WO2022087805A1

WO2022087805A1 - Chromatographic matrix and uses thereof for purification of fibronectin

Info

Publication number: WO2022087805A1
Application number: PCT/CN2020/123856
Authority: WO
Inventors: Hai Li; Hongyu NING; Tingwan XIE
Original assignee: Guangzhou Bioseal Biotech Co., Ltd.
Priority date: 2020-10-27
Filing date: 2020-10-27
Publication date: 2022-05-05
Also published as: CN116635144A

Abstract

Chromatographic matrices comprised of an acrylic polymer, and a collagen or a derivative thereof with the acrylic polymer and the collagen or its derivative being coupled in an amount of more than 1.17 and up to about 16 mg collagen or its derivative per g acrylic polymer are disclosed. Processes of preparing the chromatographic matrices are also disclosed. Methods for purification of fibronectin from a source solution, using the chromatographic matrices, are also disclosed.

Description

CHROMATOGRAPHIC MATRIX AND USES THEREOF FOR PURIFICATION OF FIBRONECTIN

FIELD OF THE INVENTION

The present invention relates, inter alia, to a new chromatographic matrix and uses thereof in a method for purification of fibronectin.

BACKGROUND OF THE INVENTION

Affinity chromatography has become a valuable tool for separating biological materials from a fluid medium in a mobile phase, e.g., an aqueous solution. The basic principle of affinity chromatography involves immobilization of a molecule of interest in a fluid medium (e.g., aqueous solution) to an insoluble support within a stationary phase. Thus, typically, a two-phase system is employed, one mobile and the other stationary. Separation occurs during passage of the mobile phase through the stationary phase. The more strongly a compound is adsorbed, the more it distributes to the stationary phase and the slower it moves with respect to the mobile phase.

In order to enhance the separation capacity of the solid phase matrix for a target compound or target compounds, it is commonplace to chemically modify the matrix. Such modifications include the covalent attachment of ligands to the surface of the matrix which ligands are selected to enhance the sorption of target compounds to the solid stationary matrix during the sorption stage. Covalent ligand attachment is typically achieved by use of reactive functionalities on the solid support matrix such as hydroxyl, carboxyl, thiol, amino groups, and the like. Conventional chemistry permits the formation of covalent amine, ether, thio ether, amide, carbamate, urea, carboxyl, and ester bonds with such functionalities.

Elution of the desired component can then be achieved by various procedures which result in disassociation of the complex. Thus, various substances such as enzymes, hormones, specific proteins, etc. can be separated on the basis of the specific interactions with the insoluble support.

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

U.S. Patent No. 5,043,062 discloses a high performance affinity chromatography separation device comprising: a chromatographic column containing: a packing material; wherein the packing material is a plurality of non-porous, nonodisperse polymeric particles having i) a particle size in the range of 0.01 to about 5 micrometers and ii) surface reactive groups which are directly or indirectly reactive with free amino groups, sulfhydryl groups, carboxy groups or aldehyde groups of biological ligands.

U.S. Patent No. 4,722,790 discloses a pliable compressible, liquid impervious bag having at least one integral part with a bag hole extending entirely therethrough. The bag includes a structure defining a bag aperture for communicating with the inside of the bag. A plurality of beads is positioned in the bag, and the beads are covalently coupled with a binder.

SUMMARY OF THE INVENTION

The invention relates, inter alia, to a new chromatographic matrix, its preparation, and uses thereof for purification of fibronectin. The present inventors have developed, inter alia, a new rigid matrix monodisperse made of porous ploy (methyl metharcylate) (PMMA) microspheres attached to denatured collagen, which can be readily prepared. The present inventors have surprisingly uncovered that such a matrix allows to obtain highly purified fibronectin from the respective mixtures in a single step of affinity chromatography.

According to an aspect of the present disclosure, there is provided a chromatographic matrix comprising: (i) an acrylic polymer, and (ii) a collagen or a derivative thereof; wherein the acrylic polymer and the collagen or derivative thereof are coupled, the collagen or derivative thereof being coupled is in an amount of more than 1.17 and up to about 16 mg collagen or a derivative thereof per g acrylic polymer.

According to some embodiments, the acrylic polymer, and the collagen or its derivative are at least partially covalently coupled.

According to some embodiments of any aspect, the acrylic polymer comprises polymethyl methacrylate (PMMA) .

According to some embodiments of any aspect, the derivative of the collagen comprises gelatin.

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

According to some embodiments of any aspect, the acrylic polymer comprises epoxy groups or their derivative.

According to some embodiments of any aspect, the collagen its derivative is coupled to the acrylic polymer upon reaction with at least part of said epoxy groups.

According to some embodiments, 1 g of the chromatographic matrix is capable of binding fibronectin at an amount ranging from 6 to 13 mg.

According to some of any embodiments, the chromatographic matrix is for use in chromatography, optionally under pressure of up to 3 bar, for binding thereto fibronectin from a source solution comprising fibronectin.

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

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

According to some embodiments, the buffer medium has pH of about 7 to about 8.

According to some embodiments, the collagen or its derivative is present in the buffer medium at a concentration ranging from 0.25 to 3%, optionally above 2%, by weight.

According to another aspect of the present disclosure, there is provided a method for purification of fibronectin from a source solution comprising the step of passing the source solution comprising fibronectin through a column comprising the chromatographic matrix as described in any of the embodiments herein under conditions that allow binding fibronectin to the matrix; and optionally eluting the fibronectin bound to the chromatographic matrix, and collecting the eluted fibronectin.

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

According to some embodiments, the source solution comprises plasma.

According to some embodiments, the step of passing the source solution comprising fibronectin through the column is carried out at a column pressure of up to 3 bar.

According to some embodiments, the step of passing the source solution comprising fibronectin through the column is carried out at a flow rate of 0.5 to 3 ml/min.

According to some embodiments, the step of eluting the fibronectin bound to the chromatographic matrix is carried out with an elution buffer comprising urea at a concentration ranging from 3 to 8 M.

According to some embodiments, there is provided purified fibronectin obtained by the method for purification of fibronectin from a source solution.

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 the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawing. With specific reference now to the drawing in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawing makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawing:

FIG. 1 presents a photographic image showing observed purity of fibronectin over a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel (using tris-glycine electrophoresis buffer) in a plate; percent refers to gelatin concentration prior to their coupling to polymethyl methacrylate (PMMA; see Table 3) ; arrows mark fibronectin molecular weights according to the marker in the 4 ^th lane from left. The purity of fibronectin was evaluated based on densitometry of the SDS-PAGE gel results using Imagelab (Bio-Rad software) .

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

An object of the present invention is to provide an efficient method for purification of fibronectin from a solution.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

In an aspect of the present disclosure, there is provided a chromatographic matrix comprising: (i) a polymer, and (ii) a 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 the derivative thereof is coupled in an amount allowing effective absorption of fibronectin thereon/thereto. Typically, the polymer is rigid and may be served as a solid phase, typically packed in a column, for the immobilization of the collagen or its derivative. In some embodiments, the polymer is selected from, without being limited thereto, an acrylic polymer, polystyrene, their derivatives, or their combination (e.g., a co-polymer or a mixture thereof) . Typically, but not exclusively, "rigid polymer" , or "rigid matrix" , denotes a polymer or a matrix that has a modulus of elasticity, either in flexure or in tension, greater than 7,000 kgf/cm ² (100,000 psi) at room temperature.

As disclosed herein, the term "chromatographic matrix" may refer to any type of particulate sorbent, resin or other solid phase, such as a membrane, which, in a purification process, acts as the absorbent to separate the molecule to be purified from other molecules present in a mixture. The matrix or resin may be, in some embodiments, in the form of columns, packed in a column or, in some embodiments, in the form of membrane adsorbents. As disclosed herein, a "membrane adsorbent" refers to a flat sheet of acrylic polymer comprising or incorporating functional groups such as affinity groups.

The term "resin" or "chromatographic resin" refers to a material capable of chromatographically separating a target compound from a mixture of compounds. The resin may comprise a support matrix, a ligand capable of binding a target compound when coupled to the matrix and optionally a linker arm for covalently attaching the ligand to the support matrix. Herein, the ligand may comprise the collagen or its derivative.

As used herein, the term “mixture” refers, but is not limited to, a combination of components in any physical form, e.g., blend, solution, suspension, dispersion, or the like.

In some embodiments, the chromatographic matrix is affinity chromatography ( "AC" ) matrix. As used herein, the term "affinity chromatography matrix" , or "AC matrix" , is intended to refer to a solid phase medium, typically a resin, that can be used for separation of biochemical mixtures based on a highly specific binding interaction between a protein of interest (e.g., fibronectin) and the AC matrix. Thus, the solid phase medium comprises a target to which the protein of interest is capable of reversibly affixing, depending upon the buffer conditions.

Currently known of immobilized or solid phase media that can comprise the AC matrix include a gel matrix, such as agarose beads (such as commercially available Sepharose matrices) , and porous beads.

Binding of the protein of interest to the AC matrix typically is achieved by column chromatography. That is, the AC matrix is formed or packed into a column, and a biochemical mixture containing a protein of interest is flowed through the column, followed by washing of the column by flowing through the column a wash solution, followed by elution of the protein of interest from the column by flowing through the column an elution buffer.

Thus, in some embodiments, the disclosed chromatographic matrix is for use in a step of chromatography.

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

The term "porous microspheres" as used herein refers to micro-sized particles that comprise pores, holes, or voids. Although the term “microsphere” is used throughout the specification, it will be appreciated that this term is intended to include any substantially spherical microparticle, including microparticles that are not true geometric spheres. In some embodiments, the median pore size is about 10 to 500 nm. In exemplary embodiments, the median pore size is about 100 nm.

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

By "size, it is meant to refer to at least one dimension of the microsphere, e.g., diameter or length.

The term "acrylic polymer" refers to a polymer comprised of a polymeric backbone which comprises at least one acrylic group (also referred to as: "acrylic monomeric unit" ) .

The term "polymer" describes an organic substance composed of a plurality of repeating structural units (monomeric units) covalently connected to one another.

In some embodiments, the term "polymer backbone" generally refers to a polymer comprising monomeric units. It is to be understood that in the context of the present invention, the term "polymeric backbone" refers to the main chain of polymeric skeleton together with chain branches projecting from the polymeric skeleton.

The term "monomeric unit" refers to the repeat units, derived from the corresponding monomer. The polymer comprises or is made of the monomeric units. By "derived from" it is meant to refer to the compound obtained upon a polymerization process.

In some embodiments, the acrylic polymer comprises a plurality of monomeric units selected from acrylate, or any derivative thereof.

In some embodiments, the acrylate is selected from, without being limited thereto, methacrylate, and methyl methacrylate (MMA) . Examples of non-functional acrylate and methacrylate monomers that may optionally be polymerized to provide the present polymers may further include, but are not limited to, methyl acrylate, ethyl acrylate, n-propyl acrylate, i-propyl acrylate, n-butyl acrylate, s-butyl acrylate, i-butyl acrylate, t-butyl acrylate, n-amyl acrylate, i-amyl acrylate, isobornyl acrylate, n-hexyl acrylate, 2-ethylbutyl acrylate, 2-ethylhexyl acrylate, n-octyl acrylate, n-decyl acrylate, methylcyclohexyl acrylate, cyclopentyl acrylate, cyclohexyl acrylate, methyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate, n-propyl methacrylate, n-butyl methacrylate, i-propyl methacrylate, i-butyl methacrylate, n-amyl methacrylate, n-hexyl methacrylate, i-amyl methacrylate, s-butyl-methacrylate, t-butyl methacrylate, 2-ethylbutyl methacrylate, methylcyclohexyl methacrylate, cinnamyl methacrylate, crotyl methacrylate, cyclohexyl methacrylate, cyclopentyl methacrylate, 2-ethoxyethyl methacrylate, and isobornyl methacrylate, and any derivative or combination thereof.

In exemplary embodiments, the acrylate comprises methyl methacrylate. Accordingly, in exemplary embodiments, the acrylic polymer comprises polymethyl methacrylate (PMMA) .

The term "derivative" , or "chemical derivative" , refers to subject molecules which has been chemically modified but retain a major portion thereof unchanged, e.g., subject molecules which are substituted by additional or different substituents, subject molecules in which a portion thereof has been oxidized or hydrolyzed, and the like. Such derivatized molecules include, for example, those molecules in which free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives.

In some embodiments, the acrylic polymer, e.g., PMMA, comprises 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, comprises at least one epoxy group (also referred to as "activated acrylic PMMA" ) .

Broadly defined, the term "epoxy" refers to a chemical group comprising an oxygen atom bonded with two carbon atoms already united in some other way. A simple example of an epoxy group is the compound ethylene oxide. Herein, the term "epoxy" is also meant to include its derivative upon its reaction or binding to collagen or its derivative.

In some embodiments, the acrylic polymer which comprises epoxy groups is selected from glycidyl acrylate and glycidyl methacrylate.

While there are imposed no particular restrictions as to its molecular weight, monomeric composition and epoxy equivalent and may be broadly varied. In general, suitably used is one having a number average molecular weight ranging from about 500 to about 10,000, e.g., from about 1000 to about 5000. In some embodiments, the average glass transition temperature (T _g) of the resulting polymer is advantageously chosen such that it ranges from -40° to 40℃.

In some embodiments, the collagen or its derivative (also referred to as "the ligand" ) is physically attached to the acrylic polymer. In some embodiments, the collagen or its derivative is covalently attached to the acrylic polymer. The collagen or its derivative may allow the absorption and thereby specific isolation of fibronectin from protein solutions.

Herein, the term "coupled" (also referred to as: "linked" , "attached" or "bound" ) may refer to describe a target biomolecule having a covalent and/or non-covalent intermolecular interactions that couple the polymer to the ligand (e.g., collagen or its derivative) . Noncovalent interactions may include, e.g., electrostatic bonding, hydrogen bonding, van der Waals interaction, hydrophobic force, etc. The term "non-covalently coupled" is also referred to as "physically coupled" .

As used herein, the term “at least partially” means partially or completely. The term "partially" means to some degree. Typically, but not exclusively, such term is meant to be at least 50%, at least 75%, at least 80%, at least 90%, or at least 95%.

The term "covalently coupled" refers to a chemical bond characterized by the sharing of pairs of electrons between atoms. For example, in some embodiments, the acrylic polymer or functional groups thereon form chemical bonds with the collagen or its derivative, as compared to attachment of the collagen to the surface of the polymer via other means, for example, non-covalent intermolecular interactions. It will be appreciated that polymers that are covalently attached to a ligand can also be bound via means in addition to covalent attachment.

The term “collagen” is intended to mean any known collagen of porcine, bovine or human origin, for example natural collagen, esterified collagen, such as methylated, ethylated or alternatively succinylated collagen, or one of its derivatives, which may or may not be heated, which may or may not be oxidized, or alternatively, for example, which may be crosslinked with another compound. For the purpose of the present disclosure, the term “natural collagen” is intended to mean collagen which has not been chemically modified, other than a possible treatment with e.g., pepsin in order to digest the telemeric peptides. Thus, for the purpose of the present invention, the term “non-denatured collagen” is intended to mean collagen which has not lost its helical structure.

In exemplary embodiments, the derivative of the collagen comprises 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 cause it to become denatured. Denaturation results in the irreversible transformation of collagen into a random coiled structure, which is the gelatin. Gelatin may be derived from one or more sources of collagen and may be derived from one or more types of collagen, such as, but not limited to, types I, II, III, and/or VI. Exemplary sources from which gelatin is derived include, but are not limited to, sea cucumber dermis collagen, bovine, caprine, porcine, ovine or other suitable donor mammal collagen, and marine animal collagen such as chinoderms. The gelatin may be derived from collagen obtained from mammalian cells synthesized in vitro. The gelatin may be derived from collagen obtained from molecularly engineered constructs and synthesized by bacterial, yeast or any other molecularly manipulated cell type.

In some embodiments, the collagen or derivative thereof (e.g., gelatin) is coupled to the acrylic polymer via or upon reaction with the functional groups, e.g., epoxy groups, on the polymer. Without being bound by any particular mechanism, it is assumed that the epoxy group on the PMMA surface can react with amino group of gelatin and form covalent linking therebetween.

By "effective absorption of fibronectin" it is meant to refer to fibronectin absorption efficacy of at least 10 mg fibronectin/g resin. In some embodiments, this term also refers to obtaining fibronectin purity of above 90%. Reference is made in this regard to Table 3 in the Examples section that follows, showing that an amount of above 1.17 gelatin/g PMMA provides fibronectin absorption efficacy of above 10 (mg fibronectin/g resin) with an estimate purity of more than 90 %, without the gelatin being gelated at room temperature (i.e. about 20 to 35 ℃) .

Accordingly, in another aspect, there is provided chromatographic matrix comprising: (i) an acrylic polymer, and (ii) a collagen or a derivative thereof; wherein the acrylic polymer and the collagen or derivative thereof are at least partially covalently coupled, and wherein the collagen or derivative thereof is coupled in an amount of more than 1.17 and up to about 16 mg collagen or derivative thereof per g acrylic polymer.

By "more than 1.17 and up to about 16 mg collagen or derivative thereof per g acrylic polymer" it is meant to refer, for example, to 1.18, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 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, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, or 16 mg collagen or derivative thereof (e.g., gelatin) per g acrylic polymer, including any value and range therebetween.

In some embodiments, the collagen or derivative thereof is in an amount of above 1.17 to about 15.4 per g acrylic polymer. In some embodiments, the collagen or derivative thereof is in an amount of about 11 to about 15.4 per g acrylic polymer.

In some embodiments, the chromatographic matrix is characterized in that 1 g of the chromatographic matrix is capable of binding fibronectin in an amount of at least 1 mg. In some embodiments, the chromatographic matrix is characterized in that 1 g of the chromatographic matrix is capable of binding fibronectin at an amount of at least 2 mg. In some embodiments, the chromatographic matrix is characterized in that 1 g of the chromatographic matrix is capable of binding fibronectin in an amount of at least 3 mg. In some embodiments, the chromatographic matrix is characterized in that 1 g of the chromatographic matrix is capable of binding fibronectin in an amount of at least 4 mg. In some embodiments, the chromatographic matrix is characterized in that 1 g of the chromatographic matrix is capable of binding fibronectin in an amount of at least 5 mg. In some embodiments, the chromatographic matrix is characterized in that 1 g of the chromatographic matrix is capable of binding fibronectin in an amount of at least 6 mg.

In some embodiments, the chromatographic matrix is characterized in that 1 g of the chromatographic matrix is capable of binding fibronectin in an amount ranging from 1 to 20 mg. In some embodiments, the chromatographic matrix is characterized in that 1 g of the chromatographic matrix is capable of binding fibronectin in an amount ranging from 5 to 20 mg. In some embodiments, the chromatographic matrix is characterized in that 1 g of the chromatographic matrix is capable of binding fibronectin in an amount ranging from 6 to 20 mg. In some embodiments, the chromatographic matrix is characterized in that 1 g of the chromatographic matrix is capable of binding fibronectin in an amount ranging from 5 to 14 mg. In some embodiments, the chromatographic matrix is characterized in that 1 g of the chromatographic matrix is capable of binding fibronectin at an amount ranging from 6 to 13 mg. In some embodiments, the chromatographic matrix is characterized in that 1 g of the chromatographic matrix is capable of binding fibronectin in an amount of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg, including any value and range therebetween. Reference is made in this regard to Table 3 in the Examples section that follows, showing the amount of gelatin coupled to the acrylic polymer (mg gelatin/g PMMA, as obtained from various concentrations of gelatin solution) . In some embodiments, the term "binding" refers to at least partially covalently binding.

Accordingly, in another aspect, there is provided chromatographic matrix comprising: (i) an acrylic polymer, and (ii) a collagen or a derivative thereof; wherein the acrylic polymer and the collagen or derivative thereof are at least partially covalently coupled, and wherein the collagen or derivative thereof is coupled in an amount of more than about 0.275 and up to about 3.8 mg collagen or derivative thereof per cm ³ acrylic polymer.

By "more than about 0.275 and up to about 3.8 mg collagen or derivative thereof per cm ³ acrylic polymer" it is meant to refer, for example, to 0.275, 0.28, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, or about 3.8, mg collagen or derivative thereof (e.g., gelatin) per cm ³ acrylic polymer, including any value and range therebetween.

In some embodiments, the collagen or derivative thereof is in an amount of above 0.27 to about 3.6 per cm ³ acrylic polymer. In some embodiments, the collagen or derivative thereof is in an amount of about 2.6 to about 3.6 per cm ³ acrylic polymer.

In some embodiments, the chromatographic matrix is characterized in that 1 cm ³ of the chromatographic matrix is capable of binding fibronectin in an amount of at least 0.8 mg. In some embodiments, the chromatographic matrix is characterized in that 1 cm ³ of the chromatographic matrix is capable of binding fibronectin in an amount of at least 1.5 mg. In some embodiments, the chromatographic matrix is characterized in that 1 cm ³ of the chromatographic matrix is capable of binding fibronectin at an amount of at least 2 mg. In some embodiments, the chromatographic matrix is characterized in that 1 cm ³ of the chromatographic matrix is capable of binding fibronectin in an amount of at least 3 mg. In some embodiments, the chromatographic matrix is characterized in that 1 cm ³ of the chromatographic matrix is capable of binding fibronectin in an amount of at least 3.5 mg.

In some embodiments, the chromatographic matrix is characterized in that 1 cm ³ of the chromatographic matrix is capable of binding fibronectin in an amount ranging from 0.8 to about 4 mg. In some embodiments, the chromatographic matrix is characterized in that 1 cm ³ of the chromatographic matrix is capable of binding fibronectin in an amount ranging from 0.9 to 3.6 mg. In some embodiments, the chromatographic matrix is characterized in that 1 cm ³ of the chromatographic matrix is capable of binding fibronectin in an amount of about 1 1.5, 2, 2.5, 3, 3.5, or about 4 mg, including any value and range therebetween.

In some embodiments, the chromatographic matrix is for use in chromatography, e.g., in a method for purification of fibronectin from a source solution, i.e. for binding fibronectin from a source solution comprising fibronectin. In some embodiments, the chromatographic matrix is for use in a medium-pressure liquid chromatography. The chromatographic matrix of the present invention is rigid enough to bear medium to high pressure and/or high flow rate in the process of chromatography, as compared to soft matrix such as Sepharose which is crosslinked, beaded-form of agarose. Thus, while Sepharose is widely use as chromatography medium this is a soft medium and could not bear high pressure. In order to avoid build-up of back pressure and collapse of the column bed, the rigid chromatography matrix such as PMMA was employed.

The term "fibronectin" refers to a disulfide linked dimeric glycoprotein which is present in a soluble form in blood plasma and other body fluids, and is deposited in a fibrillary form as a major constituent of the extracellular matrix of loose connective tissue. It is composed of three different structural motifs, termed type I, II, and III homologies resulting in a modular organization of the fibronectin molecule in which its several biological activities can each be attributed to specific domains.

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

In an aspect of the present disclosure, there is provided a composition ("the composition aspect" ) comprising: (i) an acrylic polymer; (ii) a collagen or a derivative thereof, and a (iii) buffer medium comprising borate.

As is known in the art, buffers are commonly used to adjust the pH to a desirable range. Typically, but not exclusively, pH of about 6 to about 8 is desired, however, this may need to be adjusted due to considerations such as the stability or solubility of the agents in a solution. Many buffers including salts of inorganic acids such as phosphate, borate, and sulfate are known. Although any buffer may be used in the compositions disclosed herein, in certain situations it is particularly useful to use a borate buffer in the composition disclosed herein. Reference is made to Table 2 in the Examples section that follows, showing that a borate buffer (typically at a concentration of about 0.2 M) is preferred for coupling gelatin to PMMA prior to applying the formed matrix for the fibronectin purification.

The terms “buffer medium comprising borate” , or “borate buffer” , may refer, in some embodiments, to any combination of boric acid and one or more of the conjugate bases such that the pH is adjusted to the desired range. While not intending to limit the scope of the invention in any way, or to be bound in any way by a particular theory, it is believed that the borate buffer may assist the coupling of collagen, e.g., gelatin to the acrylic polymer, e.g., PMMA. In some embodiments, the pH of the buffer is adjusted to about 8.

Embodiments described hereinabove e.g., with regard to of collagen, gelatin, acrylic polymer, epoxy groups, are further incorporated to the composition aspect.

The collagen or gelatin may present at a concentration ranging from 0.05 to 5%, e.g., 0.1 to 5% (v/w) or 0.1 up to below about 4%. In some embodiments, the collagen-or gelatin concentration 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 value and range therebetween.

As shown in the Examples section that follows, the inventor successfully utilized the composition comprising: (i) an acrylic polymer; (ii) a collagen or a derivative thereof, and a (iii) buffer medium comprising borate, so as to form the herein disclosed chromatographic matrix. Herein, by "successfully utilized" it is meant that the acrylic polymer (e.g., PMMA) and the collagen or derivative thereof (e.g., gelatin) are coupled, e.g., at least partially covalently coupled, with the collagen or its derivative is coupled in a desired amount allowing effective absorption of fibronectin on the matrix, e.g., in an amount of more than 1.17 and up to about 16 mg collagen or derivative thereof per g acrylic polymer.

Accordingly, in another aspect, there is provided a method ( "the preparation method" ) for preparing a chromatographic matrix, the method comprising contacting: (i) an acrylic polymer with (ii) a collagen or derivative thereof dissolved in a buffer medium comprising borate; thereby obtaining an acrylic polymer coupled to the collagen or a derivative thereof.

Embodiments described hereinabove e.g., with regard to of collagen, gelatin, acrylic polymer, epoxy groups, and the term "coupled" , are further incorporated in this aspect of the preparation method.

In some embodiments, the buffer medium has pH of about 7 to about 8, e.g., pH of 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 therebetween.

In some embodiments, the collagen or its derivative (e.g., gelatin) is present in the buffer medium at a concentration ranging from about 0.2 to about 4%, or, in some embodiments, 0.25 to 3%, by weight, e.g., 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, or 3%, by weight, including any value and range therebetween. In some embodiments, the collagen or its derivative (e.g., gelatin) is present in the buffer medium at a concentration of above 2%, by weight. In some embodiments, the collagen or its derivative (e.g., gelatin) is present in the buffer medium at a concentration ranging from above 2%to about 4%, or in some embodiments, above 2%to about 3%, by weight.

In another aspect, there is provided a method for purification ( "the purification method" ) of fibronectin from a source solution comprising the step of passing the source solution comprising fibronectin through a column comprising the herein disclosed chromatographic matrix under conditions that allow binding fibronectin to the matrix.

The term "passing solution through" , or any grammatical inflection thereof, refers to the solution entering a component (e.g., a column) , moving through at least part of the interior of the component, and, at times, exiting at least part of the component.

In some embodiments the column is an affinity column. The term "affinity column" refers to a molecular fractionation device comprising beads with specialized chemistry for separating or reacting with biomolecules. In some embodiments, the beads comprise the herein disclosed chromatographic matrix in an embodiment thereof. It should be noted that the use of the term "affinity column" does not imply the need for a traditional physically constrained cylindrical or vertical column. Conditions that allow binding fibronectin to the matrix relate to, without being limited thereto, pH of the buffer by which the column is equilibrated, the temperature of the source solution, the flow rate of the recharging solution, the pressure applied to/within the column ( "column pressure" ) , or any combination thereof. For example, the temperature may be controlled to between above 0 and below 100 ℃, and include ranges such as, without limitation, 10 to 50 ℃, 45 to 50 ℃, 20 to 40 ℃, or 20 to 30 ℃.

In some embodiments, the pressure is up to 5 bar. In some embodiments, the pressure of is up to 4 bar. In some embodiments, the pressure is up to 3 bar. In some embodiments, the pressure is between 1 and 20 bar, in some embodiments, between 1 and 10 bar, or, in some embodiments, between 1 and 5 bar. In some embodiments, the pressure is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bar, including any value and range therebetween.

In some embodiments the column pressure is control by a pump. The term "pump" refers to any device that causes the movement of fluids by applying suction or pressure.

In some embodiments, the column is equilibrated with a puffer ( "equilibration buffer" ) . In some embodiments, the pH of the equilibration buffer ranges from 6.5 to 8, or, in some embodiments from about 7 to about 7.4, e.g., 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 therebetween. The pH value may not necessarily maintain a substantially consistent value during the purification. In exemplary embodiments, the equilibration buffer comprises phosphate buffered saline (PBS) .

In some embodiments, the method comprises removing contaminants by washing the chromatographic matrix e.g., using a washing buffer. In exemplary embodiments, the equilibration buffer comprises phosphate buffered saline (PBS) . In some embodiments, the pH of the washing buffer ranges from 6.5 to 8, or, in some embodiments from about 7 to about 7.4, e.g., 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 therebetween.

Additionally, the flow rate of the source solution, typically with the loading buffer (e.g., PBS) in the mobile phase, may be any one of 0.1 to 10 ml/min, 0.1 to 6 ml/min, 0.2 to 10 ml/min, 0.25 to 5 ml/min, or 0.5 to 3 ml/min, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 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, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 ml/min, including any value and range therebetween.

In some embodiments, the method comprises eluting the fibronectin bound to the chromatographic matrix, e.g., using an elution buffer ( "the eluting step" ) . The eluting step may be carried out sequentially or simultaneously to the step of passing the source solution in the column.

The term “eluting” , or any grammatical inflection thereof, is used herein to mean the release of the adsorbed fibronectin from the chromatographic matrix. Oftentimes, the term “elution” as disclosed herein is interchangeable with the term “desorption” . In some embodiments, this term relates to the release of at least 80%, at least 85%, least 90%, or at least 95%, of the fibronectin. The elution may be carried out under certain elution conditions. Typically, but not exclusively, elution conditions include using a non-isocratic condition e.g., a solution or a condition different from the solution or condition used e.g., to load the source solution comprising the fibronectin, and/or different from the solution used in a previous step.

The term “simultaneously” used hereinthroughout does not necessarily mean that the whole relevant steps are carried out at same time, and may also refer, for example, to a case of first starting to carry out the washing step and immediately thereafter passing the mobile phase in the column, or, for example, to a case of first passing the mobile phase in the column and, immediately thereafter, carrying out the eluting step. In some embodiments, by "immediately" it is meant to refer to within 0 to 20 sec, 0 to 10 sec, or 0 to 2, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 sec, including any value and range therebetween.

The elution buffer may be used to elute the fibronectin from the chromatography matrix. In exemplary embodiments, the elution buffer comprises urea, typically, and without limitation, at a concentration ranging from 4 to 6 M. In some embodiments, the eluted fibronectin is able to be collected. Thus, the purification method may comprise a step of collecting the eluted fibronectin.

The term "source solution" broadly refers to a combination, mixture and/or admixture of ingredients having at least one liquid component and a protein of interest. Solutions typically include at least one solvent in greater quantity or volume than a solute. Typical solvents include water. In some embodiments, the source solution comprises a mixture of proteins. In some embodiments, the source solution comprises plasma, typically blood plasma or a fraction thereof. In some embodiments, the plasma comprises oxalated plasma. In some embodiments, the source solution comprises plasma harvested from a mammal. In some embodiments, the mammal is selected from a human, an equine, a bovine and a porcine. In exemplary embodiments, the source solution comprises porcine plasma.

In some embodiments, the eluted fibronectin is purified to at least 90%, by weight. The term “purified” as used herein refers to material that has been isolated under conditions that reduce or eliminate unrelated materials, i.e., contaminants. For example, a purified protein is substantially free of other proteins with which it is associated in the source solution. As used herein, the term “substantially free” is used operationally, in the context of analytical testing of the material. Thus, material substantially free of contaminants is at least 50%pure, at least 90%pure, at least 97%pure, or optionally at least 99%pure, by weight. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art. In some embodiments, the eluted fibronectin is purified to a range of about 90%to about 97%, by weight. Reference is made in this regard to Table 3 in the Examples section that follows and the SDS-PAGE gel results presented in Fig. 1. The Fibronectin purity was evaluated by Imagelab (Bio-Rad software) .

In some embodiments, the fibronectin is concentrated by 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, the fibronectin was concentrated by a purification factor of about 50 to about 80.In some embodiments, the fibronectin was concentrated by a purification factor of about 50, about 60, about 70, or about 80, including any value and range therebetween. An exemplary method for calculating the purification factor is described in the Examples section that follows (see Table 5) . In some embodiments, the herein disclosed chromatographic matrix can re-used e.g., for fibronectin binding, at least 1, at least 2, or at least 3 times, e.g., 1 to 3 times, without having the purification factor reduced for more than 20%upon re-applying the matrix at the same conditions.

Accordingly, there is provided fibronectin obtained by the purification method, as employed in any embodiment thereof.

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

As used herein the term “about” refers to ± 10 %.

The terms “comprises” , “comprising” , “includes” , “including” , “contains” , “containing” , “has” , “having” , and their conjugates mean ” including but not limited to” . The term “consisting of” means “including and limited to” . The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

The word “exemplary” is used herein to mean “serving as an example, instance or illustration” . Any embodiment described as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments and/or to exclude the incorporation of features from other embodiments.

The word “optionally” is used herein to mean “is provided in some embodiments and not provided in other embodiments” . Any particular embodiment of the invention may include a plurality of “optional” features unless such features conflict.

As used herein, the singular form “a” , “an” , and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, analytical, pharmacological, biological, biochemical and medical arts.

As used herein, and unless stated otherwise, the terms “by weight” , “w/w” , “weight percent” , or “wt. %” , which are used herein interchangeably describe the concentration of a particular substance out of the total weight of the corresponding mixture, solution, formulation or composition.

In those instances where a convention analogous to “at least one of A, B, and C, etc. ” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a composition having at least one of A, B, and C” would include but not be limited to compositions that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc. ) . It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” , “B” , or “A and B” .

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

EXAMPLE 1: PURIFICATION OF FIBRONECTIN BY PMMA-GELATIN MATRIX

Materials

Gelatin (Sigma Aldrich, catalog No. V900863, CAS: 9000-70-8) ;

Activated medium comprising porous PMMA microspheres (i.e. having epoxy groups on the surface of the PMMA; product name: UniEpoxy-50L, provided by Suzhou Nano-Micro Technology Co. Ltd, having a Catalog No. 15302-06132-01012; also referred to as: "activated PMMA" ) . The weight average molecular weight (MW) of the PMMA is in the range of 500,000 to 1,000,000. Table 1 below summarizes the main technical specification of the PMMA.

Table 1: Main Technical Specification of PMMA

The gelatin was purchased from Sigma Aldrich (catalog No. V900863, CAS: 9000-70-8) .

Source of fibronectin: porcine plasma. Technical data for porcine plasma: total protein: 55 mg/ml; Fibronectin concentration: 0.15 mg/ml.

Methods

Coupling of activated PMMA to gelatin: in exemplary procedures, gelatin was dissolved in borate buffer (pH 7.6) in various concentrations (by weight) : 0.1%, 0.25%, 0.5%, 1%, 2%, 3%, and 4%. Next, activated PMMA was added to the gelatin solution at 1: 1 of weight ratio followed by gentle shaking for 16 hours at 37 ℃. Thereafter, the obtained resin (gelatin coupled to PMMA) was collected by centrifugation at 3000 rpm for 10 mins. The resin was then washed with purified water and centrifuged at 3000 rpm for 10 minutes. This step of washing followed by centrifugation was repeated once again.

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

Additional exemplary procedures were carried out using the above procedures but with other buffers instead of borate: NaHCO ₃, sulfate solution, sodium sulfate and PBS with gelatin concentration of 1%by weight.

Fibronectin Purification: in exemplary procedures, 5-10 g of wet PMMA material, and equal weight of 0.25%to 3%gelatin solution were used to form a matrix used for the fibronectin purification. The fibronectin was purified from porcine plasma by a single step of affinity liquid chromatography (LC) using the gelatin-PMMA matrix obtained by the above procedures. The process was carried out in a pressure chromatography using chromatography system. The PMMA-gelatin resin was packed into the column with a compress factor of 1: 1 (the compress factor is the settling volume of resin divided by the volume of resin after packing; since PMMA-gelatin is a rigid resin its volume did not change after being packed into a column) . The column was then equilibrated by PBS, pH 7.0 to7.4, at room temperature (25 ±2 ℃) before loading the starting material.

Additional general conditions for loading:

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

ii. Flow rate: 1 ml/min;

iii. Temperature: about 25 ℃;

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

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

vi. Equilibration buffer: PBS

vii. pH 7-7.4.

The washing buffer was the same as equilibration buffer (PBS, pH 7.0-7.4) .

Specific conditions for elution:

i. Volume of eluent: depend on the elution peak, 1.5-3 CV normally up to 20 ml

ii. Flow rate: 1 ml/min;

i. Elution buffer: 4-6 M urea.

The LC apparatus used was

pure 150 system, which is manufactured by GE healthcare Co., Ltd.

pure chromatography system is a highly versatile, modular system with a number of design features to facilitate reliable purification. The system comprises the

pure instrument and UNICORN 6 control software, and has a modular design with all valves, monitors, and columns mounted on the forward facing wet side of the system, to allow easy interaction with the instrument modules.

Results

Linking PMMA-to-Gelatin in different Buffers: the results are summarized in Table 2 below.

Table 2: Results of Linking of PMMA to Gelatin Using Various Buffers

It is shown that when using NaHCO ₃ or PBS buffer as compared to borate buffer, the coupling experiments provided poor coupling of gelatin per PMMA and therefore less preferred resin. The coupling experiments also failed when using ammonium sulfate solution, and sodium sulfate solution buffers. Therefore, the borate buffer was selected for coupling gelatin to PMMA prior to its use the fibronectin purification procedures.

Fibronectin Purification: The results are presented in Table 3 below;

Table 3: Various Ratios of PMMA-to-Gelatin using Borate Buffer

*Fibronectin purity was evaluated from the SDS-PAGE (see Figure 1) by Imagelab (Bio-Rad software) ;

**Using the 4%of gelatin solution resulted in gelation at room temperature and the coupling reaction could not be performed;

***The values can be converted to mg gelatin/cm ³ resin (or mg fibronectin/cm ³ resin) upon dividing by 4.255 (1cm ³ of wet PMMA resin contain 0.235g of PMMA dried powder, so 1g of PMMA dried powder contains in 4.255cm ³ of wet PMMA resin; for example, 3.83 mg gelatin/g PMMA gives 0.9 mg gelatin/cm ³ PMMA (and 12.34 mg fibronectin/g resin gives 2.9 mg fibronectin/cm ³ resin) .

For Gelatin concentrations of 0.25%and above the purity of fibronectin was over 90%as is also observed based on SDS-PAGE results (see Figure 1) .

It is noteworthy that although Sepharose is widely use as chromatography medium this is a soft medium and could not bear high pressure. Reference is made to Table 4 below, presenting comparative parameters obtained upon using

4B. Specifically, the maximal flow rate was 1.6 ml/min (when performing chromatography in a 1 cm diameter of column) , and the fibronectin binding capacity was about 1mg/cm ³ resin (compared to 3.1 mg/cm ³ resin (= 13.19 mg/g) using the PMMA) .

Table 4: Main Technical Specification of Sepharose-gelatin

General Protocol for SDS-PAGE test: the gel plates containing 10%of SDS-PAGE gel are attached to the apparatus. Next, 400 ml of tris-glycine electrophoresis buffer are poured into the upper and lower chambers. The protein samples with loading buffer are then mixed following by boiling for 5 minutes in a water bath. The samples and protein standard are thereafter loaded to the gel. Each lane can be loaded for 5-10ul of sample.

Next, electrophoresis is run at a constant voltage of 100V. After the dye front enters the resolving gel, the voltage is adjusted to 150V. It typically takes about 1-2 hours for a run. Next, the plates are removed from the apparatus and the spacers. The plates are pried off by inserting a spatula and twisting the plate up. The gel should be stuck to one of the plates. The gel is then floated off the plates in a container containing coomassie blue solution.

Next, the gel is incubated in coomassie blue solution for 30 minutes followed by destaining the gel with a destain buffer for 3-4 times (30 minutes for each time) . After the gel background gets clear, photographic images of the gel are taken by gel imaging system, followed by analyzing the purity using the ImageLab software.

The results show that with the increase of gelatin concentration an increase in coupling was found, and the purity of fibronectin was increased accordingly. If the ligand (gelatin) coupling efficiency was low e.g., upon using gelatin concentration of 0.1%or lower, the purification performance of the gelatin-PMMA was unsatisfied accordingly (e.g., much less than 90%) .

Determination of fibronectin absorption efficacy: for determination of fibronectin absorption efficacy, the PMMA-gelatin resin (13.19 mg/g; see Table 3) was packed into a chromatography column and the column volume (known as CV) was calculated. Porcine plasma containing fibronectin was overloaded (usually over 20CVs of porcine plasma) . The flow-through fraction was then collected, followed by measuring the fibronectin concentration in the flow through and the loading the material in an Enzyme-Linked Immunosorbent Assay (ELISA) kit. The total absorption amounts of fibronectin can then be calculated by dividing the total absorption amount of fibronectin by CV, thus the determining the fibronectin absorption efficacy of each ml of resin. The wet resin was then freeze dried and the fibronectin absorption efficacy per gram of dry resin was also determined.

Purification Factor Data: Purification factor is calculated by dividing fibronectin specification in the elution fraction by that of porcine plasma as shown in Table 5 below.

Table 5: Purification Factor Data of Single Step Affinity Chromatography using PMMA-Gelatin

As shown in Table 5 above, the fibronectin was concentrated by a factor of about 71.66.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

A chromatographic matrix comprising: (i) an acrylic polymer, and (ii) a collagen or a derivative thereof; wherein the acrylic polymer and the collagen or derivative thereof are coupled in an amount of more than 1.17 and up to about 16 mg collagen or derivative thereof per g acrylic polymer.
The chromatographic matrix of claim 1, wherein the acrylic polymer and the collagen or the derivative thereof are at least partially covalently coupled.
The chromatographic matrix of claim 1 or 2, wherein the acrylic polymer comprises polymethyl methacrylate (PMMA) .
The chromatographic matrix of any one of claims 1 to 3, wherein the derivative of the collagen comprises gelatin.
The chromatographic matrix of any one of claims 1 to 4, wherein the acrylic polymer is in the form of porous microspheres having a median size ranging from 40 to 80 μm.
The chromatographic matrix of any one of claims 1 to 5, wherein said acrylic polymer comprises functional groups, optionally epoxy groups, allowing to bind thereto the collagen or derivative thereof.
The chromatographic matrix of claim 6, wherein the collagen or derivative thereof is coupled to said acrylic polymer upon reaction with at least part of said functional groups.
The chromatographic matrix of any one of claims 1 to 7, wherein 1 g of the chromatographic matrix is capable of binding fibronectin at an amount ranging from 6 to 13 mg.
The chromatographic matrix of any one of claims 1 to 8, for use in chromatography, optionally under pressure of up to 3 bar, for binding fibronectin from a source solution comprising fibronectin.
A composition comprising: (i) an acrylic polymer; (ii) a collagen or a derivative thereof, and a (iii) buffer medium comprising borate.
The composition of claim 10, wherein the derivative of the collagen comprises gelatin.
The composition of claim 10 or 11, wherein said acrylic polymer comprises functional groups, optionally epoxy groups, allowing to bind thereto the collagen or derivative thereof.
The composition of claim 12, wherein the collagen or derivative thereof is coupled to said acrylic polymer upon reaction with said functional groups.
The composition of any one of claims 10 to 13, wherein the acrylic polymer comprises polymethyl methacrylate (PMMA) .
A method for preparing a chromatographic matrix, the method comprising contacting: (i) an acrylic polymer with (ii) a collagen or derivative thereof dissolved in a buffer medium comprising borate, thereby obtaining an acrylic polymer coupled to the collagen or a derivative thereof.
The method of claim 15, wherein the acrylic polymer is at least partially covalently coupled to the collagen or a derivative thereof.
The method of claim 15 or 16, wherein said acrylic polymer comprises functional groups, optionally epoxy groups, allowing to bind thereto the collagen or derivative thereof.
The composition of any one of claims 15 to 17, wherein the derivative of the collagen comprises gelatin.
The method of any one of claims 15 to 18, wherein the collagen or derivative thereof is coupled to said acrylic polymer via said functional groups.
The method of any one of claims 15 to 19, wherein the buffer medium has pH of about 7 to about 8.
The method of any one of claims 15 to 20, wherein the collagen or derivative thereof is present in the buffer medium at a concentration ranging from 0.25 to 3%, optionally above 2%, by weight.
A method for purification of fibronectin from a source solution comprising the steps of:

(i) passing the source solution comprising fibronectin through a column comprising the chromatographic matrix of any one of claims 1 to 9 under conditions that allow binding fibronectin to the matrix;

(ii) eluting the fibronectin bound to the chromatographic matrix, and

(iii) collecting the eluted fibronectin.
The method of claim 22, wherein the eluted fibronectin is purified to at least 90%by weight.
The method of claim 22 or 23, wherein the eluted fibronectin is purified to at least 97%by weight, optionally by a single chromatographic step.
The method of any one of claims 22 to 24, wherein the source solution comprises plasma.
The method of any one of claims 22 to 25, wherein step (i) is carried out at a column pressure of up to 3 bar.
The method of any one of claims 22 to 26, wherein step (i) is carried out at a flow rate of 0.5 to 3 ml/min.
The method of any one of claims 22 to 27, wherein the eluting in step (ii) is carried out with an elution buffer comprising urea at a concentration ranging from 3 to 8 M.
A purified fibronectin obtained by the method of any one of claims 22 to 28.