Chromatography matrix and its use for purifying fibronectin

Abstract

A chromatography matrix consisting of an acrylic polymer and collagen or a derivative thereof, wherein the acrylic polymer and the collagen or a derivative thereof are coupled in an amount of greater than 1.17 mg and up to about 16 mg collagen or a derivative thereof per g acrylic polymer. Also disclosed is a method of making the chromatography matrix. Also disclosed is a method for purifying fibronectin from a source solution using the chromatography matrix.

Description

Technical Field

[0001] This invention relates in particular to novel chromatographic matrices and their use in methods for purifying fibronectin. Background Technology

[0002] Affinity chromatography has become a valuable tool for separating biological materials from fluid media (e.g., aqueous solutions) in a mobile phase. The basic principle of affinity chromatography involves immobilizing molecules of interest from a fluid medium (e.g., an aqueous solution) onto an insoluble support within a stationary phase. Therefore, a two-phase system is typically employed, with one phase being the mobile phase and the other the stationary phase. Separation occurs during the passage of the mobile phase through the stationary phase. The stronger the adsorption of a compound, the more it is distributed onto the stationary phase, and the slower its movement relative to the mobile phase.

[0003] To enhance the separation capability of a solid matrix for one or more target compounds, the matrix is ​​typically chemically modified. Such modifications involve covalent attachment of ligands to the matrix surface, where the ligands are selected to enhance the adsorption of the target compound onto the solid immobilized matrix during the adsorption phase. Covalent ligand attachment is usually achieved by using reactive functional groups (such as hydroxyl, carboxyl, mercapto, amino, etc.) on the solid support matrix. Conventional chemistry allows the formation of covalent amines, ethers, thioethers, amides, carbamates, ureas, carboxyl groups, and ester bonds with these functional groups.

[0004] The desired elution of the components can then be achieved through various procedures that lead to the dissociation of the complex. Thus, various substances (such as enzymes, hormones, specific proteins, etc.) can be separated based on specific interactions with the insoluble carrier.

[0005] Fibronectin plays a major role in many important physiological processes, such as wound healing, hemostasis, and thrombosis.

[0006] US Patent 5,043,062 discloses a high-performance affinity chromatography separation device, which includes: a chromatographic column containing: a packing material; wherein the packing material is a plurality of non-porous, monodisperse polymer particles having i) a particle size in the range of 0.01 micrometers to about 5 micrometers and ii) surface reactive groups that react directly or indirectly with free amino groups, thiol groups, carboxyl groups or aldehyde groups of biological ligands.

[0007] U.S. Patent 4,722,790 discloses a flexible, compressible, liquid-impermeable bag having at least one integral portion having a bag opening extending fully through it. The bag includes a structure defining a bag opening for communication with the bag's interior. A plurality of beads are positioned within the bag, and the beads are covalently coupled to an adhesive. Summary of the Invention

[0008] This invention relates particularly to novel chromatographic matrices, their preparation, and their use in purifying fibronectin. The inventors have specifically developed a novel rigid monodisperse matrix made of porous poly(methyl methacrylate) (PMMA) microspheres attached to denatured collagen, which can be readily prepared. The inventors have unexpectedly discovered that this matrix allows for the acquisition of highly purified fibronectin from respective mixtures in a single step of affinity chromatography.

[0009] According to one aspect of this disclosure, a chromatographic matrix is ​​provided comprising: (i) an acrylic polymer, and (ii) collagen or a derivative thereof; wherein the acrylic polymer and the collagen or a derivative thereof are coupled, the collagen or a derivative thereof being coupled in an amount greater than 1.17 mg and at most about 16 mg collagen or a derivative thereof / g acrylic polymer.

[0010] According to some implementation schemes, acrylic polymers and collagen or its derivatives are at least partially covalently coupled.

[0011] According to some embodiments of any aspect, acrylic polymers include polymethyl methacrylate (PMMA).

[0012] According to some implementation schemes of any aspect, collagen derivatives include gelatin.

[0013] According to some embodiments of any aspect, the acrylic polymer is in the form of porous microspheres having a median size in the range of 40 μm to 80 μm.

[0014] According to some embodiments of any aspect, the acrylic polymer contains epoxy groups or their derivatives.

[0015] According to some embodiments of any aspect, collagen and its derivatives are coupled to an acrylic polymer when reacting with at least a portion of the epoxy groups.

[0016] According to some implementation schemes, 1g of chromatographic matrix can bind amounts of fibronectin ranging from 6mg to 13mg.

[0017] According to some embodiments of any implementation, the chromatographic matrix is ​​used for chromatography, optionally at a pressure of up to 3 bar, for binding to fibronectin from a source solution containing fibronectin.

[0018] According to another aspect of this disclosure, a composition is provided comprising: (i) an acrylic polymer; (ii) collagen or a derivative thereof; and (iii) a buffer medium comprising a borate.

[0019] According to another aspect of this disclosure, a method for preparing a chromatographic matrix is ​​provided, the method comprising: (i) contacting an acrylic polymer with (ii) collagen or a derivative thereof dissolved in a buffer medium containing a borate, thereby obtaining an acrylic polymer coupled to collagen or a derivative thereof.

[0020] According to some implementation schemes, the buffer medium has a pH of about 7 to about 8.

[0021] According to some embodiments, collagen or its derivatives are present in the buffer medium at a concentration ranging from 0.25% by weight to 3% by weight, optionally greater than 2% by weight.

[0022] According to another aspect of this disclosure, a method for purifying fibronectin from a source solution is provided, the method comprising the steps of: passing a source solution containing fibronectin through a column containing a chromatographic matrix as described in any of the embodiments herein, under conditions that allow fibronectin to bind to a matrix; optionally eluting fibronectin bound to the chromatographic matrix; and collecting the eluted fibronectin.

[0023] According to some embodiments, the eluted fibronectin is purified to at least 90% by weight. According to some embodiments, the eluted fibronectin is optionally purified to at least 97% by weight by a single chromatographic step.

[0024] According to some implementation schemes, the source solution contains plasma.

[0025] According to some implementation schemes, the step of passing the source solution containing fibronectin through the column is carried out at a column pressure of up to 3 bar.

[0026] According to some implementation schemes, the step of passing the source solution containing fibronectin through the column is carried out at a flow rate of 0.5 ml / min to 3 ml / min.

[0027] According to some implementation schemes, the step of eluting fibronectin bound to the chromatographic matrix is ​​performed using an elution buffer containing urea at a concentration in the range of 3M to 8M.

[0028] According to some implementation schemes, purified fibronectin is provided by a method for purifying fibronectin from a source solution.

[0029] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While methods and materials similar to or equivalent to those described herein may be used in the operation or testing of embodiments of the invention, the methods and materials described below are exemplary. In case of any conflict, the patent specification and its definitions shall prevail. Furthermore, materials, methods, and examples are exemplary only and are not intended to impose necessary limitations. Attached Figure Description

[0030] This document describes some embodiments of the invention by way of example only, in conjunction with the accompanying drawings. Referring now specifically to the drawings, it should be emphasized that the details shown are by way of example and for the purpose of discussing embodiments of the invention in an illustrative manner. In this respect, the description taken in conjunction with the drawings will enable those skilled in the art to understand how to practice embodiments of the invention.

[0031] In the attached diagram:

[0032] Figure 1 Photographs showing the purity of fibronectin observed on a sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (using tris-glycine electrophoresis buffer) in slabs are provided; percentages refer to the gelatin concentration before they are coupled with polymethyl methacrylate (PMMA; see Table 3). Arrows indicate the molecular weight of fibronectin determined according to the markers in lane 4 from the left. The purity of fibronectin was assessed using an optical densitometer based on the SDS-PAGE gel results using Imagelab (Bio-Rad software). Detailed Implementation

[0033] The purpose of this invention is to provide an efficient method for purifying fibronectin from solution.

[0034] Before detailing at least one embodiment of the present invention, it should be understood that the invention is not necessarily limited in its application to the details set forth in the following description or examples. The invention can have other embodiments, or can be practiced or implemented in various ways.

[0035] In one aspect of this disclosure, a chromatographic matrix is ​​provided comprising: (i) a polymer, and (ii) collagen or a derivative thereof; wherein the acrylic polymer and the collagen or derivative thereof are at least partially coupled, and wherein the collagen or derivative thereof is coupled in an amount that allows for efficient uptake of fibronectin thereon / on / there. Typically, the polymer is rigid and capable of serving as a solid phase, typically packed within a column, for immobilizing the collagen or derivative thereof. In some embodiments, the polymer is selected from, but not limited to, acrylic polymers, polystyrene, derivatives thereof, or combinations thereof (e.g., copolymers or mixtures thereof). Typically, but not exclusively, "rigid polymer" or "rigid matrix" means having a strength greater than 7,000 kgf / cm² in bending or stretching at room temperature. 2 Polymers or matrices with an elastic modulus of (100,000 psi).

[0036] As disclosed herein, the term "chromatographic matrix" can refer to any type of particulate adsorbent, resin, or other solid phase, such as a membrane, which acts as an absorbent during purification to separate the molecule to be purified from other molecules present in a mixture. In some embodiments, the matrix or resin can be in the form of a column packed within a column, or in some embodiments, in the form of a membrane adsorbent. As disclosed herein, "membrane adsorbent" refers to an acrylic polymer flat sheet containing or doped with functional groups, such as affinity groups.

[0037] The term "resin" or "chromatographic resin" refers to a material capable of separating a target compound from a mixture of compounds by chromatography. A resin may comprise a support matrix, a ligand (which binds to the target compound when coupled to the matrix), and optionally a linker arm (for covalently attaching the ligand to the support matrix). In this document, the ligand may include collagen or a derivative thereof.

[0038] As used herein, the term “mixture” refers to, but is not limited to, a combination of components in any physical form (e.g., blends, solutions, suspensions, dispersions, etc.).

[0039] In some embodiments, the chromatographic matrix is ​​an affinity chromatography (“AC”) matrix. As used herein, the terms “affinity chromatography matrix” or “AC matrix” are intended to refer to a solid-phase medium, typically a resin, capable of separating biochemical mixtures based on highly specific binding interactions between the protein of interest (e.g., fibronectin) and the AC matrix. Thus, the solid-phase medium contains a target to which the protein of interest can be reversibly immobilized (depending on buffer conditions).

[0040] Currently known immobilized or solid-phase media capable of containing AC matrices include gel matrices, such as agarose beads (e.g., commercially available Sepharose matrix) and porous beads.

[0041] The binding of proteins of interest to the AC matrix is ​​typically achieved via column chromatography. That is, the AC matrix is ​​formed or packed into a column, and a biochemical mixture containing the protein of interest is passed through the column. The column is then washed by passing a wash solution through the column, and the protein of interest is eluted from the column by passing an elution buffer through the column.

[0042] Therefore, in some embodiments, the disclosed chromatographic matrix is ​​used in chromatographic steps.

[0043] In some embodiments, the matrix or at least the acrylic polymer is in the form of porous microspheres.

[0044] As used herein, the term "porous microsphere" refers to micron-sized particles containing pores, holes, or voids. While the term "microsphere" is used throughout the specification, it should be understood that the term is intended to include any generally spherical microparticles, including those that are not geometrically spherical. In some embodiments, the median pore size is from about 10 nm to 500 nm. In an exemplary embodiment, the median pore size is about 100 nm.

[0045] In some embodiments, the porous microspheres have a median size in the range of 20 μm to 90 μm, or in some embodiments, a median size in the range of 40 μm to 80 μm. In some embodiments, the porous microspheres have a median size of 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, or 90 μm, including any value and range between them.

[0046] The term "size" refers to at least one dimension of the microsphere, such as diameter or length.

[0047] The term "acrylic polymer" refers to a polymer backbone consisting of at least one acrylic group (also known as "acrylic monomer unit").

[0048] The term "polymer" describes an organic substance composed of multiple repeating structural units (monomer units) covalently linked to each other.

[0049] In some embodiments, the term "polymer backbone" generally refers to a polymer comprising monomer units. It should be understood that, in the context of this invention, the term "polymer backbone" refers to the main chain of the polymer backbone and the chains extending from the polymer backbone (i.e., branches).

[0050] The term "monomer unit" refers to a repeating unit derived from a corresponding monomer. Polymers contain monomer units or are made from monomer units. "Derived from" means that the compound is obtained during the polymerization process.

[0051] In some embodiments, the acrylic polymer comprises a plurality of monomer units selected from acrylates or any derivative thereof.

[0052] In some embodiments, the acrylate is selected from, but not limited to, methacrylates and methyl methacrylate (MMA). Examples of nonfunctionalized acrylate and methacrylate monomers that can be optionally polymerized to provide the polymers of the present invention may further include, but are not limited to, methyl acrylate, ethyl acrylate, n-propyl acrylate, isopropyl acrylate, n-butyl acrylate, sec-butyl acrylate, isobutyl acrylate, tert-butyl acrylate, n-pentyl acrylate, isoamyl acrylate, isobornyl acrylate, n-hexyl acrylate, 2-ethylbutyl acrylate, 2-ethylhexyl acrylate, n-octyl acrylate, n-decyl acrylate, methylcyclohexyl acrylate, cyclopentyl acrylate, cyclohexyl acrylate, and methyl methacrylate. Ethyl methacrylate, 2-hydroxyethyl methacrylate, n-propyl methacrylate, n-butyl methacrylate, isopropyl methacrylate, isobutyl methacrylate, n-pentyl methacrylate, n-hexyl methacrylate, isoamyl methacrylate, sec-butyl methacrylate, tert-butyl methacrylate, 2-ethylbutyl methacrylate, methylcyclohexyl methacrylate, cinnamyl methacrylate, crotonyl methacrylate, cyclohexyl methacrylate, cyclopentyl methacrylate, 2-ethoxyethyl methacrylate, and isobornyl methacrylate, and any derivatives or combinations thereof.

[0053] In an exemplary embodiment, the acrylate includes methyl methacrylate. Therefore, in an exemplary embodiment, the acrylic polymer includes polymethyl methacrylate (PMMA).

[0054] The term "derivative" or "chemical derivative" refers to a subject molecule that has been chemically modified but retains most of its original shape, such as a subject molecule substituted with other or different substituents, a subject molecule in which a portion has been oxidized or hydrolyzed, and so on. Such derivatized molecules include, for example, those in which a free carboxyl group can be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. A free hydroxyl group can be derivatized to form O-acyl or O-alkyl derivatives.

[0055] In some embodiments, the acrylic polymer (e.g., PMMA) contains at least one functional group capable of binding fibronectin (also referred to as "activated acrylic polymer"). In exemplary embodiments, the acrylic polymer (e.g., PMMA) contains at least one epoxy group (also referred to as "activated acrylic PMMA").

[0056] In a broad sense, the term "epoxy" refers to a chemical group containing an oxygen atom bonded to two carbon atoms (which are also bonded in some other way). A simple example of an epoxy group is the compound ethylene oxide. In this document, the term "epoxy" also means derivatives including those reacted with or incorporated into collagen or its derivatives.

[0057] In some embodiments, the acrylic polymer containing epoxy groups is selected from glycidyl acrylate and glycidyl methacrylate.

[0058] While there are no particular limitations on the molecular weight, monomer composition, and epoxy equivalent of the polymer, and these can be varied widely, polymers with a number average molecular weight typically in the range of about 500 to about 10,000, for example, in the range of about 1,000 to about 5,000, are suitable. In some embodiments, the average glass transition temperature (T0) of the resulting polymer is... g This allows for an advantageous selection of the temperature range, from -40°C to 40°C.

[0059] In some embodiments, collagen or its derivatives (also referred to as "ligands") are physically attached to acrylic polymers. In some embodiments, collagen or its derivatives are covalently attached to acrylic polymers. Collagen or its derivatives enable the absorption of fibronectin from protein solutions, thereby specifically isolating fibronectin.

[0060] In this document, the term "coupling" (also known as "connection," "attachment," or "binding") can refer to a target biomolecule having covalent and / or non-covalent intermolecular interactions that couple a polymer to a ligand (e.g., collagen or a derivative thereof). Non-covalent interactions can include, for example, electrostatic bonding, hydrogen bonding, van der Waals interactions, hydrophobic forces, etc. The term "non-covalent coupling" is also referred to as "physical coupling."

[0061] As used herein, the term "at least partially" means partially or completely. The term "partially" means to a certain extent. Generally, but not exclusively, the term means at least 50%, at least 75%, at least 80%, at least 90%, or at least 95%.

[0062] The term "covalent coupling" refers to a chemical bond characterized by the sharing of electron pairs between atoms. For example, in some embodiments, acrylic polymers or their functional groups form chemical bonds with collagen or its derivatives, whereas collagen is attached to the polymer surface via other means (e.g., non-covalent intermolecular interactions). It should be understood that polymers covalently attached to ligands can also bind via means other than covalent attachment.

[0063] The term "collagen" is intended to refer to any known collagen of porcine, bovine, or human origin, such as natural collagen, esterified collagen, or a derivative thereof such as methylated, ethylated, or alternatively succinylated collagen, which may or may not be heated, oxidized, or alternatively, crosslinked with another compound. For the purposes of this disclosure, the term "natural collagen" is intended to refer to collagen that has not been chemically modified except for possible treatment with, for example, pepsin to digest telomeres. Therefore, for the purposes of this invention, the term "undenatured collagen" is intended to refer to collagen that has not lost its helical structure.

[0064] In exemplary embodiments, collagen derivatives include gelatin. The term "gelatin" refers to denatured collagen. Gelatin can be derived from collagen in a well-known manner or can be obtained from commercial suppliers. An exemplary method of obtaining gelatin is by heating collagen at a suitable temperature to denature it. Denaturation causes the collagen to irreversibly transform into a random coil structure, which is gelatin. Gelatin can be derived from one or more collagen sources and can be derived from one or more types of collagen, such as, but not limited to, type I, II, III, and / or VI collagen. Exemplary sources of derived gelatin include, but are not limited to, sea cucumber dermal collagen, bovine, goat, porcine, sheep, or other suitable donor mammalian collagen, and marine animal collagen (such as echinodermal collagen). Gelatin can be derived from collagen obtained from mammalian cells synthesized in vitro. Gelatin can be derived from collagen obtained from molecularly engineered constructs and synthesized by bacteria, yeast, or any other molecularly manipulated cell type.

[0065] In some embodiments, collagen or its derivatives (e.g., gelatin) are coupled to an acrylic polymer via functional groups (e.g., epoxy groups) on the polymer or after reacting with functional groups on the polymer. Without being bound by any particular mechanism, it is assumed that the epoxy groups on the PMMA surface are capable of reacting with the amino groups of gelatin and forming a covalent bond between them.

[0066] The term "effective absorption of fibronectin" refers to a fibronectin absorption efficiency of at least 10 mg fibronectin / g resin. In some embodiments, this term also refers to obtaining a fibronectin purity greater than 90%. In this regard, referring to Table 3 in the following Examples section, which shows that an amount greater than 1.17 gelatin / g PMMA provides a fibronectin absorption efficiency greater than 10 (mg fibronectin / g resin), with an estimated purity greater than 90%, while the gelatin does not gel at room temperature (i.e., approximately 20°C to 35°C).

[0067] Therefore, in another aspect, a chromatographic matrix is ​​provided comprising: (i) an acrylic polymer, and (ii) collagen or a derivative thereof; wherein the acrylic polymer and the collagen or a derivative thereof are at least partially covalently coupled, and wherein the collagen or a derivative thereof is coupled in an amount greater than 1.17 mg and at most about 16 mg of collagen or a derivative thereof / g of acrylic polymer.

[0068] The phrase "greater than 1.17 mg and at most about 16 mg collagen or its derivatives / g acrylic polymer" means, for example, 1.18 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, 3 mg, 3.1 mg, 3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9 mg, 4 mg, 4.1 mg, 4.2 mg, 4.3 mg per gram of acrylic polymer. g, 4.4mg, 4.5mg, 4.6mg, 4.7mg, 4.8mg, 4.9mg, 5mg, 5.1mg, 5.2mg, 5.3mg, 5.4mg, 5.5mg, 5.6mg, 5.7mg, 5.8mg, 5.9mg, 6mg, 6.1mg, 6.2mg, 6.3mg, 6.4m g, 6.5mg, 6.6mg, 6.7mg, 6.8mg, 6.9mg, 7mg, 7.1mg, 7.2mg, 7.3mg, 7.4mg, 7.5mg, 7.6mg, 7.7mg, 7.8mg, 7.9mg, 8mg, 8.1mg, 8.2mg, 8.3mg, 8.4mg, 8.5m g, 8.6mg, 8.7mg, 8.8mg, 8.9mg, 9mg, 9.1mg, 9.2mg, 9.3mg, 9.4mg, 9.5mg, 9.6mg, 9.7mg, 9.8mg, 9.9mg, 10mg, 10.1mg, 10.2mg, 10.3mg, 10.4mg, 10.5m g, 10.6mg, 10.7mg, 10.8mg, 10.9mg, 11mg, 11.1mg, 11.2mg, 11.3mg, 11.4mg, 11.5mg, 11.6mg, 11.7mg, 11.8mg, 11.9mg, 12mg, 12.1mg, 12.2mg, 12.3m g, 12.4mg, 12.5mg, 12.6mg, 12.7mg, 12.8mg, 12.9mg, 13mg, 13.1mg, 13.2mg, 13.3mg, 13.4mg, 13.5mg, 13.6mg, 13.7mg, 13.8mg, 13.9mg, 14mg, 14.1m g.9 mg or 16 mg of collagen or its derivatives (e.g., gelatin), including any values ​​and ranges in between.

[0069] In some embodiments, the amount of collagen or its derivatives is greater than 1.17 to about 15.4 / g acrylic polymer. In some embodiments, the amount of collagen or its derivatives is about 11 to about 15.4 / g acrylic polymer.

[0070] In some embodiments, the chromatographic matrix is ​​characterized in that 1 g of the chromatographic matrix can bind at least 1 mg of fibronectin. In some embodiments, the chromatographic matrix is ​​characterized in that 1 g of the chromatographic matrix can bind at least 2 mg of fibronectin. In some embodiments, the chromatographic matrix is ​​characterized in that 1 g of the chromatographic matrix can bind at least 3 mg of fibronectin. In some embodiments, the chromatographic matrix is ​​characterized in that 1 g of the chromatographic matrix can bind at least 4 mg of fibronectin. In some embodiments, the chromatographic matrix is ​​characterized in that 1 g of the chromatographic matrix can bind at least 5 mg of fibronectin. In some embodiments, the chromatographic matrix is ​​characterized in that 1 g of the chromatographic matrix can bind at least 6 mg of fibronectin.

[0071] In some embodiments, the chromatographic matrix is ​​characterized in that 1 g of the chromatographic matrix can bind fibronectin in an amount ranging from 1 mg to 20 mg. In some embodiments, the chromatographic matrix is ​​characterized in that 1 g of the chromatographic matrix can bind fibronectin in an amount ranging from 5 mg to 20 mg. In some embodiments, the chromatographic matrix is ​​characterized in that 1 g of the chromatographic matrix can bind fibronectin in an amount ranging from 6 mg to 20 mg. In some embodiments, the chromatographic matrix is ​​characterized in that 1 g of the chromatographic matrix can bind fibronectin in an amount ranging from 5 mg to 14 mg. In some embodiments, the chromatographic matrix is ​​characterized in that 1 g of the chromatographic matrix can bind fibronectin in an amount ranging from 6 mg to 13 mg. In some embodiments, the chromatographic matrix is ​​characterized in that 1 g of the chromatographic matrix can bind fibronectin in amounts of 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg, including any values ​​and ranges between them. In this regard, refer to Table 3 in the following Examples section, which shows the amount of gelatin coupled to the acrylic polymer (mg gelatin / g PMMA, as obtained from gelatin solutions of various concentrations). In some embodiments, the term "bonded" means at least partially covalently bonded.

[0072] Therefore, in another aspect, a chromatographic matrix is ​​provided comprising: (i) an acrylic polymer, and (ii) collagen or a derivative thereof; wherein the acrylic polymer and the collagen or a derivative thereof are at least partially covalently coupled, and wherein the collagen or a derivative thereof is present in a concentration greater than about 0.275 mg and at most about 3.8 mg collagen or a derivative thereof / cm³. 3 Quantitative coupling of acrylic polymers.

[0073] The term "greater than approximately 0.275 mg and at most approximately 3.8 mg collagen or its derivatives / cm" is used. 3 "Acrylic polymers" means, for example, 0.275 mg, 0.28 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, 3 mg, 3.1 mg, 3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, or about 3.8 mg of collagen or its derivatives (e.g., gelatin) per cm³. 3 Acrylic polymers, including any values ​​and ranges between them.

[0074] In some embodiments, the amount of collagen or its derivatives is greater than 0.27 to about 3.6 / cm³. 3 Acrylic polymers. In some embodiments, the amount of collagen or its derivatives is about 2.6 to about 3.6 / cm³. 3 Acrylic polymers.

[0075] In some implementations, the chromatographic matrix is ​​characterized by 1 cm 3 The chromatographic matrix is ​​capable of binding at least 0.8 mg of fibronectin. In some embodiments, the chromatographic matrix is ​​characterized by a 1 cm... 3 The chromatographic matrix is ​​capable of binding at least 1.5 mg of fibronectin. In some embodiments, the chromatographic matrix is ​​characterized by a 1 cm... 3 The chromatographic matrix is ​​capable of binding at least 2 mg of fibronectin. In some embodiments, the chromatographic matrix is ​​characterized by a 1 cm... 3 The chromatographic matrix is ​​capable of binding at least 3 mg of fibronectin. In some embodiments, the chromatographic matrix is ​​characterized by a 1 cm... 3 The chromatographic matrix can bind at least 3.5 mg of fibronectin.

[0076] In some implementations, the chromatographic matrix is ​​characterized by 1 cm3 The chromatographic matrix is ​​capable of binding fibronectin in amounts ranging from 0.8 mg to approximately 4 mg. In some embodiments, the chromatographic matrix is ​​characterized by a 1 cm... 3 The chromatographic matrix is ​​capable of binding fibronectin in amounts ranging from 0.9 mg to 3.6 mg. In some embodiments, the chromatographic matrix is ​​characterized by a 1 cm... 3 The chromatographic matrix can bind amounts of fibronectin of 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, or about 4 mg, including any values ​​and ranges in between.

[0077] In some embodiments, the chromatographic matrix is ​​used in chromatographic processes, such as methods for purifying fibronectin from a source solution, i.e., for binding fibronectin from a source solution containing fibronectin. In some embodiments, the chromatographic matrix is ​​used in medium-pressure liquid chromatography. Compared to soft matrices (such as Sepharose, which is cross-linked, bead-like agarose), the chromatographic matrix of the present invention has sufficient rigidity to withstand medium to high pressures and / or high flow rates during chromatographic processes. Therefore, although Sepharose is widely used as a chromatographic medium, it is a soft medium and cannot withstand high pressures. To avoid the accumulation of back pressure and column bed collapse, a rigid chromatographic matrix, such as PMMA, is employed.

[0078] The term "fibronectin" refers to a disulfide-linked diglycoprotein that exists in soluble form in blood plasma and other body fluids, and is deposited as fibrils as a major component of the extracellular matrix of loose connective tissue. It consists of three distinct structural motifs, known as type I, II, and III homology, which produce a modular organization of fibronectin molecules, in which several of its biological activities can be attributed to specific structural domains.

[0079] In some embodiments, the pressure applied in the medium-pressure liquid chromatography is up to 8 bar. In some embodiments, the pressure applied in the medium-pressure liquid chromatography is up to 7 bar. In some embodiments, the pressure applied in the medium-pressure liquid chromatography is up to 6 bar. In some embodiments, the pressure applied in the medium-pressure liquid chromatography is up to 5 bar. In some embodiments, the pressure applied in the medium-pressure liquid chromatography is up to 4 bar. In some embodiments, the pressure applied in the medium-pressure liquid chromatography is up to 3 bar. In some embodiments, the pressure applied in the medium-pressure liquid chromatography is between 1 bar and 20 bar; in some embodiments, between 1 bar and 10 bar; or in some embodiments, between 1 bar and 5 bar. In some implementations, the pressure applied in the medium-pressure liquid chromatography is 1 bar, 2 bar, 3 bar, 4 bar, 5 bar, 6 bar, 7 bar, 8 bar, 9 bar, 10 bar, 11 bar, 12 bar, 13 bar, 14 bar, 15 bar, 16 bar, 17 bar, 18 bar, 19 bar, or 20 bar, including any value and range in between.

[0080] In one aspect of this disclosure, a composition (“Composition Aspect”) is provided, comprising: (i) an acrylic polymer; (ii) collagen or a derivative thereof; and (iii) a buffer medium comprising a borate.

[0081] As is known in the art, a buffer solution is typically used to adjust the pH to a desired range. Generally, but not exclusively, a pH of about 6 to about 8 is desirable; however, this may need to be adjusted due to considerations such as the stability or solubility of the reagent in solution. Many buffer solutions containing salts of inorganic acids, such as phosphates, borates, and sulfates, are known. While any buffer solution can be used in the compositions disclosed herein, the use of a borate buffer solution is particularly useful in certain circumstances. Referring to Table 2 in the Examples section below, which shows that a borate buffer solution (typically at a concentration of about 0.2 M) is preferred for coupling gelatin with PMMA prior to applying the formed matrix to fibronectin purification.

[0082] In some embodiments, the term "borate-containing buffer medium" or "borate buffer" can refer to any combination of boric acid and one or more conjugate bases, such that the pH is adjusted to a desired range. While not intended to limit the scope of the invention in any way, or to restrict its scope by any particular theory, it is believed that borate buffers can facilitate the coupling of collagen (e.g., gelatin) with acrylic polymers (e.g., PMMA). In some embodiments, the pH of the buffer is adjusted to approximately 8.

[0083] The embodiments described above, such as those relating to collagen, gelatin, acrylic polymers, and epoxy groups, are further incorporated into the composition aspect.

[0084] Collagen or gelatin can be present at concentrations ranging from 0.05% to 5%, for example, from 0.1% to 5% (v / w) or from 0.1% down to less than about 4%. In some embodiments, the concentration of collagen or gelatin is 0.05%, 0.1%, 0.25%, 0.5%, 1%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%, or 5% (v / w), including any values ​​and ranges between them.

[0085] As shown in the following Examples section, the inventors have successfully utilized a composition to form the chromatographic matrix disclosed herein, the composition comprising: (i) an acrylic polymer; (ii) collagen or a derivative thereof; and (iii) a buffer medium comprising a borate. In this document, “successfully utilized” means that the acrylic polymer (e.g., PMMA) and collagen or a derivative thereof (e.g., gelatin) are coupled, for example, at least partially covalently coupled, wherein the collagen or derivative thereof is coupled in a desired amount that allows fibronectin to be efficiently absorbed onto the matrix, for example, in an amount greater than 1.17 mg and at most about 16 mg collagen or a derivative thereof / g acrylic polymer.

[0086] Therefore, in another aspect, a method for preparing a chromatographic matrix (“preparation method”) is provided, the method comprising: (i) contacting an acrylic polymer with (ii) collagen or a derivative thereof dissolved in a buffer medium containing a borate; thereby obtaining an acrylic polymer coupled to collagen or a derivative thereof.

[0087] The embodiments described above, such as those relating to collagen, gelatin, acrylic polymers, epoxy groups, and the term "coupling," are further incorporated into this aspect of the preparation method.

[0088] In some implementations, the buffer medium has a pH of about 7 to about 8, such as 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4 or 8.5, including any value and range between them.

[0089] In some embodiments, collagen or its derivatives (e.g., gelatin) are present in the buffer medium at a concentration ranging from about 0.2 wt% to about 4 wt%, or in some embodiments, at a concentration ranging from 0.25 wt% to 3 wt%, such as 0.25 wt%, 0.5 wt%, 0.75 wt%, 1 wt%, 1.25 wt%, 1.5 wt%, 1.75 wt%, 2 wt%, 2.25 wt%, 2.5 wt%, 2.75 wt%, or 3 wt%, including any values ​​and ranges between them. In some embodiments, collagen or its derivatives (e.g., gelatin) are present in the buffer medium at a concentration higher than 2 wt%. In some embodiments, collagen or its derivatives (e.g., gelatin) are present in the buffer medium at a concentration ranging from higher than 2 wt% to about 4 wt%, or in some embodiments, at a concentration higher than 2 wt% to about 3 wt%.

[0090] In another aspect, a method (“purification method”) for purifying fibronectin from a source solution is provided, the method comprising the step of passing a source solution containing fibronectin through a column containing a chromatographic matrix disclosed herein, under conditions that allow fibronectin to bind to a matrix.

[0091] The term “to pass through” or any grammatical variation thereof refers to a solution entering a component (e.g., a column), moving through at least a portion of the component’s interior, and sometimes leaving at least a portion of the component.

[0092] In some embodiments, the column is an affinity column. The term "affinity column" refers to a molecular fractionation device comprising beads having specific chemical properties for separating or reacting with biomolecules. In some embodiments, the beads comprise, in one embodiment, the chromatographic matrix disclosed herein. It should be noted that the use of the term "affinity column" does not imply a requirement for a conventionally physically constrained cylindrical or vertical column. Conditions that allow fibronectin to bind to the matrix include, but are not limited to, the pH of the buffer solution for equilibrating the column, the temperature of the source solution, the flow rate of the refill solution, the pressure applied at / within the column ("column pressure"), or any combination thereof. For example, the temperature can be controlled between above 0°C and below 100°C, and includes ranges such as, but not limited to, 10°C to 50°C, 45°C to 50°C, 20°C to 40°C, or 20°C to 30°C.

[0093] In some implementations, the pressure is at most 5 bar. In some implementations, the pressure is at most 4 bar. In some implementations, the pressure is at most 3 bar. In some implementations, the pressure is between 1 bar and 20 bar; in some implementations, between 1 bar and 10 bar; or in some implementations, between 1 bar and 5 bar. In some implementations, the pressure is 1 bar, 2 bar, 3 bar, 4 bar, 5 bar, 6 bar, 7 bar, 8 bar, 9 bar, 10 bar, 11 bar, 12 bar, 13 bar, 14 bar, 15 bar, 16 bar, 17 bar, 18 bar, 19 bar, or 20 bar, including any value and range between them.

[0094] In some implementations, the column pressure is controlled by a pump. The term "pump" refers to any device that moves fluid by applying suction or pressure.

[0095] In some embodiments, the column is equilibrated with a buffer solution (“equilibration buffer”). In some embodiments, the pH of the equilibration buffer is in the range of 6.5 to 8, or in some embodiments, in the range of about 7 to about 7.4, such as 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8, including any value and range in between. During purification, the pH value does not need to be maintained at a substantially consistent value. In an exemplary embodiment, the equilibration buffer comprises phosphate-buffered saline (PBS).

[0096] In some embodiments, the method includes removing contaminants by washing the chromatographic matrix (e.g., using a wash buffer). In exemplary embodiments, the equilibration buffer comprises phosphate-buffered saline (PBS). In some embodiments, the pH of the wash buffer is in the range of 6.5 to 8, or in some embodiments, in the range of about 7 to about 7.4, such as 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8, including any values ​​and ranges therebetween.

[0097] In addition, the flow rate of the source solution (usually PBS) is 0.1 ml / min to 10 ml / min, 0.1 ml / min to 6 ml / min, 0.2 ml / min to 10 ml / min, 0.25 ml / min to 5 ml / min or 0.5 ml / min to 3 ml / min. n intermediate, for example, 0.1ml / min, 0.2ml / min, 0.3ml / min, 0.4ml / min, 0.5ml / min, 0.6ml / min, 0.7ml / min, 0.8ml / min, 0.9ml / min, 1ml / min, 1.1ml / min, 1.2ml / m in, 1.3ml / min, 1.4ml / min, 1.5ml / min, 1.6ml / min, 1.7ml / min, 1.8ml / min, 1.9ml / min, 2ml / min, 2.1ml / min, 2.2ml / min, 2.3ml / min, 2.4ml / min, 2.5m l / min, 2.6ml / min, 2.7ml / min, 2.8ml / min, 2.9ml / min, 3ml / min, 3.1ml / min, 3.2ml / min, 3.3ml / min, 3.4ml / min, 3.5ml / min, 3.6ml / min, 3.7ml / min, 3 .8ml / min, 3.9ml / min, 4ml / min, 4.1ml / min, 4.2ml / min, 4.3ml / min, 4.4ml / min, 4.5ml / min, 4.6ml / min, 4.7ml / min, 4.8ml / min, 4.9ml / min, 5ml / min, 5.1ml / min, 5.2ml / min, 5.3ml / min, 5.4ml / min, 5.5ml / min, 5.6ml / min, 5.7ml / min, 5.8ml / min, 5.9ml / min, 6ml / min, 6.1ml / min, 6.2ml / min, 6.3ml / m in, 6.4ml / min, 6.5ml / min, 6.6ml / min, 6.7ml / min, 6.8ml / min, 6.9ml / min, 7ml / min, 7.1ml / min, 7.2ml / min, 7.3ml / min, 7.4ml / min, 7.5ml / min, 7.6m l / min, 7.7ml / min, 7.8ml / min, 7.9ml / min, 8ml / min, 8.1ml / min, 8.2ml / min, 8.3ml / min, 8.4ml / min, 8.5ml / min, 8.6ml / min, 8.7ml / min, 8.8ml / min, 8.9 ml / min, 9 ml / min, 9.1 ml / min, 9.2 ml / min, 9.3 ml / min, 9.4 ml / min, 9.5 ml / min, 9.6 ml / min, 9.7 ml / min, 9.8 ml / min, 9.9 ml / min, or 10 ml / min, including any value and range in between.

[0098] In some implementations, the method includes eluting fibronectin bound to the chromatographic matrix, for example, using an elution buffer (“elution step”). The elution step can be performed sequentially or simultaneously with the step of passing the source solution through the column.

[0099] The term “elution” or any grammatical variation thereof is used herein to refer to the release of adsorbed fibronectin from the chromatographic matrix. Generally, the term “elution” as disclosed herein is interchangeable with the term “desorption.” In some embodiments, the term refers to the release of at least 80%, at least 85%, at least 90%, or at least 95% of the fibronectin. Elution may be performed under certain elution conditions. Generally, but not exclusively, elution conditions include the use of non-isotropic conditions, for example, solutions or conditions different from those used for, for example, loading a source solution containing fibronectin and / or solutions or conditions different from those used in previous steps.

[0100] The term "simultaneously" as used herein does not necessarily mean that all related steps are performed simultaneously, and can also refer to, for example, starting the washing step first and then immediately passing the mobile phase through the column, or, for example, passing the mobile phase through the column first and then immediately performing the elution step. In some embodiments, "immediately" means within 0 to 20 seconds, 0 to 10 seconds, or 0 to 2 seconds, such as 0 seconds, 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 11 seconds, 12 seconds, 13 seconds, 14 seconds, 15 seconds, 16 seconds, 17 seconds, 18 seconds, 19 seconds, or 20 seconds, including any value and range therebetween.

[0101] Elution buffers can be used to elute fibronectin from a chromatographic matrix. In exemplary embodiments, the elution buffer contains urea, typically but not limited to, urea in the concentration range of 4M to 6M. In some embodiments, the eluted fibronectin can be collected. Therefore, purification methods can include the step of collecting the eluted fibronectin.

[0102] The term "source solution" broadly refers to a combination, mixture, and / or admixture of components having at least one liquid component and a protein of interest. Solutions typically contain at least one solvent in a larger quantity or volume than the solute. Typical solvents include water. In some embodiments, the source solution contains a mixture of proteins. In some embodiments, the source solution contains plasma, typically blood plasma or a fraction thereof. In some embodiments, the plasma comprises oxalate-treated plasma. In some embodiments, the source solution contains plasma harvested from mammals. In some embodiments, the mammals are selected from humans, horses, cattle, and pigs. In an exemplary embodiment, the source solution contains porcine plasma.

[0103] In some embodiments, the eluted fibronectin is purified to at least 90% by weight. As used herein, the term "purified" means that the substance has been separated under conditions that reduce or eliminate extraneous substances (i.e., contaminants). For example, purified protein is substantially free of other proteins associated with it in the source solution. As used herein, the term "substantially free" is used operationally in the context of analytical testing of the material. Thus, a material substantially free of contaminants is at least 50% by weight pure, at least 90% by weight pure, at least 97% by weight pure, or optionally at least 99% by weight pure. Purity can be assessed by chromatography, gel electrophoresis, immunoassay, compositional analysis, bioassay, and other methods known in the art. In some embodiments, the eluted fibronectin is purified to the range of about 90% by weight to about 97% by weight. For this purpose, refer to Table 3 in the Examples section below and... Figure 1 The SDS-PAGE gel results provided are shown in the image. Fibronectin purity was assessed using Imagelab (Bio-Rad software).

[0104] In some embodiments, fibronectin is concentrated with a purification factor of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, or at least 70. In some embodiments, fibronectin is concentrated with a purification factor of about 50 to about 80. In some embodiments, fibronectin is concentrated with a purification factor of about 50, about 60, about 70, or about 80 (inclusive of any values ​​and ranges between them). Exemplary methods for calculating the purification factor are described in the following Examples section (see Table 5). In some embodiments, the chromatographic matrix disclosed herein can be reused, for example for fibronectin binding, at least once, at least twice, or at least three times, for example, once to three times, without a reduction in the purification factor of no more than 20% after repeated application of the matrix under the same conditions.

[0105] Therefore, fibronectin obtained by purification methods is provided, as used in any of its embodiments.

[0106] In some embodiments, the eluted fibronectin is purified to, for example, at least 97% by weight by a single chromatographic step. Typically, a single chromatographic step does not rely on multiple wash buffers or multiple elution buffers (e.g., buffers containing urea) to recover the bound fibronectin.

[0107] As used in this article, the term “about” means ±10%.

[0108] The terms “comprising,” “including,” “containing,” “containing,” “implying,” “integrating,” “having,” “having,” and variations thereof mean “including but not limited to.” The term “composed of” means “including and limited to.” The term “essentially composed of” means that a composition, method, or structure may contain additional ingredients, steps, and / or components, provided that such additional ingredients, steps, and / or components do not substantially alter the essential and novel characteristics of the composition, method, or structure protected by the claims.

[0109] The term "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations and / or excludes features of other implementations.

[0110] The term "optionally" is used herein to mean "provided in some embodiments but not in others." Any specific embodiment of the invention may include several "optional" features unless such features conflict.

[0111] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural references. For example, the terms “a compound” or “at least one compound” can include multiple compounds (including mixtures thereof).

[0112] Throughout this application, various embodiments of the invention may be presented in a scope format. It should be understood that the scope format is merely for convenience and brevity and should not be construed as a fixed limitation on the scope of the invention. Therefore, a scope description should be considered to have all clearly disclosed possible sub-scopes and the respective numerical values ​​within those scopes. For example, a scope such as 1 to 6 should be considered to have clearly disclosed sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the respective numerical values ​​within those scopes, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the scope.

[0113] Whenever this document indicates a range of numbers, it means that any enumerated number (fraction or integer) within the indicated range is included. The phrases “range is” between the first and second indicated numbers and “range is from the first indicated number to the second indicated number” are used interchangeably in this document and refer to all fractions and integers including the first and second indicated numbers and those in between.

[0114] As used herein, the term “method” refers to the manner, means, techniques and procedures used to accomplish a given task, including but not limited to those manner, means, techniques and procedures known to or readily developed from known manner, means, techniques and procedures by practitioners in the fields of chemistry, analysis, pharmacology, biology, biochemistry and medicine.

[0115] As used herein, and unless otherwise indicated, the terms “by weight,” “w / w,” “weight percentage,” or “weight %” are used interchangeably in this document and describe the concentration of a particular substance in the total weight of the respective mixture, solution, formulation, or composition.

[0116] In cases where conventions such as "at least one of A, B, and C" are used, such structures are generally intended to have a meaning that those skilled in the art will understand (e.g., "a composition having at least one of A, B, and C" will include, but is not limited to, compositions having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art should also understand that, in practice, whether in the detailed description, claims, or drawings, transitional words and / or phrases presenting two or more alternative terms should be understood to cover the possibility of including one of the terms, any one of the terms, or both of the terms. For example, the phrase "A or B" will be understood to include the possibility of "A," "B," or "A and B."

[0117] It should be understood that, for clarity, certain features of the invention described in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various structures of the invention described in the context of a single embodiment may also be provided individually or in any suitable sub-combination, or as appropriately provided in any other said embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiments would not function without those elements.

[0118] The various embodiments and aspects of the invention as described above and claimed in the claims section below are experimentally supported in the following examples.

[0119] Example

[0120] The following embodiments, together with the foregoing description, illustrate some implementations of the invention in a non-limiting manner.

[0121] Example 1: Purification of fibronectin via PMMA-gelatin matrix

[0122] Material

[0123] Gelatin (Sigma Aldrich, catalog number V900863, CAS: 9000-70-8);

[0124] The activated medium comprises porous PMMA microspheres (i.e., PMMA with epoxy groups on its surface; product name: UniEpoxy-50L, supplied by Suzhou Nano-Micro Technology Co., Ltd., catalog number 15302-06132-01012; also known as "activated PMMA"). The weight-average molecular weight (MW) of PMMA ranges from 500,000 to 1,000,000. Table 1 below summarizes the main technical specifications of PMMA.

[0125] Table 1: Main Technical Specifications of PMMA

[0126]

[0127] Gelatin was purchased from Sigma Aldrich (product catalog number V900863, CAS: 9000-70-8).

[0128] Fibronectin source: porcine plasma. Technical data of porcine plasma: Total protein: 55 mg / ml; fibronectin concentration: 0.15 mg / ml.

[0129] method

[0130] Coupling of activated PMMA with gelatin: In an exemplary procedure, gelatin was dissolved in borate buffer (pH 7.6) at various concentrations (by weight): 0.1%, 0.25%, 0.5%, 1%, 2%, 3%, and 4%. Activated PMMA was then added to the gelatin solution at a 1:1 weight ratio, followed by gentle shaking at 37°C for 16 hours. Subsequently, the obtained resin (gelatin coupled with PMMA) was collected by centrifugation at 3000 rpm for 10 minutes. The resin was then washed with purified water and centrifuged at 3000 rpm for 10 minutes. This washing step and subsequent centrifugation were repeated once more.

[0131] The resin was then incubated in 0.2 M ethanolamine solution for 5 hours to block the active functionalized (epoxy) groups on the PMMA, and the resin was collected by centrifugation at 3000 rpm for 10 minutes, followed by washing with phosphate-buffered saline (PBS) (pH 7.4).

[0132] Use the above procedure to perform other exemplary procedures, but use other buffers instead of borate: NaHCO3, sulfate solution, sodium sulfate, and PBS with a gelatin concentration of 1% by weight.

[0133] Fibronectin Purification: In an exemplary procedure, a matrix for fibronectin purification is formed using 5-10 g of wet PMMA material and an equal weight of 0.25% to 3% gelatin solution. Using the gelatin-PMMA matrix obtained through the above procedure, fibronectin is purified from porcine plasma by single-step affinity liquid chromatography (LC). This process is performed using pressure chromatography with a chromatographic system. PMMA-gelatin resin is packed into a column with a 1:1 compressibility factor (the compressibility factor is the sedimentation volume of the resin divided by the volume of the resin after packing; since PMMA-gelatin is a rigid resin, its volume remains unchanged after packing). The column is equilibrated with PBS at pH 7.0 to 7.4 at room temperature (25°C ± 2°C) and then the starting material is loaded.

[0134] Other general loading conditions:

[0135] i. Column pressure: 0.1MPa-0.3MPa (1 bar-3 bar);

[0136] ii. Flow rate: 1 ml / min;

[0137] iii. Temperature: Approximately 25°C;

[0138] iv. Column volume (CV): 1 ml - 3 ml;

[0139] v. Volume of loaded material: 15CV-20CV;

[0140] vi. Equilibration buffer: PBS

[0141] vii. pH 7-7.4.

[0142] The washing buffer is the same as the equilibration buffer (PBS, pH 7.0-7.4).

[0143] Specific elution conditions:

[0144] i. Volume of eluent: depends on the elution peak, 1.5CV-3CV, typically up to 20ml

[0145] ii. Flow rate: 1 ml / min;

[0146] i. Elution buffer: 4M-6M urea.

[0147] The LC equipment used was manufactured by GE Healthcare Co., Ltd. Pure 150 system, Pure chromatography systems are highly versatile and modular systems with many design features that facilitate reliable purification. The system includes... Pure instrumentation and UNICORN 6 control software, with a modular design in which all valves, monitors and columns are mounted on the front-facing wet side of the system to allow easy interaction with the instrumentation module.

[0148] result

[0149] PMMA was conjugated with gelatin in different buffer solutions: the results are summarized in Table 2 below.

[0150] Table 2: Results of PMMA-gelatin linkage using various buffer solutions

[0151]

[0152] The results showed that the gelatin / PMMA coupling experiments were less effective with NaHCO3 or PBS buffer compared to borate buffer, making them the second-best resins. Coupling experiments also failed with ammonium sulfate and sodium sulfate buffers. Therefore, borate buffer was chosen for gelatin-PMMA coupling prior to fibronectin purification procedures.

[0153] Fibronectin purification: Results are provided in Table 3 below;

[0154] Table 3: Various ratios of PMMA to gelatin using borate buffer

[0155]

[0156] *Fibronectin purity assessed from SDS-PAGE using Imagelab (Bio-Rad software) (see [link]). Figure 1 );

[0157] **A gel is produced at room temperature using a 4% gelatin solution, and the coupling reaction cannot proceed;

[0158] ***Convert the value to mg gelatin / cm³ after dividing by 4.255. 3 Resin (or mg fibronectin / cm) 3 (resin)(1cm) 3 The wet PMMA resin contains 0.235g of dry PMMA powder, therefore 1g of dry PMMA powder contains 4.255cm³ of dry PMMA powder. 3(in wet PMMA resin); for example, 3.83 mg gelatin / g PMMA yields 0.9 mg gelatin / cm³. 3 PMMA (and 12.34 mg fibronectin / g resin yielded 2.9 mg fibronectin / cm) 3 (resin).

[0159] For gelatin concentrations of 0.25% and higher, the purity of fibronectin exceeds 90%, as observed separately based on SDS-PAGE results (see [link to SDS-PAGE results]). Figure 1 ).

[0160] It is worth noting that although Sepharose is widely used as a chromatographic medium, it is a soft medium and cannot withstand high pressure. Refer to Table 4 below for usage instructions. The comparison parameters obtained in 4B were as follows: Specifically, the maximum flow rate was 1.6 ml / min (when performing chromatography on a 1 cm diameter column), and the fibronectin binding capacity was approximately 1 mg / cm³. 3 Resin (with PMMA at 3.1 mg / cm) 3 Compared to resin (=13.19mg / g).

[0161] Table 4: Main Technical Specifications of Sepharose Gelatin

[0162] Fibronectin binding capacity 1 mg / mL resin color White Ligand group density 4.5mg~8mg gelatin / mL resin diameter ~90μm pH stability 3-10 Maximum pressure (MPa) 0.2 Maximum flow rate 75cm / h (1.6ml / min)

[0163] The general procedure for SDS-PAGE testing is as follows: Attach a gel plate containing 10% SDS-PAGE gel to the instrument. Then, pour 400 ml of Tris-glycine electrophoresis buffer into both the upper and lower chambers. Next, mix the protein sample with the loading buffer and boil in a water bath for 5 minutes. Then, load the sample and protein standards onto the gel. Each lane can hold 5 μl–10 μl of sample.

[0164] Then, run electrophoresis at a constant voltage of 100V. After the dye front enters the separating gel, adjust the voltage to 150V. The run typically takes about 1-2 hours. Next, remove the plates from the apparatus and spacers. Pry the plates open by inserting a scraper and twisting the plate upwards. The gel should adhere to one of the plates. Then, float the gel off the plates in a container containing Coomassie blue solution.

[0165] The gel was then incubated in Coomassie blue solution for 30 minutes, followed by destaining with destaining buffer 3-4 times (30 minutes each time). After the gel background became transparent, a photographic image of the gel was taken using a gel imaging system, and then the purity was analyzed using ImageLab software.

[0166] The results showed that coupling increased with increasing gelatin concentration, and the purity of fibronectin increased accordingly. If the ligand (gelatin) coupling efficiency was low, for example when using a gelatin concentration of 0.1% or lower, the purification performance of gelatin-PMMA was correspondingly unsatisfactory (e.g., well below 90%).

[0167] Determination of fibronectin absorption efficiency: To determine fibronectin absorption efficiency, PMMA-gelatin resin (13.19 mg / g; see Table 3) was packed into a chromatographic column and the column volume (CV) was calculated. Porcine plasma containing fibronectin was overloaded (typically exceeding 20 CV). The fractions were then collected, and the fibronectin concentration in the fraction was measured. The material was then loaded into an enzyme-linked immunosorbent assay (ELISA) kit. The total fibronectin absorption was then calculated by dividing the total fibronectin absorption by the CV, thus determining the fibronectin absorption efficiency per ml of resin. The wet resin was then freeze-dried, and the fibronectin absorption efficiency per gram of dry resin was separately determined.

[0168] Purification factor data: The purification factor is calculated by dividing the fibronectin specification in the elution fraction by the fibronectin specification in porcine plasma, as shown in Table 5 below.

[0169] Table 5: Purification coefficient data of single-step affinity chromatography using PMMA-gelatin

[0170]

[0171] As shown in Table 5 above, the coefficient of fibronectin concentration is approximately 71.66.

[0172] Although the invention has been described in conjunction with its specific embodiments, many alternatives, modifications, and variations will obviously be apparent to those skilled in the art. Therefore, this document is intended to cover all such alternatives, modifications, and variations that fall within the substantive and broad scope of the appended claims.

Claims

1. A composition for preparing a chromatographic matrix, the composition comprising: (i) polymethyl methacrylate (PMMA); (ii) collagen or a derivative thereof; and (iii) a buffer medium comprising a borate, wherein the chromatographic matrix comprises: (i) polymethyl methacrylate (PMMA) in the form of porous microspheres, and (ii) collagen or a derivative thereof; wherein the polymethyl methacrylate (PMMA) contains functional groups that allow the collagen or a derivative thereof to bind to the polymethyl methacrylate (PMMA), wherein the polymethyl methacrylate (PMMA) and the collagen or a derivative thereof are coupled in an amount greater than 1.17 mg and at most 16 mg collagen or a derivative thereof / g polymethyl methacrylate (PMMA), and wherein 1 g of the chromatographic matrix is ​​capable of binding fibronectin in an amount ranging from 6 mg to 13 mg.

2. The composition according to claim 1, wherein the collagen derivative comprises gelatin.

3. The composition according to claim 1 or 2, wherein the functional group comprises an epoxy group.

4. A method for preparing a chromatographic matrix, the method comprising contacting (i) polymethyl methacrylate (PMMA) with (ii) collagen or a derivative thereof dissolved in a buffer medium containing borate, thereby obtaining polymethyl methacrylate (PMMA) coupled to said collagen or a derivative thereof, wherein said chromatographic matrix comprises the following components: (i) polymethyl methacrylate (PMMA) in the form of porous microspheres, and (ii) collagen or a derivative thereof; wherein the polymethyl methacrylate (PMMA) contains functional groups that allow the collagen or a derivative thereof to bind to the polymethyl methacrylate (PMMA), wherein the polymethyl methacrylate (PMMA) and the collagen or a derivative thereof are coupled in an amount greater than 1.17 mg and at most 16 mg collagen or a derivative thereof / g polymethyl methacrylate (PMMA), and wherein 1 g of the chromatographic matrix is ​​capable of binding fibronectin in an amount ranging from 6 mg to 13 mg.

5. The method according to claim 4, wherein the functional group comprises an epoxy group.

6. The method according to claim 4 or 5, wherein the collagen derivative comprises gelatin.

7. The method according to claim 4 or 5, wherein the buffer medium has a pH of 7 to 8.

8. The method according to claim 4 or 5, wherein the collagen or its derivative is present in the buffer medium at a concentration ranging from 0.25% by weight to 3% by weight.

9. The method according to claim 4 or 5, wherein the collagen or its derivative is present in the buffer medium at a concentration greater than 2% by weight and not greater than 3% by weight.

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