1, The above polymer support is a polymer support having a core-shell structure used for separation or purification of proteins or nucleic acids.

10. In paragraph 9, A polymer support having a core-shell structure used for the separation or purification of nucleic acids, wherein the nucleic acids include at least one selected from mRNA, tRNA, rRNA, miRNA, single-stranded RNA, double-stranded RNA, or siRNA.

11. In paragraph 9, A polymer support having a core-shell structure, characterized in that the separation or purification of the protein or nucleic acid is performed in a solution containing 10 to 30 volume% of ethanol and 1 to 500 mM of NaCl.

12. Non-reactive core layer manufactured from polymer resin; A first shell layer cross-linked with the above core; A second shell layer formed on the outer surface of the first shell layer and including an activator on the outer surface; and A first spacer attached to the outside of the shell layer; A resin for purifying biological materials, including:

13. In paragraph 12, A resin for purifying a biomaterial, wherein the first spacer comprises a structure represented by the following chemical formulas 1 to 4. [Chemical Formula 1] -[CO(CH2) n [VIEW] m But (In the above chemical formula 1, n and m are integers from 1 to 10) [Chemical Formula 2] -CO(CH2) n O- (In the above chemical formula 2, n is an integer from 1 to 10) [Chemical Formula 3] [Chemical Formula 4] -[NH-CO(CH2) n CO] m But (In the above chemical formula 4, n and m are integers from 1 to 10) 14. In paragraph 12, The terminal of the above first spacer further includes an external linker, A resin for purifying a biomaterial, wherein the external linker comprises a structure represented by the following chemical formulas 3 to 4. [Chemical Formula 3] [Chemical Formula 4] -[NH-CO(CH2) n CO] m But (In the above chemical formula 4, n and m are integers from 1 to 10) 15. Non-reactive core layer manufactured from polymer resin; a first shell layer cross-linked with the core; and A second shell layer formed on the outer surface of the first shell layer and including an activator on the outer surface; In a polymer support including: The above polymer support is a polymer support having a core-shell structure used in a spin-down process.

16. In paragraph 15, A polymer support having a core-shell structure, wherein the above spin-down is performed at 10,000 rpm or more.

17. Non-reactive core layer manufactured from polymer resin; A first shell layer cross-linked with the above core; A second shell layer formed on the outer surface of the first shell layer and including an activator on the outer surface; a first spacer attached to the outside of the shell layer; and A second spacer, ligand or antibody attached to the first spacer; A resin for purifying biological materials, including:

18. In paragraph 17, The above biomaterial purification resin is a biomaterial purification resin used in a spin-down process or a chromatography process.

19. In paragraph 17, A resin for purifying a biomaterial, wherein the first and second spacers comprise structures represented by the following chemical formulas 1 and 2. [Chemical Formula 1] -CO(CH2) n NH- (In the above chemical formula 1, n is an integer from 1 to 10) [Chemical Formula 2] -CO(CH2) n O- (In the above chemical formula 2, n is an integer from 1 to 10) 20. In paragraph 17, The above ligand is a resin for purifying a biomaterial, which is graphene oxide, nucleic acid, nucleic acid derivative, peptide or peptoid.

21. In paragraph 17, A resin for purifying a biological substance, wherein the antibody is bound to mRNA, tRNA, rRNA, miRNA, dsRNA or siRNA.

22. In paragraph 21, The above antibody is a resin for purifying biological substances, which is a J2 antibody.

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