Chromatographic media and methods

Abstract

A chromatography medium for separating a sample in solution is provided, wherein the chromatography medium comprises a matrix material having an average flowpore size of 0.1 to 2.0 μm, and the matrix material is functionalized with a thiophilic aromatic ligand to a ligand concentration of up to 1500 μmol / g of matrix material. A method for separating a sample in solution using the chromatography medium is also provided. The present invention is useful for separating different plasmid DNA isoforms.

Description

Technical Field

[0001] The present disclosure relates to a chromatography medium for separating analytes in solution and a method for separating analytes using such a chromatography medium.

Background Art

[0002] Plasmids are raw materials for the production of mRNA and viral vectors, and thus there is a demand for highly purified plasmid DNA. In the purification of supercoiled plasmid DNA, complete removal of other cellular components such as host proteins, endotoxins, chromosomal DNA, RNA, open circular and nicked form plasmid DNA is required.

[0003] Other chromatography methods such as size exclusion chromatography, gel filtration, hydroxyapatite, ion exchange chromatography, reverse phase chromatography, and hydrophobic interaction chromatography have been used for the purification of plasmid DNA. Most of the said methods lack the possibility of separating supercoiled plasmid DNA from other forms of plasmids.

[0004] R. Lemmens et al., J. Chromatogr. B 784 (2003) 291-300 describes a three-step method for the purification of plasmid DNA including i) group separation, ii) thiophilic aromatic chromatography using various thioether-containing adsorbents bound to Sepharose 6 Fast Flow chromatography resin, and iii) anion exchange chromatography.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

[0006] Therefore, there is a need for improved methods for separating plasmid DNA isoforms. [Means for solving the problem]

[0007] The object of the present invention is to provide a chromatography medium for separating samples in solution, such as superhelical plasmid DNA. It is also an object to provide a method for separating samples in solution.

[0008] The present invention is defined by the appended independent claims. Non-limiting embodiments arise from the dependent claims, the appended drawings and the following description.

[0009] In the first aspect, a chromatography medium for separating a sample in solution is provided, comprising a matrix material having an average flowpore size of 0.1 to 2.0 μm, wherein the matrix material is functionalized with a thiophilic aromatic ligand to a ligand concentration of up to 1500 μmol / g of the matrix material.

[0010] The term "thiophilic" in this specification refers to the interaction between a ligand and a target based on thiophilic aromatic chromatography. The ligand concentration may be 10-1500 μmol / g, 50-1400 μmol / g, 100-1300 μmol / g, 150-1200 μmol / g, 200-1100 μmol / g, 250-1000 μmol / g, 300-1000 μmol / g, 400-1000 μmol / g, 500-1000 μmol / g, 600-1000 μmol / g, 700-1000 μmol / g, 800-1000 μmol / g, or 300-900 μmol / g relative to the matrix material.

[0011] The sample can be any polynucleotide, such as plasmid DNA or RNA, genomic DNA, or sheared nucleotide sequences. The sample is preferably 20 kbp or less in size. In particular, the sample may be an isoform of a specific plasmid DNA.

[0012] Standard resins used in chromatography have limited and diffusion-dependent access to the pores of large objects such as plasmids. This chromatography medium, containing a matrix material functionalized with thiophilic and aromatic ligands and featuring an average flow pore size of 0.1–2.0 μm, enables higher flow rates, leading to reduced processing times. Furthermore, the medium increases adsorption capacity compared to standard resins (e.g., by more than five times). This matrix material provides high separation capability for large samples / objects such as plasmids.

[0013] This matrix material can be a convective material, and the flow through such a material is convective rather than diffusive. A convective matrix material includes any matrix in which perfusion of the matrix is ​​brought about by applying a pressure difference between the inflow and outflow of the matrix, and the transport of material into or out of the matrix is ​​achieved substantially convectively, and occurs at high flow rates and very rapidly.

[0014] Examples of convection matrix materials include porous adsorption membranes and monolithic materials. Adsorption membranes may be polymer membranes, such as those containing polyethersulfone. Another example of adsorption membranes is polymer nanofiber membranes, such as cellulose, cellulose acetate, and cellulose fibers, which are optionally treated or derivatized for use as adsorbents. The polymers used for polymer membranes or polymer nanofiber membranes may be natural or synthetic polymers, including derivatized polymers such as hydroxylated polymers. Adsorption membranes may instead be monolithic materials or conventional membranes made by emulsification. Another alternative is 3D printed materials.

[0015] In the matrix material of the present invention, there is no need for the sample to diffuse into the pore. With the matrix material of the present invention, the retention time can be measured in seconds, thus enabling rapid loading of low-titer samples. The sample is attracted to and binds to the thiophilic aromatic ligand in the functionalized matrix material. The use of thiophilic aromatic ligands enables the purification of plasmids, such as superhelical plasmid DNA, for use in gene therapy, DNA vaccines, and research applications.

[0016] The matrix material may be a fibrous substance. The fibrous substance may be a nonwoven polymer material. The matrix material may contain or consist of cellulose and / or other polymers other than cellulose. A fibrous cellulose matrix material may be produced by electrospinning of cellulose acetate through multiple reaction steps.

[0017] Thiophilic aromatic ligands can be amine-derived aromatic ligands. Amine-derived aromatic ligands may contain an aromatic ring having at least one nitrogen atom as a ring element.

[0018] The ligand may include a thioether moiety bonded to an aromatic ring, the aromatic ring preferably containing at least one nitrogen atom as a ring element.

[0019] Thiophilic aromatic ligands may include any group selected from pyridyl, phenyl, benzyl, toluyl, phenethyl, naphthyl, imidazolyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridadinyl, piperidinyl, morpholinyl, piperazinyl, indolyl, quinolinyl, and purinyl groups, and any substituted versions thereof.

[0020] Such substituted groups can be, for example, substituted phenyl groups having one or more electron-withdrawing groups. One example is nitrophenyl. The electron-withdrawing groups may be nitrogen-containing groups such as -CF3, -NO2, or -CN.

[0021] The thiophilic aromatic ligand or a part of the ligand has the formula:

Chemical formula

[0022] The electron-withdrawing group can be -CF3, or a nitrogen-containing group such as -NO2 or -CN.

[0023] In an embodiment where A is N, B may not exist.

[0024] Instead, the matrix material M has the formula:

Chemical formula

[0025] The electron-withdrawing group may be a nitrogen-containing group such as -CF3, -NO2, or -CN.

[0026] Preferably, at least one A is N.

[0027] In embodiments where A is N, B may not exist.

[0028] The thiophyllic aromatic ligand can be selected from 2-mercaptopyridine, 4-mercaptopyridine, and 2-mercaptoethylpyridine.

[0029] The linker may include a portion resulting from a reaction that binds the ligand to the matrix material. Well-known coupling chemistry is known to those skilled in the art and may include, for example, the reaction with divinyl sulfone (DVS) as described herein, or the reaction using epichlorohydrin (ECH) or allyl glycidyl ether (AGE). Thus, the linker may include a portion obtained by the reaction of an optionally activated or derivatized matrix material with divinyl sulfone, epichlorohydrin, or allyl glycidyl ether, and may be bound to the matrix material via thereafter. For example, the linker may include a vinyl sulfone portion.

[0030] Optionally, the linker Z may also include a spacer or extender portion, as described elsewhere in this specification.

[0031] The ligand can optionally bind to the matrix material via oxygen.

[0032] Ligands can bind to the matrix material via extender groups selected from polysaccharide and polymer structures. The extenders are separated from the substrate through polymerization (undetermined length).

[0033] According to a second embodiment, a chromatography unit containing the above-mentioned chromatography medium is provided.

[0034] The chromatography unit is suitable for use in chromatography. Generally, the chromatography unit contains one or more solutions at its inlet and outlet. In the chromatography unit, the chromatography medium is arranged so that the solution passes through the medium. A pump coupled to the chromatography unit can create the flow rate of the solution passing through the medium in the unit.

[0035] The chromatography unit may be a disinfected / cleaned unit, preferably in accordance with Good Manufacturing Practice (GMP), and involves a reduction in bioburden and endotoxin levels by exposure to 0.5 M NaOH and 40% isopropyl alcohol for up to 4 hours.

[0036] In a further aspect of the present invention, the chromatography medium or the chromatography unit may be used for the separation of different isoforms of plasmid DNA present in solution, such as for the purification of superhelical plasmid DNA.

[0037] A further embodiment provides a method for separating a sample in a solution, comprising the steps of: obtaining a solution containing the sample; adding the solution to a chromatographic medium described herein in a binding buffer having a conductivity of 240 to 270 mS / cm; eluting the sample from the chromatographic medium by contacting the chromatographic medium with an elution buffer having a conductivity of 0 to 240 mS / cm; and collecting the resulting eluate containing the sample.

[0038] The aforementioned solution may contain different plasmid DNA isoforms, such as superhelical plasmid DNA (pDNA), ring-open form of plasmid DNA, and nickeld form, and the sample of interest is generally a superhelical plasmid DNA isoform.

[0039] The binding buffer enables or facilitates the binding of the ligand on the matrix material of the medium to the sample. The selected conductivity is 240-270 mS / cm, depending on the characteristics of the sample. Elution of the sample from the medium is performed using an elution buffer with a lower conductivity than the binding buffer, 0-240 mS / cm. Elution may be a stepwise gradient elution, in which the chromatographic medium is brought into contact with an elution buffer whose conductivity decreases.

[0040] The conductivity of the binding buffer can be optimized so that unwanted plasmid DNA isoforms do not bind to the unit and are collected in the flow-through fraction along with RNA and other impurities, while the target sample, superhelical plasmid DNA, is retained on the chromatography medium by binding to the ligand and subsequently eluted with the elution buffer. Such a single-step elution method may be advantageous in terms of yield.

[0041] In another embodiment, a method for separating different isoforms of plasmid DNA in a solution is provided, comprising the steps of: obtaining a solution containing plasmid DNA isoforms in a solution; adding the solution to a chromatographic medium in a binding buffer having a conductivity of 240 to 270 mS / cm; contacting the chromatographic medium with a first elution buffer having a conductivity of 200 to 240 mS / cm; collecting the eluate containing the resulting first plasmid DNA isoform; and subsequently contacting the chromatographic medium with a second elution buffer having a conductivity of 0 to 200 mS / cm, which is lower than the conductivity of the first elution buffer; and collecting the eluate containing the resulting second plasmid DNA isoform.

[0042] The solution may contain different plasmid DNA isoforms, such as superhelical plasmid DNA, ring-open and nickeld form plasmid DNA, as well as impurities such as RNA. The sample of interest is generally a superhelical plasmid DNA isoform. The first elution contains the first plasmid DNA isoform, which contains the purified superhelical plasmid DNA of interest. The second elution contains unwanted plasmid DNA isoforms, such as ring-open and / or nickeld form plasmid DNA, along with RNA and other impurities. This two-step method can therefore be used to separate plasmid DNA isoforms, separating the product of interest in the first step and everything else in the second step. Such a process can be used on an industrial scale.

[0043] The binding buffer used should facilitate the binding of the sample to the ligand in the matrix material of the medium. The conductivity of the binding buffer can be greater than 200 mS / cm, such as 240-270 mS / cm, and the conductivity to be selected depends on the characteristics of the sample being studied. Examples of binding buffers include: sodium sulfate, ammonium sulfate (base buffer), TE buffer: 10-20 mM Tris / 1 mM EDTA / 2.0-2.4 M ammonium sulfate, 2.4-2.0 M ammonium sulfate / 0.3 M NaCl / 100 mM Tris / 10 mM EDTA buffer.

[0044] Elution of a sample from a chromatography medium may involve stepwise or gradient elution, in which the chromatography medium comes into contact with an elution buffer whose conductivity decreases.

[0045] The elution buffer used should elute the sample from the medium. The conductivity of the elution buffer can range from 0 to 240 mS / cm, and the choice of elution buffer depends on sample recovery and sample stability in solution. Some samples may require specific salts to maintain stability. Examples of elution buffers include those containing 10-20 mM Tris / 1 mM EDTA / 1.7 M ammonium sulfate. Buffers with low or no ammonium sulfate content (including water alone) can also remove bound components.

[0046] When the first and second elution buffers are used to elute plasmid DNA from the chromatography medium, the chromatography medium first comes into contact with the first elution buffer, which has a conductivity of 200–240 mS / cm. Such a first elution buffer may be, for example, 1.7 M ammonium sulfate / 0.3 M NaCl / 100 mM Tris / 10 mM EDTA. After recovering the resulting eluate containing the first plasmid DNA isoform, the chromatography medium comes into contact with the second elution buffer, which has a lower conductivity than the first elution buffer, with a conductivity of 0–200 mS / cm, and the resulting eluate containing the second plasmid DNA isoform is recovered.

[0047] By this method, for example, superhelical DNA can be purified and concentrated to over 90% of its original starting solution.

[0048] The method may further include a step of adding the eluate to the multimodal chromatography resin after the elution step.

[0049] The method described above involves washing the chromatographic medium after eluate recovery, and may include washing with the addition of a 0.1–2 M NaOH solution to the chromatographic medium. Alternatively, washing the chromatographic medium after eluate recovery may include adding a 0.1–0.5 M HCl or phosphoric acid solution to the chromatographic medium. Washing ensures that the chromatographic medium can be used for multiple chromatography operations. [Brief explanation of the drawing]

[0050] [Figure 1a-d] Figures 1a-d illustrate different reaction schemes for the preparation of matrix materials for chromatography media. Matrix materials containing fibrous nonwoven polymer material with vinyl sulfone reactive groups were functionalized with thiophilic aromatic ligands. Figure 1a: Addition of 2-((2-(pyridine-2-yl)ethyl)thio)ethane-1-amine dihydrochloride to the activated crosslink substrate; Figure 1b: Reduction with dithiothreitol following the addition of cystamine dihydrochloride to the substrate, followed by the addition of 2-vinylpyridine; Figure 1c: Addition of 2,2'-(ethylenedioxy)diethaneethiol followed by 2-vinylpyridine to the activated crosslink substrate; Figure 1d: Addition of 2-vinylpyridine following the addition of di(2-mercaptoethyl) ether to the activated crosslink substrate. [Figure 2] Figure 2 is a bar graph showing the dynamic binding capacity for each route described in Figures 1a to 1d. Routes 1a to 1d yield similar dynamic binding capacities (determined by the QB10 method (dynamic binding capacity at 10% pass-through volume (QB10))), but routes 1c and 1d showed higher recovery rates (100%) compared to routes 1a and 1b. [Figure 3] Figure 3 shows variants of thiophilic aromatic ligands used to functionalize the matrix material. [Figure 4] Figure 4 shows a chromatogram demonstrating the separation of plasmid DNA from RNA in solution using a matrix material functionalized with a thiophyllic aromatic ligand at a ligand concentration of 900 μmol / g, and a flow rate of 5 MV / min (membrane volume per minute). [Figure 5] Figure 5 shows the size range for plasmid purification and recovery using matrix materials with three different ligand densities and plasmids of various sizes. [Figure 6]Figure 6 shows agarose gels loaded with samples from the washing (Figure 6, A3 W), elution (Figure 6, A3 E), and stripping (Figure 6, A3 S) stages of plasmid DNA purification using a matrix material functionalized with a thiophilic aromatic ligand at a ligand concentration of 900 μmol / g1. Samples filtered by tangental flow filtration (TFF, cutoff value 300 kDa) (Figure 6, Post TFF1) and samples added to a chromatography column (Figure 6, PS Load) were also loaded onto the gels. [Figure 7a-b] Figures 7a-b show bar graphs illustrating the percentage of impurities in each stage: washing, elution, and strip. [Modes for carrying out the invention]

[0051] Since plasmids are the raw materials for the production of mRNA and viral vectors, there is a demand for highly purified plasmid DNA. Superhelical plasmid DNA can be expressed in cells of any origin. Most commonly, microorganisms such as bacteria, including E. coli, are used to culture plasmids, but the use of host cells is not restricted and can be prokaryotic or eukaryotic. Host cells harboring plasmids can be cultured using several methods well known in the art, such as incubators, bioreactors, and fermenters. Plasmids can be virtually any size, for example, ranging from 1 kb to 500 kb. Plasmids can be high or low copy numbers and can carry any gene, either genomic or synthetic, encoding a target protein or peptide from any source. The cultivation of host cells, as well as the use of plasmids for gene therapy, is well known in the art.

[0052] After culturing host cells containing plasmids, the cells are recovered, for example, by centrifugal filtration. The cells can be stored, for example, in a freezer, or processed immediately. Following a cell breakdown process, such as by lysis, for example, alkaline lysation, a solution is obtained containing superhelical plasmids as well as ring-open and nickeld forms of plasmids, RNA, host proteins, endotoxins, and any impurities such as chromosomal DNA.

[0053] Purification of superhelical plasmid DNA requires the complete removal of other cellular components such as host proteins, endotoxins, chromosomal DNA, RNA, and open-ring and nickeld forms of plasmid DNA. Isolating different plasmid DNA isoforms is a major challenge in plasmid DNA purification.

[0054] The term "superhelical plasmid DNA" refers to plasmid DNA in a circular form that has a helical topology. DNA can be positively or negatively superhelical. For example, superhelical plasmid DNA may have terminal loops and form a right-handed helical structure with two origins.

[0055] The term "open-ring plasmid DNA" refers to plasmid DNA in which one strand of the DNA double helix is ​​broken, releasing at least a portion of the strain on the double helix that causes the plasmid to form a superhelical structure. As a result, the open-ring pDNA forms a non-helical closed ring, but maintains the open-ring topology.

[0056] Chromatographic media for separating samples in solution, such as plasmid DNA or RNA, genomic DNA, and any polynucleotide, including seeded nucleotide sequences, are described below. The chromatographic media of the present invention can facilitate the production of highly purified superhelical plasmid DNA for use in gene therapy and DNA vaccine applications.

[0057] The matrix material of the chromatography medium used is a substance having an average flowpore size of 0.1 to 2.0 μm, and the matrix material is functionalized with thiophilic aromatic ligands to a ligand concentration of up to 1500 μmol / g of matrix material.

[0058] The matrix material can be a convective matrix material. Such a material may be, for example, an adsorbent film through which the flow through such material is convective rather than diffusive. The convective matrix material includes any matrix through which perfusion of the matrix is ​​brought about by applying a pressure difference between the inflow and outflow of the matrix, and the transport of material into or out of the matrix is ​​achieved substantially convectively, and occurs at high flow rates and very rapidly.

[0059] Examples of convection matrix materials include porous adsorption membranes and monolithic materials. Adsorption membranes may be, for example, polyethersulfone membranes, polymer nanofiber membranes, or polymer membranes treated for use as adsorption membranes, such as cellulose, cellulose acetate, and cellulose fibers. Treatment may include one or more ligand cross-linking, derivatization, and coupling. Matrix materials may be nonwoven materials containing fibers such as cellulose. Such fibrous substrates are obtained based on electrospinning of polymer fibers or cellulose fibers and may have cross-sectional diameters of 10-1000 nm, such as 20-800 nm, 200-400 nm, or 300-400 nm. Such matrix materials can be seen in the HiTrap Fibro unit Cytiva, Sweden.

[0060] The polymers used in polymer films or polymer nanofiber films may be natural or synthetic polymers, including derivatized polymers. For example, hydroxylated polymers may be used to provide hydroxylated polymer films. Hydroxylated polymers may include sugar polymers. In addition to the aforementioned cellulose and polyethersulfone, polymers useful for adsorption films include polymers based on polytetrafluoroethylene (PTEE), polypropylene, polyamide, or polyacrylamide.

[0061] The adsorption membrane can instead be a conventional membrane made from monolithic material or by emulsification. Another alternative is 3D printed material.

[0062] The average flow pore (MFP) size is an indicator of a substance's flow characteristics and is measured by capillary flow porometry, which involves displacing a wet liquid with known surface tension from the sample pore by flowing a gas while increasing pressure. A higher MFP results in a greater liquid flow through the substance at any given pressure. The average flow pore size is calculated when 50% of the flow has passed through the sample. The average flow pore size therefore coincides with the pore size calculated at the pressure where the wet curve and half-dry curve intersect.

[0063] In an alternative definition, the average flow pore size of the matrix material of the present invention may be seen as the effective pore size, which is defined as the size of the largest sphere that can pass through the pore.

[0064] The average flowpore size of the matrix material may be 0.1-2.0 μm, 0.1-1.8 μm, 0.1-1.6 μm, 0.1-1.4 μm, 0.1-1.2 μm, 0.1-1.0 μm, 0.1-0.8 μm, 0.1-0.6 μm, 0.1-0.4 μm, 0.1-0.2 μm, 0.2-2.0 μm, 0.4-2.0 μm, 0.6-2.0 μm, 0.8-2.0 μm, 1.0-2.0 μm, 1.2-2.0 μm, 1.4-2.0 μm, 1.6-2.0 μm, 1.8-2.0 μm, or 0.5-1.5 μm.

[0065] The matrix material can be functionalized with ligands to ligand concentrations of 10–1500 μmol / g of matrix material. The matrix material can also be functionalized with thiophilic aromatic ligands to ligand concentrations of up to 1500 μmol / g of matrix material. Under clearly defined conditions, these ligands can isolate superhelical plasmid DNA from its ring-open isoforms.

[0066] Thiophilic aromatic ligands (see Figure 3) may include aryl groups selected from pyridyl, phenyl, benzyl, toluyl, phenethyl, naphthyl, imidazolyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridadinyl, piperidinyl, morpholinyl, piperazinyl, indolyl, quinolinyl, and purinyl groups, and any substituted versions of these groups. Such substituted groups may be, for example, substituted phenyl groups having one or more electron-withdrawing groups. One example is nitrophenyl. As an example, the thiophilic aromatic ligand may be 2-mercaptoethylpyridine.

[0067] Thiophilic aromatic ligands can be bound to a matrix material via extender groups selected from polysaccharide and polymer structures. These extender groups may be, for example, dextran, acrylamide, or polyglycerol. When dextran is used as the extender, it may have a molecular weight ranging from 5,000 to 2,000,000 daltons. The extender groups are separated from the substrate through polymerization (undetermined length).

[0068] In some embodiments, extender groups are not used, and ligands are directly immobilized / bonded to the matrix material using linkers, such as crosslinkers. Linkers can be formed by reagents used to activate the matrix material and enable ligand coupling. Established methods include, for example, the introduction of epoxy groups via the use of epochlorohydrin or 1,4-butanediol diglycidyl ether. Other examples include the introduction of double bonds in the matrix material using reagents such as divinyl sulfone. Crosslinkers composed of these reagents can be further extended by novel functionalizations that enable ligand coupling.

[0069] Spacers, such as 2-12 carbon alkyl, linear or branched, or 2-12 carbon ethers, can be used between thiophyllic aromatic ligands and crosslinkers such as vinyl sulfones. Linkers and crosslinkers can also react with extender groups and link to ligands or spacers. The spacer is the length of carbon between the linker and ligand and is used to improve the base stability of the linker-ligand bond.

[0070] Figures 1a-1d illustrate different exemplary reaction schemes for functionalizing fibrous nonwoven polymer materials containing fibers having vinyl sulfone reactive groups. The resulting matrix material contains vinyl sulfone reactive groups that are functionalized with thiophilic aromatic ligands.

[0071] Figure 1a illustrates that 2-((2-(pyridine-2-yl)ethyl)thio)ethane-1-amine dihydrochloride (0.14 M) reacts with a fibrous nonwoven polymer material containing vinyl sulfone reactive groups at pH 9.6, enabling the formation of a functionalized matrix material having thiophilic aromatic ligands.

[0072] Figure 1b illustrates an exemplary reaction scheme in which a fibrous nonwoven polymer material containing divinyl sulfone-crosslinked and activated fibers is functionalized with a pH 9 aqueous solution of cystamine dihydrochloride (0.22 M), followed by reduction of the disulfide bonds with a dithiothreitol aqueous solution (0.66 M) to obtain a cysteamine derivatized substrate. The exposed sulfhydryls on the thus obtained substrate further react with 2-vinylpyridine (30 mM) to form a functionalized matrix material having thiophilic aromatic ligands. Figure 1c illustrates an exemplary reaction scheme in which a fibrous nonwoven polymer material containing divinyl sulfone-crosslinked and activated fibers is functionalized with a 2,2'-(ethylenedioxy)diethaneethiol aqueous solution (0.15 M), followed by reaction of terminal sulfhydryls with 2-vinylpyridine (30 mM) to form a functionalized matrix material having thiophilic aromatic ligands.

[0073] Figure 1d illustrates an exemplary reaction scheme in which a fibrous nonwoven polymer material containing divinyl sulfone-crosslinked and activated fibers is functionalized with an aqueous solution of di(2-mercaptoethyl) ether (0.15 M), followed by the reaction of terminal sulfhydryls with 2-vinylpyridine (30 mM) to form a functionalized matrix material having thiophyllic aromatic ligands.

[0074] A solution containing a sample, such as plasmid DNA, is added to a functionalized matrix material of the chromatography medium in a binding buffer.

[0075] The sample is attracted to and bound to the thiophilic aromatic ligand of the functionalized matrix material.

[0076] As is well known in this industry, washing may be performed after adsorption and before elution to remove retained unwanted substances.

[0077] Once the sample has adsorbed to the ligand, a washing and elution process may follow. Elution may be carried out by contacting a chromatography medium with an elution buffer, and the resulting eluate containing the sample is collected. Elution may also be carried out using a conductivity gradient of aqueous solutions with decreasing salt concentrations (stepwise or gradient elution).

[0078] The dissolution process can be carried out as a dynamic or batch procedure. Dissolution is conveniently carried out according to widely known principles, such as a decreasing conductivity gradient.

[0079] Generally, elution of plasmid DNA from chromatography media containing matrix materials that are convection-based fibrous substrates, such as fibrous nonwoven polymer materials, can be reduced to flow rates of several seconds, i.e., 60 MV / min, and can be increased to several minutes (up to 6 minutes), i.e., 0.2 MV / min, if necessary. The optimal retention time for such fibrous matrix materials is 5–20 MV / min. For resins, the typical retention time is 1–8 minutes, and the optimal retention time is often 4 minutes.

[0080] After eluate recovery, the chromatography medium can be washed with 0.5 M NaOH, up to 2 M NaOH, before reuse. Acidic solutions are also an option for in-situ washing to reduce any accumulated contaminants. Due to the short retention time (6–12 seconds), only a short washing stop of one minute per cycle is required.

[0081] Standard resins used in chromatography have limited and diffusion-dependent access to the pore for large objects such as plasmids. This chromatography medium, containing a matrix material of nonwoven fibrous material functionalized with thiophilic aromatic ligands, allows for higher flow rates, leading to reduced processing times. Furthermore, the medium increases adsorption capacity compared to standard resins (more than 5 times). This matrix material provides high separation capability for large samples / objects such as plasmids. There is no need for diffusion to the pore, and low-titer samples are quickly loaded with retention times of a few seconds.

[0082] Therefore, the above method and chromatographic materials can be used, for example, for the purification of plasmids for use in gene therapy, DNA vaccines, and laboratory research related to gene therapy. [Examples]

[0083] The chromatographic material and its use described herein are provided as illustrative examples only and should not be construed as limiting the scope of the invention as defined by the appended claims.

[0084] Plasmid preparation Plasmids were prepared using a conventional plasmid preparation protocol, which involves purification with Sepharose 6FF resin following the use of alkaline lysate. A pellet of E. coli derived from DH10b cells was first lysed in buffer (10 ml / g pellet, 50 mM Tris, 50 mM glucose, 10 mM EDTA, pH 7.5), and the cells were then lysed for up to three minutes by the slow addition of 1 volume of lysis buffer (0.2 M NaOH, 1% SDS) with gentle agitation. After three minutes, the alkaline lysate was neutralized by the addition of 1 volume of neutralization buffer (3 M KAc, 2.07 M HAc). The neutralized flocculated lysate was then brought to the surface by the addition of ammonium bicarbonate (8 g / L) and incubated overnight for degassing and the formation of a stable flocculated cake. The neutralized lysate was then purified by deep filtration and subsequently concentrated 15-fold by tangental flow filtration (TFF) with hollow fiber. Finally, plasmids were purified from RNA and protein residues by group isolation using Sepharose 6FF resin.

[0085] Preparation of matrix material A solution of cellulose acetate (CA) with a relative molecular weight of 29,000 g / mol was dissolved in a common solvent prior to electrospinning to produce fibers with diameters in the range of 300-600 nm. Optimal conditions for nanofiber production can be found, for example, in O. Hardick, et al, J. Mater. Sci. 46(2011)3890. Sheets of approximately 20 g / m2 of the material were layered and subjected to a combination of heating and pressurizing treatments.

[0086] Sections (100 × 155 mm²) of the formed CA material were placed between polypropylene gauze and loaded into a flow reactor. The sections were washed three times with deionized water for 20 minutes each, and then left for 16 hours after the final rinse.

[0087] The CA material was saponified and expanded in situ to form cellulose bonded with branched polyglycerol. Wash water was drained from the flow reactor. Potassium hydroxide (156 g) was dissolved in DI H2O (3.5 L) in a 15 L container. Glycidol (950 mL) was added to the basic solution and stirred for 4 minutes. The reaction mixture was added to the flow reactor containing the CA sheet. The recirculation pump was powered on and the mixture was recirculated for 6 hours. After 6 hours of recirculation, the pump was stopped and the flow reactor was drained. The material was washed four times with 6 L of distilled water, with each wash lasting 15 minutes.

[0088] Subsequently, divinyl sulfone derivatization of polyglycerol-modified cellulose was carried out. In an 8 L beaker / container, deionized water (2.963 L) was added followed by Na2CO3 (222 g). The contents were stirred until the base was completely dissolved. Acetonitrile (HPLC for gradient analysis; >99.9%; 0.886 L) was added, and the mixture was stirred for 2 minutes before being loaded into the flow reactor. The recirculation pump was powered on, and the mixture was recirculated for 2 minutes (>99%, 0.684 L). The reaction mixture was recirculated at room temperature for 6 hours. The reaction mixture was drained, and the substance was washed four times for 20 minutes each with recirculation of 1:1 water / acetone (6 L) at 24-26°C. The substance was then rinsed twice for 15 minutes each with recirculation of distilled water (6 L). The substance was then used.

[0089] Functionalization of matrix materials using thiophyllic aromatic ligands, Route 1a (Figure 1a) A sheet of Fibro-VS (a fibrous nonwoven polymer material containing fibers with vinyl sulfone reactive groups) was placed on a polypropylene tray and covered with 25 mL of a solution of 2-((2-(pyridine-2-yl)ethyl)thio)ethane-1-amine dihydrochloride (0.884 g) in 25 mL of deionized water adjusted to pH 9.6. The tray was then placed in an orbital shaker at low speed at room temperature for 16 hours. After this time, the solution was removed by decantation, and the sheet was washed three times by covering it with 50 mL of deionized water, placing it in the orbital shaker for 5 minutes, and then removing the washing solution by decantation. After the final wash, the sheet was removed, and any excess water was wiped off with a blue disposable roll towel.

[0090] Blocking of vinyl sulfone reaction groups A thioglycerol phosphate buffer (2.5% thioglycerol, pH 8.3) was prepared by dissolving sodium phosphate dibasic dodecahydrate (3.58 g) and EDTA disodium dihydrate (37 mg) in water (95 mL) with stirring. Thioglycerol (2.5 mL) was added, and the resulting solution was basicized to pH 8.3 with 49% NaOH solution and diluted to 100 mL. Sheets of functionalized material were placed in a tray and immersed in 25 mL of buffered thioglycerol solution. The system was gently shaken in an orbital shaker for 16 hours (150 rpm) before being washed three times in water (50 mL) for 15 minutes each time.

[0091] Functionalization of matrix materials using thiophyllic aromatic ligands, Route 1b (Figure 1b) A sheet of Fibro-VS was placed in a polypropylene tray and treated with a solution of cystamine dihydrochloride (1.25 g) in deionized water (25 mL) adjusted to pH 9.0. The tray was gently shaken in an orbital shaker set to 250 rpm at room temperature for 18 hours. The solution was removed by decantation, and the sheet was rinsed four times with deionized water for 20 minutes each time.

[0092] Reduction of Fibro-cystamine to Fibro-cysteamine using DTT The Fibro-cystamine sheets were placed in a polypropylene tray and treated with a reducing agent solution prepared from dithiothreitol (2.56 g), sodium chloride (781 mg), EDTA (33 mg), NaHCO3 (749 mg), and Na2CO3 (94 mg) in deionized water (25 mL). The tray was sealed and shaken gently in an orbital shaker for 4 hours. The solution was removed, and the sheets of material were rinsed four times with deionized water for 20 minutes each.

[0093] Addition of 2-vinylpyridine to Fibro-cysteamine The fibro-cysteamine sheet was placed in a polypropylene tray and then treated with the addition of 2-vinylpyridine (80 μL) in deionized water (25 mL) to obtain a 30 mM solution. The sheet was gently shaken at room temperature for 16 hours. The reaction solution was removed by decantation, and the sheet was rinsed twice with deionized water for 20 minutes each, then three times with a 1:1 acetone / water mixture for 20 minutes each, and then three times with deionized water for 20 minutes each.

[0094] Functionalization of matrix materials using thiophyllic aromatic ligands, Route 1c (Figure 1c) 2,2'-(ethylenedioxy)diethaneethiol (EDDE; 0.625 mL) was added to 25 mL of DI water in a 100 mL Duran bottle with stirring to obtain a 2.5% v / v solution. Tetrabutylammonium fluoride (92 mg) was added to adjust the pH to 9.6. Sheets of Fibro VS were dried on an adsorbent and placed in a sealable polypropylene tray. The dithiol solution was added to the sheets, the tray was sealed and placed in an orbital shaker in a fume hood in a sealed container for 16 hours. After this time, the solution was removed by decantation in bleach and replaced with 50 mL of deionized water, and the tray was gently shaken in an orbital shaker for 20 minutes. This rinsing step was repeated four times before removing the final rinse solution.

[0095] Addition of 2-vinylpyridine to Fibro-EDDE K2HPO4 (16.73 g) and KH2PO4 (0.523 g) were dissolved in 900 mL of deionized water, and then deionized water QSAD was added until the total volume reached 1000 mL to prepare a 0.1 M phosphate buffer solution at pH 8. The resulting Fibro-Linker-SH sheet was placed in a polypropylene tray and rinsed twice with the freshly prepared pH 8 phosphate buffer solution before drying. The sheet was then treated in a fume hood to prepare a 30 mL solution by adding 25 mL of pH 8 phosphate buffer solution followed by 80 μL of 2-vinylpyridine. The polypropylene tray was covered, placed in a large zip bag, and then sealed. The system was shaken overnight at room temperature (20-23°C). The reaction solution was decanted in bleach, and the sheet was rinsed with DI water (6 × 20 mins) before drying and testing.

[0096] Functionalization of matrix materials using thiophyllic aromatic ligands, Route 1d (Figure 1d) Bis(2-mercaptoethyl) ether (BMEE; 0.625 mL) was added to 25 mL of deionized water in a 100 mL Duran bottle with stirring to obtain a 2.5% v / v solution. Tetrabutylammonium fluoride (92 mg) was added to adjust the pH to 9.6. Sheets of Fibro VS were dried on an adsorbent and placed in a sealable tray. A dithiol solution was added to the sheets, and the sealed tray was placed in a sealed container and gently shaken in an orbital shaker in a fume hood for 16 hours. After this time, the solution was decanted in bleach and replaced with 50 mL of deionized water, and the tray was returned to the orbital shaker for 20 minutes. This rinsing step was repeated four times before removing the final rinse solution.

[0097] Addition of 2-vinylpyridine to Fibro-BMEE K2HPO4 (16.73 g) and KH2PO4 (0.523 g) were dissolved in 900 mL of deionized water, and then deionized water QSAD was added until the total volume reached 1000 mL to prepare a 0.1 M phosphate buffer solution at pH 8. The resulting Fibro-Linker-SH sheet was placed in a polypropylene tray and rinsed twice with the freshly prepared pH 8 phosphate buffer solution before drying. The sheet was then treated in a fume hood to prepare a 30 mL solution by adding 25 mL of pH 8 phosphate buffer solution followed by 80 μL of 2-vinylpyridine. The polypropylene tray was covered, placed in a large zip bag, and then sealed. The system was shaken overnight at room temperature (20-23°C). The reaction solution was decanted in bleach, and the sheet was rinsed with DI water (6 × 20 mins) before drying and testing.

[0098] Thiophilic chromatography The functionalized matrix materials described above were used as thiophilic interaction chromatography (TIC) media in the following experiments to isolate superhelical plasmid DNA from other components, RNA, in solution.

[0099] A membrane volume of 0.4 mL was used, and the system was operated at 5 MV / min, which is equivalent to a flow rate of 2 ml / min or a retention time of 12 seconds.

[0100] The binding buffer used consisted of 2.0 M ammonium sulfate, 100 mM Tris, and 10 mM EDTA at pH 7.5, containing the impure plasmid sample.

[0101] The washing buffer used consisted of 2.0 M ammonium sulfate, 100 mM Tris, and 10 mM EDTA at pH 7.5, and passed through the unit at 10 MV.

[0102] The elution buffer used consisted of 1.7 M ammonium sulfate, 100 mM Tris, 10 mM EDTA, and 300 mM NaCl, and passed through the unit at 20 MV.

[0103] The strip buffer used to remove (strip) any remaining bound components from the column consisted of 100 mM Tris and 10 mM EDTA at pH 7.5, with the units passing through at 15 MV.

[0104] The CiP (Cleaning in Place) solution was 0.5 M NaOH, and it was left to stand in contact with the unit for 1 hour.

[0105] From the resulting chromatogram (explained in Figure 4), the dynamic binding capacity (DBC) result can be calculated. The chromatogram demonstrates the separation of plasmid DNA from RNA in solution.

[0106] Functionalization of the above matrix materials using different ligand concentrations of 0.28, 0.14, and 0.07 mmol / mL at the start of the reaction yielded ligand densities of approximately 923, 622, and 331 μmol / g, respectively (see Table 1).

[0107] [Table 1]

[0108] These different matrix materials with varying ligand densities were then tested with samples containing plasmids of various sizes (smallest at 4.3 kb, medium at 6.1 kb and 11.6 kb, and largest at 17.0 kb) in different chromatographic trials. Figure 5 shows that, in the series, the lowest ligand density (331 μmol / g) showed the lowest recovery rate for small (64%) and medium (88%) size plasmids, but the best recovery rate (50%) for the largest plasmid. The highest ligand density showed the best recovery rate for all small and medium size plasmids, indicating that ligand density can be adjusted to match the size of the target molecule.

[0109] The quality of the purified plasmids was then examined using the eluate from gel electrophoresis. This showed that the eluted fraction was rich in superhelical DNA (Figure 6). Figure 6 shows agarose gels loaded with samples from the washing (Figure 6, A3 W), elution (Figure 6, A3 E), and stripping stages (Figure 6, A3 S). Samples filtered by tangental flow filtration (TFF, cutoff value 300 kDa) (Figure 6, Post TFF1) and samples added to a chromatography column (Figure 6, PS Load) were also loaded onto the gels.

[0110] As summarized in Table 2, the impurity profile of the eluted fraction decreased compared to the initial feed (PS load) (see Figures 7a-7b). Genomic DNA decreased to 1 / 10, while RNA contamination decreased to 0.1 μg / mL. The superhelical DNA fraction increased from 85% of the load to 95% of the eluate (see Figure 7a), and the open-ring DNA fraction decreased to below the limit of detection (LOD) (see Figure 7b), confirming the selectivity of the matrix material for superhelical plasmids.

[0111] [Table 2]

[0112] The ligand stability from all routes shown in Figures 1a-d was investigated by exposing the functionalized chromatographic materials to both basic and acidic buffers for periods ranging from less than 20 minutes to 4 hours. As shown in Table 3, dynamic binding capacity (DBC at 10% breakthrough) was tested using herring sperm DNA before and after exposure to acidic / basic buffers.

[0113] [Table 3]

[0114] No drops were observed during execution, demonstrating the chemical stability of all chemical routes. As shown in Figure 2, all ligands produced from the described routes demonstrated high recovery, base stability, and low pressure at 8 MV / min with herring sperm DNA at a DBC of 7–8 mg / ml.

[0115] Consideration The chemical routes described above and in Figures 1-1d produce matrix materials for chromatographic media that can purify and separate polynucleotide species through thiophilic interaction chromatography. Depending on the length of the spacers used in the chemical routes, products are produced that can withstand repeated and prolonged exposure to harsh conditions, including but not limited to washing solutions of 0.1-2.0 M NaOH or 0.1-0.5 M HCl or phosphoric acid.

[0116] This matrix material can purify different plasmid isoforms from impurities such as RNA.

[0117] By combining high DBC and short retention time, productivity can be achieved that is 10 times higher than what can be obtained with comparable resins, as measured by grams of plasmid per unit time per unit volume.

Claims

1. A chromatography medium for separating a sample in solution, comprising a matrix material having an average flowpore size of 0.1 to 2.0 μm, wherein the matrix material is functionalized with a thiophilic aromatic ligand to a ligand concentration of up to 1500 μmol / g of matrix material.

2. The chromatography medium according to claim 1, wherein the thiophyllic aromatic ligand comprises an aryl group selected from pyridyl, phenyl, benzyl, toluyl, phenethyl, naphthyl, imidazolyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridadinyl, piperidinyl, morpholinyl, piperazinyl, indolyl, quinolinyl, and purinyl groups, and any substituted versions thereof.

3. Matrix material M is given by formula: 【Chemistry 1】 (In the formula, independently) M represents the matrix material. Z optionally represents a linker that includes a spacer portion. X is NH, O, or S in each existence. Y is S or CH 2 is or does not exist, A is either C or N, B is H, a C1-C6 alkyl group, for example, a C1-C3 alkyl group, or an electron-withdrawing group, or is absent. n is an integer from 0 to 12, and (m is 0 or an integer between 1 and 12) The chromatography medium according to claim 1, which is functionalized with an aromatic ligand accordingly.

4. The ligand or part of the ligand has the formula: 【Chemistry 2】 (In the formula, independently, X is NH, O, or S in each existence. Y is S or CH 2 And, A is either C or N, B is either H or an electron-withdrawing group, or it is not present. n is an integer from 0 to 12. m is an integer from 0 to 12, and p is 0 or an integer between 1 and 12. The chromatography medium according to claim 1 or 2, as described in [the relevant section].

5. The chromatography medium according to any one of claims 1 to 3, wherein the thiophyllic aromatic ligand is selected from 2-mercaptopyridine, 4-mercaptopyridine, vinylpyridine, 2-mercaptoethanol, and 2-mercapto-ethylpyridine.

6. The chromatography medium according to any one of claims 1 to 5, wherein the ligand is bound to the matrix material through an extender group selected from a polysaccharide structure and a polymer structure.

7. The chromatography medium according to any one of claims 1 to 6, wherein the matrix material is a convective flow material.

8. The chromatography medium according to any one of claims 1 to 7, wherein the matrix material is a nonwoven fabric.

9. A chromatography unit comprising a chromatography medium according to any one of claims 1 to 8.

10. A method for separating a sample in a solution, A step to obtain a solution containing the sample, A step of adding the solution to a chromatographic medium according to any one of claims 1 to 8 in a binding buffer having a conductivity of 240 to 270 mS / cm, A step of eluting a sample from a chromatography medium by contacting the chromatography medium with an elution buffer having a conductivity of 0 to 240 mS / cm, and A step to recover the eluate containing the sample formed in this manner, A method that includes this.

11. A method for separating different DNA isoforms in a solution, A step to obtain a solution containing plasmid DNA isoforms in a solution, A step of adding the solution to a chromatographic medium according to any one of claims 1 to 8 in a binding buffer having a conductivity of 240 to 270 mS / cm, A step of contacting the chromatography medium with a first elution buffer having a conductivity of 200-240 mS / cm, A step of recovering the eluate containing the first plasmid DNA isoform thus formed, and Subsequently, the chromatography medium is brought into contact with a second elution buffer having a conductivity of 0–200 mS / cm, which is lower than the conductivity of the first elution buffer. A step to recover the eluate containing the second plasmid DNA isoform formed in this manner, A method that includes this.

12. The method according to claim 10 or 11, wherein the elution of a sample from the chromatographic medium includes stepwise or gradient elution, which involves contacting the chromatographic medium with an elution buffer whose conductivity has been reduced.

13. The method according to claim 11 or 12, wherein the second plasmid DNA isoform is a superhelical plasmid DNA.

14. The method according to any one of claims 10 to 13, further comprising the step of adding the eluate to a multimodal chromatography resin after the elution step.

15. A method according to any one of claims 10 to 14, comprising washing a chromatography medium after recovering an eluate, wherein the washing comprises adding a 0.1 to 2.0 M NaOH solution to the chromatography medium.

16. A method according to any one of claims 10 to 15, comprising washing a chromatography medium after recovering an eluate, wherein the washing comprises adding a 0.1 to 0.5 M HCl or phosphoric acid solution to the chromatography medium.

17. Use of a chromatography medium according to any one of claims 1 to 8 or a chromatography unit according to claim 9 for the separation of different plasmid DNA isoforms present in a solution.

18. Use of a chromatography medium according to any one of claims 1 to 8 or a chromatography unit according to claim 9 for the purification of superhelical plasmid DNA.

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