Functionalized chromatographic media lacking surface extensions - Patents.com

Abstract

A chromatography medium is provided that includes a matrix of cellulose-based nanofibers, the nanofibers being optionally crosslinked to one another. A ligand is attached to the matrix without any intermediate extension groups. Also provided is a method for preparing the functionalized chromatography medium. The method includes (i) providing a substrate including cellulose acetate, (ii) forming a fibrous matrix / membrane by spinning nanofibers from the substrate, (iii) saponifying the nanofibers to form regenerated cellulose nanofibers, (iv) derivatizing the regenerated cellulose nanofibers with a crosslinking agent, and (v) attaching a ligand to the derivatized cellulose nanofibers, the preparation of the functionalized chromatography medium being free of any surface extensions. The chromatography medium is useful for the separation of large analytes such as viruses.

Description

[Technical field]

[0001] The present disclosure relates to chromatographic media and uses thereof, methods for preparing functionalized chromatographic media, and processes for separating analytes in solution using such functionalized chromatographic media. [Background technology]

[0002] In chromatographic purification, porous microscale beads are commonly used. These beads have functionalized surfaces to allow, for example, identification of pharmaceuticals from complex process streams. With Protein A ligands on the bead surface, purification of monoclonal antibodies (mAbs) can be mediated by affinity interactions.

[0003] An alternative to porous beads is, for example, Cytiva's HiTrap Fibro unit. Fibro material is a non-woven fibrous material derived from cellulose acetate and other materials. Fibro material uses a unique structure that overcomes the diffusion and flow limitations of packed-bed chromatographic purification systems. It also aims to address the capacity issues of chromatographic membranes, which are mostly limited to the contaminant capture step. Fibro material has the potential to improve process flexibility and robustness while meeting the demands of the rapidly changing biopharmaceutical industry.

[0004] The cellulose acetate-based Fibro membrane absorber is a highly productive chromatography format that allows for residence times of a few seconds (~3 s) while retaining good binding capacity. This is largely due to its highly porous structure with an extended surface via a glycidol-based polymeric polyol. The use of glycidol in conjunction with divinylsulfone has been shown to be necessary to obtain good binding with Protein A in mAb applications and is unique to the Fibro platform at this time, as it is a common intermediate used for next-generation products.

[0005] However, glycidol is classified as a substance that is carcinogenic, mutagenic, or toxic to reproduction (CMR), so its use should be reduced or avoided wherever possible. Summary of the Invention [Problem to be solved by the invention]

[0006] It is an object of the present disclosure to provide a method for preparing functionalized fibromaterials that ensures good analyte binding capacity without the use of glycidol in the functionalization of the material. [Means for solving the problem]

[0007] The invention is defined by the accompanying independent claims. Non-limiting embodiments will become apparent from the dependent claims, the accompanying drawings and the following description.

[0008] In one aspect of the invention, a chromatography medium is provided that includes a matrix of cellulose-based nanofibers, the nanofibers optionally being crosslinked to one another, and a ligand linked to the matrix without intermediate extending groups.

[0009] The ligands may be linked to the nanofibers of the matrix via linking groups that contain less than 10 repeating moieties or may be non-polymeric. In embodiments, the linking groups may contain 20 or fewer atoms. The linking groups may be selected from vinyl sulfone moieties and alkyne or allyl derivatives.

[0010] The matrix may have a mean flow pore size in the range of 0.1 to 2.0 μm. Advantageously, the matrix may allow convective flow of fluid through the matrix.

[0011] Crosslinks may be formed between the nanofibers and may be provided by a crosslinking agent selected from the group consisting of divinylsulfone, bisacrylamide, butanediol diglycidyl ether, epichlorohydrin, allyl glycidyl ether, allyl bromide, 1,4-dibromobutane, and bismaleimide.

[0012] In another aspect, the present invention provides the use of such a chromatographic medium for the separation of an analyte selected from the group consisting of mRNA, viruses (including viral vectors), virus-like particles, plasmids, and extracellular vesicles.

[0013] According to another aspect, there is provided a method for preparing a functionalized chromatographic medium, comprising the steps of: A method is provided that includes the steps of: (i) providing a substrate comprising cellulose acetate; (ii) forming a fibrous matrix / membrane by spinning nanofibers from the substrate; (iii) saponifying the nanofibers to form regenerated cellulose nanofibers; (iv) derivatizing the regenerated cellulose nanofibers with a crosslinking agent; and (v) linking a ligand to the derivatized cellulose nanofibers, wherein the preparation of the functionalized chromatography medium does not include surface extensions.

[0014] Cellulose acetate (CA) is an acetate ester of cellulose produced from cellulose via the process of acetylation.

[0015] Electrospinning can be used to form CA nanofibers. Such a matrix of CA nanofibers forms a fibrous matrix or membrane. Such methods are well known in the art.

[0016] For example, functionalization of cellulose acetate-based materials with the extender group glycidol together with divinyl sulfone is known in the art. Such functionalization has been shown to be sufficient for binding Protein A to fibrous matrices in mAb applications. However, glycidol is classified as a carcinogen, mutagen, or reproductive toxicant (CMR), and therefore its use should be reduced or avoided whenever possible.

[0017] The methods outlined above for functionalizing cellulose acetate-based fibromaterials are methods in which no extension groups such as glycidol are used: the ligands are linked directly to the derivatized cellulose nanofibers without the use of extension groups.

[0018] As used herein, the term "analyte" refers to a substance that is separated by a chromatographic process. When the analyte to be separated is an entity of interest that is intended to be purified, it may be referred to as a "target" or a "target entity". The phrase "large target" typically refers to an entity that is much larger than an antibody, for example, an entity with a diameter of at least 20 nm. For reference, the size of a monoclonal antibody is typically about 5 nm. Examples of large targets include mRNA, viruses (such as AAV, adenovirus, lentivirus and other enveloped viruses), and virus-based vectors, virus-like particles, plasmids, and extracellular vesicles such as exosomes.

[0019] The inventors have found that functionalized chromatographic media, in some cases, do not require extensions to link ligands to the matrix. Surprisingly, it has been shown that good binding capacities for large targets, such as mRNA, viruses, plasmids, etc., can be achieved without extension groups using functionalized chromatographic media prepared as described herein.

[0020] The glycidol polymerization used so far results in branching of the polymer chains formed, resulting in a "bush" structure. Thus, typically, one or more polymer chains are branched, and may well be hyperbranched. The different types of monomer residues in the glycidol polymer are glycerol triethers (branch points), 1,2- or 1,3-glycerol diethers (linear), and 1- or 2-glycerol monoethers (terminal). In many cases, triether and monoether residues predominate, resulting in hyperbranched polymers. Such hyperbranched polymer chains may contain at least 20 repeating moieties. In contrast, the functionalized chromatographic media disclosed herein typically lack hyperbranched structures, and may lack branched structures. Additionally, the functionalized chromatographic media disclosed herein may lack the glycerol mono-, di-, and tri-ethers described above.

[0021] Another advantage observed with the chromatographic media described herein is that the flow properties of the media may be improved, especially in larger target applications. Without wishing to be bound by theory, it is believed that as the size of the target increases, it becomes more difficult for the target entity to move freely within the support. Here, the absence of surface extensions may be beneficial, since the porosity of the support is not reduced by the extensions filling up part of the volume between the nanofibers. For large targets, high porosity is preferred, since it allows for higher flow rates without creating undesirably high back pressure. High back pressure may be particularly detrimental for sensitive target entities such as viruses, plasmids and extracellular vesicles, compared to monoclonal antibodies.

[0022] ΔP (delta pressure) refers to the differential pressure over a chromatography column or bed and is defined as the difference between the pre-column or pre-bed pressure and the post-column or post-bed pressure. In this context, the chromatography medium may correspond to the column or bed. As an example, a typical ΔP for a fibrous matrix / membrane is specified to be less than 0.5 at a flow rate of 20 mL / min. For matrices / membranes with surface extensions, ΔP may typically be in the range of ΔP of 0.2-0.4 bar for a given flow rate, e.g., 10 mL / min. In the absence of surface extensions, ΔP may instead be in the range of 0.15-0.25 bar. Thus, the use of a chromatography medium without extensions results in improved flow properties and lower column pressures. (The values ​​shown are for lentiviral applications using TRIS buffers).

[0023] The crosslinking agent used in the present invention serves to provide crosslinking of the nanofibers of the fibrous matrix. In addition, the crosslinking agent also provides derivatization of the nanofibers, which is used for the subsequent attachment of ligands. Thus, the crosslinking agent can also be considered as an activating agent that activates the matrix to allow the attachment of ligands thereto. Thus, the crosslinking agent can be an agent that acts as both a crosslinking agent and an activating agent.

[0024] The crosslinking agent may include at least two functional groups arranged to react with the hydroxyl groups of the regenerated cellulose nanofibers. The functional groups may be selected from halides, acrylamides, epoxides, tosylates, groups containing double or triple bonds that are activated or can be activated, or any combination thereof. Examples of groups containing double or triple bonds that can be activated include vinyls, alkynes, and alkenes.

[0025] The functional group containing a double bond can be, for example, vinyl.

[0026] The functional groups may be selected from divinyl sulfone, bisacrylamide, butanediol diglycidyl ether, epichlorohydrin, allyl glycidyl ether, allyl bromide, 1,4-dibromobutane, bismaleimide, or any combination thereof.

[0027] In one embodiment, the crosslinker can be divinylsulfone.

[0028] The divinylsulfone-derivatized regenerated cellulose nanofiber matrix / membrane may have a vinylsulfone content of 200-1600 μmol / g. In embodiments, the vinylsulfone content may be 200-1200 μmol / g, 200-1000 μmol / g, or preferably 200-800 μmol / g. In other embodiments, the vinylsulfone content may be 800-1600 μmol / g, for example 800-1300 μmol / g, or 1000-1300 μmol / g.

[0029] The ligand may be linked to the nanofiber matrix via a linker group, which may result from the above-mentioned saponification and derivatization with a crosslinker such as divinyl sulfone. The linker group is typically smaller than the surface extensions obtained by glycidol polymerization. As described above, the surface extensions provided by polymerization of glycidol may result in branched polymers with at least 20 repeating moieties per branched polymer. In contrast, the linker group used in the present invention may contain 20 atoms or less, such as, for example, 10 atoms or less. Typically, the linker does not contain a repeating moiety or contains at least substantially fewer than 20 repeating moieties, such as 10 repeating moieties or less, such as 5 or less, such as at most 2 repeating moieties. For example, when using divinyl sulfone as the linker, the linker group may contain 8 backbone atoms or less. As used herein, the terms "extension", "surface extension" and "extension group" refer to the polymeric structure of the repeating moiety that connects the ligand to a support such as a chromatography base matrix. Thus, expressions such as "without surface extensions", "without intermediate extension groups", "lacking surface extensions" are intended to mean that the linking of the ligand to the matrix (in this case the nanofiber) is performed without the use of a polymeric structure. "Intermediate" refers to a position between the matrix and the ligand. Instead, the linking of the ligand may be provided by a linking group that is substantially smaller than the surface extensions and is typically not formed from repeating moieties. Thus, the linker group may be non-polymeric, meaning that it does not form a polymeric structure. For example, the linking may be provided by a linker that includes 20 or fewer atoms.

[0030] The ligands may be selected from anionic ligands, cationic ligands, affinity ligands and multimodal ligands.

[0031] The ligand or portion of the ligand attached to the derivatized cellulose nanofiber has the following formula:

[0032] [ka]

[0033] (In the formula, X is independently for each occurrence H, OH or C 1~3 is selected from the group R1, R2, R3 and R4 are independently H and C 1~3 is selected from the group The C3 group is linear or branched; C 1~3 The groups are OH, OC 1~2 , S.C. 1~2 , NH, NHR, NR2, R is H and C 1~3 wherein the R is selected from the group The diamine functionality of the ligand or a portion of the ligand is linked to the derivatized cellulose nanofiber to generate at least one weak anion exchange group up to an ionic capacity of 10 to 500 μmol / mL. The ligand or a portion of the ligand may be represented by the formula above. The ligand includes a diamine functionality that generates at least one weak anion exchange group up to an ionic capacity of 10 to 500 μmol / mL.

[0034] The diamine ligand may form part of a larger structure, such as a polymer.

[0035] In the ligand or portion of the ligand containing the diamine functionality that produces at least one weak anion exchange group described by the formula above, the two amines may be separated by 2 to 4 carbon atoms, and each amine group may be substituted by two R groups, which R groups are selected from H and alkyl groups C 1~4 and may be branched and / or substituted with other groups such as hydroxyl, amine, ether and thioether, the latter being limited to 3 to 8 atoms.

[0036] Such ligands are used to create anion exchange chromatography media. This can be used, for example, for the purification of extracellular vesicles such as enveloped virus particles or exosomes. Enveloped viruses bud from host cells and have an outer lipid bilayer derived from the cell membrane that contains viral glycoproteins. Inside the enveloped particle is a protein capsid that contains the genetic material of the virus. The envelope is important for infecting host cells (binding and fusing with the host cell membrane), but is highly sensitive to, for example, shear forces, salts, pH, and detergents. The conditions during production and purification are important to preserve the infectivity of the virus and maximize the recovery of functional infectious virus. Examples of such enveloped viruses are DNA viruses such as herpesviruses, oxviruses, hepadnaviruses, and asphalviridae; RNA viruses such as flaviviruses, alphaviruses, togaviruses, coronaviruses, hepatitis D, orthomyxoviruses, paramyxoviruses, rhabdoviruses, bunyaviruses, and filoviruses; and retroviruses such as lentiviruses.

[0037] The support material is functionalized with ligands to an ionic capacity (number of charged functional groups per ml of medium (µmol / ml)) ranging from 10 to 500 µmol / mL, or 50 to 300 µmol / mL or 100 to 300 µmol / mL. There are no known affinity ligands for enveloped virus particles or exosomes. Development of affinity ligands for enveloped viruses is difficult because harsh elution conditions are typically required that can harm the virus. An ion exchange capture solution would allow for milder elution conditions, resulting in an improved process with higher recovery.

[0038] At least one weak anion exchange group may comprise a multimodal weak anion exchange group, i.e., the anion exchange group provides at least two different but cooperative sites for interacting with the compound to be bound (i.e., enveloped virus particles or exosomes). For example, one of these sites may provide an attractive type of charge-charge interaction between the ligand and the substance of interest. The other site may contribute to binding by introducing a second local charge or by increasing the local amount of solvated water, thus affecting the binding capacity.

[0039] The weak anion exchange group may be positively charged at pH 6-10 or may be partially positively charged.

[0040] Such positively or partially positively charged weak anion exchange groups can attract enveloped virus particles or exosomes that are negatively charged at neutral pH, such as lentivirus particles.

[0041] The weak anion exchange group may be positively charged or partially positively charged at pH 6-10 or 6-9.5, 6-9 or 6-8.

[0042] Weak ion exchange groups mean that they have a gradient with pH from fully charged to uncharged, with a neutral charge (equal amounts of + and -) for PI. In contrast, strong anion exchange groups based on quaternary amines are always charged. Nearly all other anion exchange groups that are not based on quaternary amines are weak, i.e., their charge varies (even to zero) within the reasonable range of pH used (e.g., pH 2-11).

[0043] The ligand may be selected from N,N,N'-triethylethylenediamine, diethylene-triamine, N,N'-dimethylethylenediamine, N-methylethylenediamine, 1,3-diaminopropane, 1,3-diamino-2-hydroxypropane, 2-methyl-1,3-propanediamine, N,N-diethylethylenediamine and diethylethylaminoethyl.

[0044] When the functionalized chromatographic medium is equipped with anionic, cationic, or multimodal ligands, the ionic capacity can be from 10 to 500 μmol / mL.

[0045] The optimum ionic capacity depends on the ligand used.

[0046] The ligand may be an affinity ligand.

[0047] The affinity ligand can be, for example, an antibody, a recombinant protein engineered to have affinity for a particular target, a peptide or smaller synthetic ligand, Protein A, Protein G, IMAC, Con A, heparin, and the like.

[0048] The affinity ligands can be coupled to the derivatized cellulose nanofibers with a ligand density (number of affinity ligands per mL of medium) of at least 150 nmol / mL.

[0049] The optimal ligand density depends on the target to be purified and the size of the affinity ligand used. The affinity ligand density can be, for example, 150 to 1000 nmol / mL, or 200 to 800 nmol / mL.

[0050] The method may further include the step of blocking non-reacted cross-linking groups, such as divinylsulfone groups.

[0051] Blocking of non-reactive groups can be done, for example, with thioglycerol or simply with NaOH.

[0052] As mentioned above, functionalized chromatography media in which the ligand is an affinity ligand can be used in affinity chromatography.

[0053] As mentioned above, functionalized chromatographic media in which the ligands are anionic, cationic, or multimodal ligands can be used in ion exchange chromatography.

[0054] According to another aspect, there is provided a method for separating an analyte in a solution, the method comprising the steps of obtaining a solution comprising the analyte, adding the solution to a functionalized chromatographic medium produced as described above, comprising an anionic, cationic or multimodal ligand in a binding buffer having a conductivity of 1.5-35.0 mS / cm, eluting the analyte from the chromatographic medium by contacting the chromatographic medium with an elution buffer having a conductivity of 20-105 mS / cm, and recovering the eluate thus formed, comprising the analyte. According to a third aspect, there is provided a method for separating an analyte in a solution, the method comprising the steps of obtaining a solution comprising the analyte, adding the solution to a functionalized chromatographic medium produced as described above, comprising an affinity ligand in a binding buffer having a conductivity of 1.5-35.0 mS / cm, eluting the analyte from the chromatographic medium by contacting the chromatographic medium with an elution buffer having a conductivity of 20-105 mS / cm, and recovering the eluate thus formed, comprising the analyte.

[0055] The binding buffer may be, for example, 20-50 mM TRIS buffer, and the elution buffer may be, for example, 20-50 mM TRIS buffer 0.1-1.3 M NaCl.

[0056] In one example where a lentivirus is being isolated, the binding buffer may have a conductivity of about 20 mS / cm.

[0057] Elution may be performed using an elution buffer with a conductivity of 20-105 mS / cm, which may be achieved using an elution buffer containing 0.13-1.3 M NaCl and a pH gradient of 6.5-8.5.

[0058] In the above methods, the analyte to be separated may be selected from mRNA, viruses, virus-like particles, plasmids, exosomes, and protein complexes.

[0059] Such analytes typically have a size, e.g., diameter, of at least 10 nm, and may have a size of at least 20 nm. In contrast, the size of monoclonal antibodies is typically about 5 nm. An example of a protein complex is IgM. IgM may have a molecular weight about five times that of IgG-based monoclonal antibodies. [Brief description of the drawings]

[0060] [Figure 1] Functionalization of cellulose acetate materials with glycidol and divinyl sulfone (top of FIG. 1) and a new method to directly functionalize cellulose acetate materials with divinyl sulfone without using glycidol extensions (bottom of FIG. 1). Square brackets indicate repeating parts. "RC" stands for regenerated cellulose (CA with the acetate groups hydrolyzed). [Figure 2a] FIG. 1 shows the different steps in the functionalization of a cellulose acetate material without surface extensions using divinylsulfone with a ligand, H2N-X, for example an antibody or DAX (N,N-diethylethylenediamine). [Figure 2b] FIG. 1 shows the different steps in the functionalization of a cellulose acetate material without surface extensions using divinylsulfone with a ligand, H2N-X, for example an antibody or DAX (N,N-diethylethylenediamine). [Figure 2c] FIG. 1 shows the different steps in the functionalization of a cellulose acetate material without surface extensions using divinylsulfone with a ligand, H2N-X, for example an antibody or DAX (N,N-diethylethylenediamine). [Figure 2d]FIG. 1 shows the different steps in the functionalization of cellulose acetate material with DEAE ligands using divinyl sulfone without the use of surface extensions. [Diagram 3] FIG. 1 shows the functionalization of cellulose acetate with DEAC (N,N-diethyl-aminoethyl chloride) and DAX (N,N-diethylethylenediamine), respectively, without the use of surface extensions and where unreacted divinylsulfone groups are deactivated with thioglycerol. [Figure 4] FIG. 1 shows the results of lentivirus purification using different DAX- and DEAE-functionalized fibrous matrix / membrane chromatography media without extensions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] Cellulose acetate (CA) is an acetate ester of cellulose produced from cellulose through a process of acetylation. Electrospinning can be used to form CA nanofibers. Such CA nanofiber matrices or membranes form fibrous matrices or membranes that can be used as support materials for chromatography. The cellulose acetate fibrous membranes can be convection-based matrices and can have a mean flow pore size of 0.1-2.0 μm and a cross-sectional diameter of 10-1000 nm. Such fibrous materials (hereafter referred to as fibrous materials) can be found in Cytiva's HiTrap™ Fibro units.

[0062] A convection-based chromatography matrix includes any matrix in which the application of a hydraulic pressure difference between the inflow and outflow of the matrix forces perfusion of the matrix, achieving substantial convective transport of substances into or out of the matrix very rapidly at high flow rates.

[0063] The mean flow pore size of the membrane or matrix can 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. The mean flow pore (MFP) diameter is a measure of the flow properties of a material and is measured by capillary flow porometry, which is based on the displacement of a wetting liquid with known surface tension from the pores of a sample by the application of gas under increasing pressure. The larger the MFP diameter, the greater the flow of liquid through the material at a given pressure. The mean flow pore diameter is calculated from the point when 50% of the flow has passed through the sample. Thus, the mean flow pore diameter corresponds to the pore size calculated at the pressure where the wet and semi-dry curves intersect. In another definition, the mean flow pore diameter of the current stationary phase can be considered as the effective pore size, defined as the size of the largest sphere that can pass through the pore.

[0064] The functionalization of cellulose acetate-based materials with extension groups such as glycidol is known in the art (see FIG. 1 (top)). The use of glycidol extensions in conjunction with divinyl sulfone has been commonly used to functionalize CA, as it provides sufficient binding of Protein A for mAb applications. However, glycidol is classified as a carcinogen, mutagen, or toxic to reproduction (CMR), and therefore its use should be reduced or avoided whenever possible.

[0065] Below we describe a method for functionalizing cellulose acetate-based fibrous materials without the use of glycidol extending groups (see also Figure 1 (bottom)).

[0066] A substrate containing cellulose acetate is prepared and hydrolyzed by saponification (Figure 2a) to form regenerated cellulose. Saponification is carried out under aqueous basic conditions with or without a co-solvent such as EtOH. The pH used may be between 12-14, preferably around 12.7 (0.5M OH-). The base of choice may be 0.5M KOH.

[0067] In one example, potassium hydroxide (0.5 M) in a water / ethanol solution (2:1) is used and allowed to react at room temperature for at least 5 hours.

[0068] The regenerated cellulose is then derivatized with a crosslinking agent (Figure 2b). Such a crosslinking agent may contain at least two functional groups arranged to react with the hydroxyl groups of the regenerated cellulose nanofibers and may be selected from, for example, halides, acrylamides, epoxides, tosylates, functional groups containing double bonds such as vinyl that can be activated, or any combination thereof. The functional groups may be selected from divinyl sulfone, bisacrylamide, butanediol diglycidyl ether, epichlorohydrin, allyl glycidyl ether, allyl bromide, 1,4 dibromobutane, bismaleimide, or any combination thereof.

[0069] In Figure 2b and Figure 2d, divinyl sulfone is used as a cross-linking agent. Derivatization of divinyl sulfone can be obtained by adding hydroxyl groups of the cellulose membrane to one of the vinyl groups of divinyl sulfone under basic conditions. Part of the second vinyl moiety reacts with other hydroxyl groups on the cellulose backbone to cross-link the fibers, while the remaining vinyl sulfone is functionalized in a later step to provide bonds. The concentration of the divinyl sulfone solution used can be in the range of 0.5-4 mol / L, preferably in the range of 1.5-2.5 mol / L. The pH can be in the range of 8.2-10, preferably in the range of 8.5-9.5. In one example, regenerated cellulose is reacted in a solution of Na2CO3, acetonitrile (C2H3N) and divinyl sulfone (CH2=CH)2SO2 (or C4H6O2S). In one example, Na2CO3, (0.54 M) and divinyl sulfone in a water / acetonitrile (C2H3N) (4:1) solution are reacted with regenerated cellulose at room temperature for at least 5 hours.

[0070] Ligands are attached to vinylsulfone-derivatized cellulose nanofibers (Figure 2c (H2N-X), Figure 2d (DEAE)). The ligands can be anionic, cationic, affinity or multimodal ligands.

[0071] The ligand can be a diamine functional group that generates at least one weak anion exchange group, such as DEAE (diethylaminoethyl) (Figure 2d, Figure 3 (top)) and DAX (N,N-diethylethylenediamine) (Figure 2c, Figure 3 (bottom)).

[0072] DEAE is a known ligand for chromatography. Diamine ligands can be purchased directly, such as DAX, but can also be prepared in situ by addition of an alkylamine with a leaving group that can be added to the support and the resulting amino resin can be further reacted. DEAC ((2-chloroethyl)diethylamine), used to prepare DEAE resins, is a good example of such a reagent.

[0073] Direct coupling of polyamine ligands to vinyl groups can be carried out, for example, at a pH of 11-13 using a 0.1-0.8 molar ligand solution.

[0074] The above functionalized support materials, for example functionalized with DEAC or DAX, may be used in anion exchange chromatography, for example for the purification of enveloped virus particles or exosomes. The support materials may be functionalized with ligands up to an ionic capacity (number of charged functional groups per ml of medium (μmol / ml)) of 10 to 500 μmol / mL. There are no known affinity ligands for this type of enveloped virus particles or exosomes.

[0075] Alternatively, the ligand can be an affinity ligand, such as, for example, an antibody, a recombinant protein modified to have affinity for a particular target, a peptide or a smaller synthetic ligand (Figure 2c). The affinity ligand can be linked to the solid support without the use of an extension group, by a crosslinker such as a vinyl group, through the addition of a nucleophilic group such as an amine, or through a thiol present on the ligand. The linking conditions are similar to those used for DAX. In the case of pH-sensitive ligands, the linking is performed at a lower pH, such as around pH 11. In the case of ligands containing free thiol groups, the linking can be performed at an even lower pH, such as pH 8.3-9.5, preferably around pH 9. Such functionalized support materials can be used in affinity chromatography.

[0076] The method may further comprise the step of blocking unreacted vinyl sulfone derivatized groups, for example, by adding ethylenediamine (or 2-mercaptoethylamine hydrochloride) (FIG. 3).

[0077] The experimental section below describes specific, non-limiting examples of the generation of functionalized support materials and their use in chromatography.

[0078] experiment Lentiviral feed material LVV (lentiviral vector) was transfected at approximately 2 × 10 in HEK293 suspension cell cultures (transient / producer cell line). 6 The cells were produced at a cell density of 10 ... 6 > 5 x 10 TU / mL 9 A feed with VP / mL and impurities ranging from 4000-11000 µg / mL for total protein and 200-1500 ng / mL for DNA was used. The pH of the final feed should be 7.4-8.0 (adjust if necessary). The feed should be stored immediately at -80 °C in an aliquot suitable for chromatography (recommended: 40 mL).

[0079] Fibrous Matrix Materials A cellulose acetate (CA) solution of 29,000 g / mol relative molecular weight was dissolved in a common solvent prior to electrospinning to produce fibers with diameters in the range of 300-600 nm. Optimized conditions for nanofiber production can be found, for example, in O. Hardick et al., J. Mater. Sci. 46 (2011) 3890. 1 m of material 2 The sheets, each weighing approximately 20 g, were layered and treated with a combination of heat and pressure.

[0080] 35 strips (100 x 155 mm 2 The formed CA material was sandwiched between polypropylene gauze and introduced into the flow reactor. The strip was washed three times for 20 min with distilled water and then left overnight in the final wash.

[0081] The CA material was saponified to form regenerated cellulose (RC). A solution of KOH (132 g) in water (3,149 L) was prepared in a suitable container. Ethanol (1.574 L) was added to the KOH solution while stirring. The basic ethanoic acid solution was added to the flow reactor. The recirculation pump was switched on and the mixture was allowed to recirculate for 6 hours. After 6 hours of recirculation, the pump was stopped and the flow reactor was drained. The material was washed 4 times with 6 L of distilled water for at least 20 minutes each time, followed by washing with 6 L of acetone (2 x 20 minutes) before air drying the material in the flow reactor.

[0082] Divinylsulfone derivatization of RC was then performed. Distilled water (4.211 L) was added to an 8 L beaker / container followed by Na2CO3 (316.1 g). The contents were stirred until the base was completely dissolved. Acetonitrile (gradient analytical HPLC; >99.9%; 1.258 L) was added and the mixture was stirred for 2 min before being charged to the flow reactor. The recirculation pump was switched on and the mixture was recirculated for 2 min before adding the divinylsulfone portion (>99%, 1.350 L). The reaction mixture was recirculated at room temperature for 6 h. The reaction mixture was drained and the material was washed by recirculating 1:1 water / acetone (6 L) four times at 24-26 °C for 20 min each time. Distilled water (6 L) was then recirculated to rinse the material twice, for 15 min each time. The material was then used.

[0083] Ligand Linkage To the fibrous matrix material produced as described above, ligands were then attached to the divinylsulfone-derivatized cellulose nanofibers. A) Ligands having diamine functionality that generate at least one weak anion exchange group up to an ionic capacity of 10 to 500 μmol / mL, exemplified here by DEAE (diethylaminoethyl) and DAX (N,N-diethylethylenediamine). B) An affinity ligand, exemplified here by the AVB antibody.

[0084] DEAE (Diethylaminoethyl) Ligand For DEAE functionalization the following protocol was used.

[0085] DEAE Concatenation The above Fibro-VS strips were washed 4 times with 150 ml of DV20 in a polypropylene (PP) container to remove residual solvent, after which 2 g of KOH was dissolved in 25 ml of deionized water and added to the Fibro-VS strips for 30 min. Then 1.9 ml of DEAC (2-(diethylamino)ethyl chloride hydrochloride) (65%) was added along with 23 ml of DV20. The PP container was sealed with parafilm and placed on an orbital shaker (≈60 rpm). The reaction was allowed to continue for 16 h at room temperature. The discs were then washed with 150 ml of DV20 x 6 x 20 min. Titration gave an ionic capacity of 113.3 μmol / ml.

[0086] An inactivation solution was prepared: disodium ethylenediaminetetraacetate dihydrate (EDTA*Na2*2H2O, 61 mg) and disodium hydrogen phosphate dodecahydrate (Na2HPO4*12H2O, 5.7 g) were added to deionized water (150 ml). After stirring for 5 min, thioglycerol (12 ml) was added and the pH was adjusted to 8.3.

[0087] Fibro-VS strips were suspended in the inactivation solution and stirred at room temperature for 16 h, after which the strips were washed three times with DV20, once with 1 M NaCl, and three times with DV20, each wash using 150 mL of solution with a contact time of 20 min.

[0088] DAX (N,N-diethylethylenediamine) ligand The Fibro VS strips described above were reacted with N,N-diethylethylenediamine to form a functionalized matrix with N,N-diethylethylenediamine groups to form the Fibro DAX (diamino-exchanged) material. The following protocol was used for functionalization:

[0089] The N,N-diethylethylenediamine ligation solution was formed. 1) 1.2% (V / V) in water 2) 3% (V / V) in water

[0090] Fibro-VS strips were placed in polypropylene (PP) containers on an orbital shaker with the respective ligation solutions. The reactions were left stationary for 16 hours, washed with 150 mL of distilled water, and placed back on the orbital shaker for approximately 20 minutes. The water washing process was repeated five times. Titrations gave ionic capacities of 142 and 186 μmol / mL, respectively.

[0091] Inactivation was performed according to the procedure described above for Fibro-DEAE.

[0092] Antibody Ligand Approximately 20 mL of AVB ligand (ligand for AAV (adeno-associated virus)) solution (13 mg / ml) was placed on each of four VivaSpin filters (5000D cutoff, Sartorius Stedim) and centrifuged at 4000 rpm for 30 minutes. The 13 mg / ml solution was used to calibrate the NanoDrop spectrophotometer.

[0093] The spin-filtered AVB solution was used to prepare the immobilization conditions shown in Table 1.

[0094] [Table 1]

[0095] The ligation buffer used was 3.0 M (NH4)2SO4, 0.1 M NaHCO3, pH 9.0.

[0096] During immobilization, Fibro-VS strips prepared as described above were placed on an orbital shaker and immobilized overnight in different concentrations of AVB (see Table 1). After this time, the supernatant was collected and the strips were washed four times with distilled water for 20 min each. The AVB concentration in the supernatant was measured with a NanoDrop spectrophotometer to determine the binding level and ligand density. The strips were then immersed in 30 mM ethanolamine blocking solution (pH 9) and placed on an orbital shaker for 1-2 h. After this time, the strips were washed twice with distilled water for 20 min each, followed by two alternating washes with PBS solutions of pH=2 and pH=7.4, followed by a further wash with distilled water. The strips were agitated in 20% ethanol, 20% glycerol and 60% water.

[0097] The thickness of each strip was measured to calculate the membrane volume (mL), and the mass (mg) of immobilized AVB was calculated using the concentration in the supernatant after immobilization. From this, the ligand density under each condition was calculated using the concentration in AVB as a variable.

[0098] Anion exchange chromatography using DEAE and DAX functionalized support materials. In the following anion exchange chromatography experiments, the DEAE and DAX functionalized support materials described above were used as anion exchange chromatography media in purifying lentiviral particles.

[0099] The support material was used in a Fibro HiTrap unit of an AKTA pure 150 chromatography system with the following running conditions: Buffer A1 (running buffer): 50 mM Tris, pH 8.0 Buffer B1 (elution buffer): 50 mM TRIS, pH 8.0, 1.3 M NaCl CIP buffer (cleaning in place buffer): 1.0M NaOH Residence time: 2.4 seconds (dynamic binding capacity (DBC)>10 12 VP / mL) Pressure Limits: Delta-column maximum pressure limit is 1.0MPa (10bar). Approximate loading range: for lentivirus, 3 x 10 per device at a dwell time of 2.4 seconds 11 VP / 3×10 8 TU. However, typically about 20-200mL of NFF (normal flow filtration) material can be loaded. As a general guide, ideally the loading should be 50-85% of the dynamic binding capacity of the unit to achieve the highest recovery and purity. The actual loading amount is highly feed dependent, depending on the feed titer, HCP (host cell protein) and DNA content as well as the amount and size of any particles.

[0100] Prior to adding the lentivirus sample, the sample pump lines were washed through the waste valve with 100% Buffer A1 until the UV / conductivity stabilized, and the Fibro unit was washed with 100% Buffer A1 at 10 mL / min until the 280 nm UV and conductivity signals stabilized. The "Sample Pump Waste" outlet was then used to wash the main sample pump with the lentivirus feed.

[0101] Table 2 below summarizes the bind-elute protocol used.

[0102] [Table 2]

[0103] DAX and DEAE functionalized membranes with different ligand densities and without glycidol were tested for their ability to recover infectious virus. Different ligand densities were investigated by the method described above (Table 2). The results are summarized in Figure 4. The ionic capacity (IC) of the DAX low prototype was 41 μmol / mL, the IC of the DAX medium prototype was 186 μmol / mL, the IC of the DAX high prototype was 214 μmol / mL, the IC of the DEAE low prototype was 113 μmol / mL, and the IC of the DEAE high prototype was 247 μmol / mL. The infection yield was 54% (STD 34) for DAX low, 78% (STD 2) for DAX medium, 33% (STD 2) for DAX high, 84% (STD 14) for DEAE low, and 31% (STD 14) for DEAE high. The results show that 100-200 μmol / mL is optimal for both ligands. Using optimal conditions and membranes, an infection yield of approximately 80% can be achieved with either the ligand DAX or DEAE.

[0104] Since the binding of lentiviral particles is an electrostatic interaction, some of it occurs within a certain ionic capacity range of 10-500 μmol / mL. The optimal ionic capacity value is related to the need to have enough ligand to ensure binding, but not too high an ionic capacity that would make the binding too stiff and cause a loss of recovery. It seems that the optimal ionic capacity may be strongly dependent on the support material used (ligand density by contact surface). For fibro, the optimal IC seems to be about 100-250 μmol / mL. The capacity of the membrane is estimated by determining the total particles that need to be loaded to achieve 10% breakthrough (also called DBC or QB10% capacity), and for ligand densities of 10-500 μmol / mL, at least 1.00E+12 particles / mL would be preferred for most applications.

[0105] The above results are shown for lentiviruses, which are enveloped viruses. Similar results are obtained with other enveloped virus types, such as DNA and RNA viruses, as well as exosomes.

[0106] Affinity chromatography using support materials bearing AVB ligands

[0107] The above functionalized support material with AVB ligands was used in affinity chromatography. A Cytiva MiniPEEK column (polyetheretherketone) containing a single stack of 10-layer Fibro AVB membrane (0.343 μmol / g, membrane volume 19-20 μl) was used. Running Buffer: 20mM Tris, 500mM NaCl, 0.001% Pluronic F-68, pH 8.5 Elution buffer: 100mM NaOAc, 500mM NaCl, 0.001% Pluronic F-68, pH 2.5 Neutralization buffer: 200mM Tris, 500mM NaCl, 0.001% Pluronic F-68, pH 10.5

[0108] After equilibration with running buffer on a MiniPEEK column, approximately 1.2E13 capsids of AAV-5 (approximately 1E12 capsids / mL) were loaded at 0.5 mL / min. A 0.6 mL flow-through fraction was collected. The membrane was then washed with 10 mL of running buffer and eluted with 6 mL of elution buffer, each collected (separately) in a single tube. The eluted fraction was neutralized to pH 7.5-8.5 by adding 1 mL of neutralization buffer.

[0109] The AAV titer in each fraction was quantified using an AAV-5 titer assay ELISA kit (Progen) according to the manufacturer's instructions. The breakthrough rate (BT) of each fraction was calculated.

[0110] Membrane capacity was estimated by determining the total capsids that needed to be loaded to achieve 10% breakthrough (also known as DBC or QB10% capacity) relative to the volume of the MiniPEEK device (taking into account the specific thickness of each membrane). This value was then corrected for a membrane volume of 1 mL, assuming linear scale-up.

[0111] The AAV titer in each fraction was quantified using an AAV-5 titer ELISA kit (Progen) according to the manufacturer's instructions. When calculating the recovery rate, the elution fractions containing 10% or more of the most concentrated fraction were considered.

[0112] Samples and standards were diluted in MiliQ H2O using a Nimbus liquid handler. Protein concentration of each fraction was quantified using the Pierce™ Coomassie Plus (Bradford) Assay Kit (Thermo) according to the manufacturer's instructions.

[0113] Samples and standards were diluted using a Nimbus liquid handler. DNA concentration in each fraction was quantified using the Quantit Picogreen dsDNA kit (Thermo) according to the manufacturer's instructions.

[0114] Table 3 below shows the estimated capacities of membranes provided by different ligand densities ranging from 143 to 714 nmol / mL by determining the total capsids that need to be loaded to achieve 10% breakthrough (QB10% capacity).

[0115] [Table 3]

[0116] For most applications, a QB10 of at least 1.00E+13 capsids / ml would be preferred. As can be seen from Table 3, ligand densities below 250 nmol / mL herein resulted in capacities below 1.00E+13.

Claims

1. a matrix of cellulose-based nanofibers, the nanofibers being optionally crosslinked to one another; and Ligands that are linked to the matrix without intermediate extension groups 4. A chromatography medium comprising:

2. 2. The chromatography medium of claim 1, wherein the matrix comprises crosslinks between the nanofibers provided by a crosslinker selected from the group consisting of divinyl sulfone, bisacrylamide, butanediol diglycidyl ether, epichlorohydrin, allyl glycidyl ether, allyl bromide, 1,4-dibromobutane, and bismaleimide.

3. 3. The chromatography medium of claim 1 or 2, wherein the ligand is linked to the nanofibers of the matrix via a linking group that contains fewer than 10 repeating moieties, or via a linking group that contains 20 or fewer atoms.

4. 4. The chromatography medium according to claim 1, wherein the ligands are selected from anionic ligands, cationic ligands, affinity ligands and multimodal ligands.

5. The ligand or a portion of the ligand has the formula: 【Chemistry 1】 (In the formula, X is independently for each occurrence H, OH or C 1～3 is selected from the group R1, R2, R3 and R4 are independently H and C 1～3 is selected from the group C 3 The group is linear or branched, C 1～3 The groups are OH, OC 1～2 , S.C. 1～2 , N.H., N.H.R., N.R. 2 and R is H and C 1～3 is selected from the group 5. The chromatography medium of claim 4, wherein the diamine functionality of the ligand or part of the ligand is linked to the derivatized cellulose nanofiber to generate at least one weak anion exchange group up to an ionic capacity of 10 to 500 μmol / mL.

6. 6. A chromatography medium according to claim 5, wherein the ligand is selected from N,N,N'-triethylethylenediamine, diethylenetriamine, N,N'-dimethylethylenediamine, N-methylethylenediamine, 1,3-diaminopropane, 1,3-diamino-2-hydroxypropane, 2-methyl-1,3-propanediamine, N,N-diethylethylenediamine and diethylethylaminoethyl.

7. 7. The chromatography medium of claim 4, wherein the functionalized chromatography medium is provided with said anionic, cationic or multimodal ligands to an ionic capacity of 10 to 500 μmol / mL.

8. 5. The chromatography medium of claim 4, wherein the ligand is an affinity ligand.

9. 9. The chromatography medium of claim 8, wherein the chromatography medium has a ligand density of at least 150 nmol / mL.

10. 10. A chromatography medium according to any one of claims 1 to 9, wherein the matrix allows convective flow of fluid through the matrix.

11. 11. The chromatography medium of any one of claims 1 to 10, wherein the matrix has a mean flow pore size in the range of 0.1 to 2.0 μm.

12. 12. The chromatography medium of claim 1, wherein the ligand is linked to the nanofibers of the matrix via a linking group selected from a vinyl sulfone moiety and an alkyne or allyl derivative.

13. 1. A method for preparing a functionalized chromatographic medium, comprising the steps of: (i) providing a substrate comprising cellulose acetate; (ii) forming a fibrous matrix / membrane by spinning nanofibers from a substrate; (iii) saponification of the nanofibers to form regenerated cellulose nanofibers; (iv) derivatization of regenerated cellulose nanofibers with a crosslinking agent; (v) Linking Ligands to Derivatized Cellulose Nanofibers Including, A method, wherein the preparation of the functionalized chromatographic medium does not include any surface extensions.

14. The method of claim 13, wherein the crosslinking agent comprises at least two functional groups positioned to react with the hydroxyl groups of the regenerated cellulose nanofibers.

15. 15. The method of claim 14, wherein the functional groups are selected from halides, acrylamides, epoxides, tosylates, functional groups containing double or triple bonds that are activated or can be activated, or any combination thereof.

16. 16. The method of claim 15, wherein the functional groups are selected from divinyl sulfone, bisacrylamide, butanediol diglycidyl ether, epichlorohydrin, allyl glycidyl ether, allyl bromide, 1,4 dibromobutane, bismaleimide, or any combination thereof.

17. 15. The method of claim 14, wherein the crosslinking agent is divinyl sulfone.

18. 18. The method of claim 17, wherein the divinylsulfone derivatized regenerated cellulose nanofiber matrix / membrane has a vinylsulfone content in the range of 200 to 1600 μmol / g, such as 200 to 1000 μmol / g.

19. 19. The method according to any one of claims 14 to 18, wherein the ligand is selected from anionic ligands, cationic ligands, affinity ligands and multimodal ligands.

20. The ligand or portion of the ligand linked to the derivatized cellulose nanofiber has the following formula: 【Chemistry 2】 (In the formula, X is H, OH or C 1～3 is selected from the group R1, R2, R3 and R4 are independently H and C 1～3 is selected from the group C 3 The group is linear or branched, C 1～3 The groups are OH, OC 1～2 , S.C. 1～2 , N.H., N.H.R., N.R. 2 and R is H and C 1～3 is selected from the group 20. The method of claim 19, wherein the diamine functionality of the ligand or a portion of the ligand is linked to the derivatized cellulose nanofiber to generate at least one weak anion exchange group up to an ionic capacity of 10-500 μmol / mL.

21. 21. The method of claim 20, wherein the ligand is selected from N,N,N'-triethylethylenediamine, diethylenetriamine, N,N'-dimethylethylenediamine, N-methylethylenediamine, 1,3-diaminopropane, 1,3-diamino-2-hydroxypropane, 2-methyl-1,3-propanediamine, N,N-diethylethylenediamine and diethylethylaminoethyl.

22. 22. The method according to any one of claims 19 to 21, wherein the functionalized chromatographic medium is provided with anionic, cationic or multimodal ligands to an ionic capacity of 10 to 500 μmol / mL.

23. The method of claim 19, wherein the ligand is an affinity ligand.

24. 24. The method of claim 23, wherein the affinity ligand is linked to the derivatized cellulose nanofibers at a ligand density of at least 150 nmol / mL.

25. 1. A method for separating analytes in a solution, comprising the steps of: obtaining a solution containing an analyte; adding a solution in a binding buffer having a conductivity of 1.5 to 35.0 mS / cm to a chromatographic medium provided with an anionic, cationic or multimodal ligand according to any one of claims 4 to 12 or a chromatographic medium produced according to any one of claims 19 to 22, eluting the analyte from the chromatographic medium by contacting the chromatographic medium with an elution buffer having a conductivity of 20 to 105 mS / cm; and Recovering the eluate containing the formed analyte. A method comprising:

26. 1. A method for separating analytes in a solution, comprising the steps of: obtaining a solution containing an analyte; adding a solution in a binding buffer having a conductivity of 1.5 to 35.0 mS / cm to a functionalized chromatographic medium provided with an affinity ligand according to any one of claims 4 and 8 to 12 or a functionalized chromatographic medium produced according to any one of claims 19 and 23 to 24, eluting the analyte from the chromatographic medium by contacting the chromatographic medium with an elution buffer having a conductivity of 20 to 105 mS / cm; and Recovering the eluate containing the formed analyte. A method comprising:

27. 27. The method of claim 25 or 26, wherein the analyte is selected from the group consisting of mRNA, a virus or virus-like particle, a plasmid, an exosome, and a protein complex.

28. 28. The method of claim 27, wherein the analyte is selected from the group consisting of mRNA, a virus, a virus-like particle, a plasmid, and an exosome.

29. 28. The method of claim 27, wherein the analyte is an enveloped virus, such as a lentivirus.

30. 13. Use of a chromatography medium according to any one of claims 1 to 12 for the separation of an analyte selected from the group consisting of mRNA, viruses, virus-like particles, plasmids and extracellular vesicles, preferably viruses such as viral vectors.

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