WO2005018803A1 - Immobilization matrix for peptides and proteins - Google Patents

Abstract

The invention relates to sintered thermoplastic polymer matrices and to the uses of such matrices for the immobilization of peptides and proteins. Preferably, the polymer used is polyethylene.

Description

IMMOBILIZATION MATRIX FOR PEPTIDES AND PROTEINS

The invention relates to sintered thermoplastic polymer matrices and particularly to the uses of such matrices for the immobilization of peptides and proteins.

Following the sequencing of the human genome, the next challenge in man's quest to understand gene function is the large-scale study of proteins. Such studies are important since proteins are ultimately responsible for cellular functions. However, there are numerous problems associated with such endeavours. There is therefore a need to expand the number of existing methods and to develop new techniques to manipulate proteins, to purify proteins and to investigate protein-protein interactions and post-translational modifications.

One such technique makes use of proteins immobilized on a solid support. Such techniques have many advantages, for example they permit the stabilization of proteins, they allow better control of the experimental conditions and they facilitate the removal of modifying reagents. Methods of immobilization have previously been developed which allow simple and complex proteins to retain their native conformation [1]. This is extremely important for more advanced studies on proteins [2-6].

The most popular matrices that are currently used for the immobilization of proteins are beads of Sepharose or Sephadex [7]. Sepharose is composed of cross-linked polysaccharide agarose; Sephadex is a copolymer of allyl dextran (a water-soluble glucose polymer) and polyacrylamide (a polymer of acrylamide monomers). Sepharose is stable at pH 4-10 and at temperatures between 1°C and 40°C; Sephadex is stable at pH 2-10 and at temperatures between 1°C and 100°C [8, 9]. Other advantageous properties of Sepharose and Sephadex include (i) their high binding capacity (up to 6 mg protein per ml of matrix); (ii) the presence of reactive sites on the matrix which facilitates coupling with ligands; and (iii) they are relatively inert. For these reasons and others, Sepharose and Sephadex have for a significant time been the matrices of choice for the purification of proteins.

Whilst Sepharose and Sephadex particles are the preferred particles for use in many circumstances, particles of many other materials are known for various biochemical uses.

These include highly cross-linked macroreticular polystyrenes for ion-exchange processes and DNA synthesis; low cross-linked organic swellable polystyrenes for peptide synthesis; silicas for HPLC supports; controlled pore glass for DNA synthesis and enzyme immobilization; and graft polymers of polyethylene glycol and low cross-linked polystyrenes for organic synthesis, peptide synthesis and bead-based peptide libraries.

Particles of porous polyethylene have been used in solid-phase peptide synthesis [10, 11]; porous polyethylene is also known as a solid supporting substance in the form of membranes, filters and implants in biomedical studies relevant to cell biology and biomaterials [12-18]. The use of polyolefin particles possessing chemically oxidized surfaces for the immobilization of proteins, enzymes and whole cells via cleavable spacers are also known [19]. Furthermore, the use of macroporous polyethylene cloths which are capable of adsorbing antibodies as supports in enzyme immunoassays are known [20].

There are, however, problems which are associated with the use of particles or beads as solid supports. For example, when producing a column comprising particles or beads, the particles or beads must be allowed to settle and pack together before the column may be used. This inevitably produces delays. Furthermore, the problems of back-pressure from such columns are well known, i.e. the packing of the particles in the column results in a resistance to the flow of fluid through the column. Due to the widespread use of particles as solid supports, most of these problems are readily accepted as being inherent features of particle-based systems which are necessary and which must be tolerated in order to achieve the desired result.

However, it has now been found that by taking a radical step away from traditional particle-based solid supports, the above problems can be reduced significantly. It has been found that matrices that are produced from sintered thermoplastic polymers having modified surfaces have specific advantages over the previously-known matrices such as Sephadex and Sepharose. Such new matrices have a great potential for use in bioprocessing. The matrices are cheap, unreactive, temperature and solvent resistant, and their binding capacity can be as high as that of Sepharose due to their porous structure. One specific advantage of the matrices of the invention is that they can be moulded or cut into various shapes as required. These particular characteristics of the matrices of the invention can be very important to the development of new formats for bioprocessing and other new applications.

The invention therefore provides a porous matrix comprising sintered thermoplastic polymer particles, wherein the matrix has a modified surface which is chemically reactive.

In an alternative embodiment, the invention provides a porous matrix comprising sintered thermoplastic polymer particles, wherein the matrix has a modified surface which is functionalized. Furthermore, the invention provides a porous matrix comprising sintered thermoplastic polymer particles, wherein the matrix has a modified surface which provides pendant functional groups which are suitable for attaching a ligand to the surface, optionally via a linker. The matrices of the invention are essentially rigid.

In contrast to prior art slurries of particles (e.g. Sepharose beads), the matrices of the invention have a number of specific advantages. The matrices of the invention may have pre-defined pore sizes, shapes and/or lengths which can be predetermined as desired during the manufacture of the matrix. Thus matrices with predetermined flow characteristics can be produced and hence the problems with "back-flow pressure" which are associated with most prior art particle slurries can be avoided.

The matrices of the invention are comprised of particles of a thermoplastic polymer.

Preferably the thermoplastic polymer is a polyolefin or a vinyl polymer. Examples of such polyolefins include polyethylene and polypropylene. Examples of vinyl polymers include PVA and PVC. Preferred polymers include polyethylene or polypropylene, most preferably polyethylene.

In other embodiments of the invention, the thermoplastic polymer may be PVDF, PTFE or Nylon. As used herein, the term "polymer" generally includes, but is not limited to, homopolymers, copolymers, such as for example, block, graft, random and alternating copolymers, terpolymers, etc., and blends and modifications thereof. In addition, unless otherwise specifically limited, the term "polymer" also includes all possible geometric configurations of the molecule. These configurations include, inter alia, isotactic, syndiotactic, atactic and random symmetries.

In the context of the invention, the thermoplastic polymer is most preferably polyethylene; or a copolymer or blend which comprises polyethylene, preferably at least 80% polyethylene, particularly preferably at least 90% polyethylene and most preferably at least

95% polyethylene.

Examples of usable polyethylenes include high density polyethylene and ultra high molecular weight polyethylene, as manufactured by Porvair Technology, UK, under the tradename "Vyon".

The thermoplastic polymer may also comprise flow modifiers, additives, etc., as are usual in the art.

The thermoplastic polymer particles to be sintered to form the matrix will in general have a size in the range that is appropriate for the ultimate use of the matrix. The particles may be spherical, generally spherical or may be any other suitable regular or irregular shape.

The person skilled in the art will appreciate that the rate of fluid passage through the matrix of the invention will be determined at least in part by the sizes of the particles which comprise the matrix and the conditions under which those particles are sintered. Other variables to be taken into account in this regard include the molecular size and other properties of any material which is linked to the matrix. Merely with routine experimentation, the person skilled in the art will readily be able to produce matrices with fluid passage rates which are appropriate for a specific purpose. As used herein, the term "sintered thermoplastic polymer" refers to a number of thermoplastic polymer particles which generally have been coalesced into a single unit under the influence of heat and vibration, without actually liquefying the polymer. The matrix therefore comprises a plurality of fused thermoplastic polymer particles having a defined structure which is maintained upon the application of a fluid. The "sintered thermoplastic polymer" will also in general be essentially rigid due to the fused nature of the constituent particles, i.e. it will be essentially incompressible and it will not shrink or swell in aqueous solutions. However, some embodiments of the invention such as sheets or membranes which comprise the matrix of the invention may be flexible.

Methods of sintering thermoplastics are well known in the art. These include the methods disclosed in publications in the name of Porvair Pic [e.g. 21, 22].

The pore size of matrix post-sintering may be predetermined during its manufacture to be appropriate for the desired use. In general, the sizes of the pores in the matrix may be 1 -

1000 μm, 1-500 μm, 500-1000 μm or 200-700 μm. Preferably, the pore sizes are 2-500 μm and most preferably 5-100 μm. Preferably, the mean pore size is 5 -100 μm, and most preferably 20-40 μm or 40-80 μm.

After sintering, the matrix is modified in order to provide a chemically-reactive surface, e.g. a functionalized surface, preferably an irregular surface. This modification increases the surface area of the matrix. It also provides functional groups on the surface which facilitate the attachment of the desired material, for example, a ligand. In other terms, the chemically reactive surface is a modified surface which provides pendant functional groups which are suitable for attaching a ligand to the surface, optionally via a linker.

A number of techniques are known for the surface modification of thermoplastic polymers. Three preferred techniques which are usable in this regard are gas plasma amination, gamma-irradiation and chemical oxidation.

Gas plasma amination is based on the reaction between gas plasma-induced radicals on the surface of the thermoplastic polymer and then reaction with ammonia thus resulting in formation of surface amines. The technique is commonly used for preparation of matrices for attachment to RNA or DNA [10, 23].

In the gamma-irradiation technique, the formation of reactive radicals is induced by gamma rays, followed by a consecutive reaction with ammonia or acrylic acid. The result is a surface amination or surface overlaid with acrylic acid [10, 13, 14, 23].

Chemical oxidation techniques result in the creation of intermediate irregular reactive functions via the breaking of carbon bonds in the thermoplastic.

There are several general factors which can regulate the aggressiveness of the surface treatment. These include temperature, pressure, type of oxidizing agent and pH of the reaction in general [11]. In general, the particles are treated for a time and under conditions which are sufficient to provide the chemically-reactive surface. Examples of the time and conditions which may be used in this regard are known in the art [24],

Preferably, the surface of the matrix is modified by treatment with one or more acids selected from the group consisting of trifluoroacetic acid, trifiuorom ethane sulfonic acid, chromium trioxide and sulfuric acid; optionally in the presence of one or more peroxide salts such as K₂Cr₂O₇ or KMnO .

A number of strategies have been commonly employed for the chemical oxidation of thermoplastics. If modification of the thermoplastic surface only is desired, this can be achieved by relatively mild chemical oxidation using peroxide salts and acids such as K₂Cr O₇ or KMnO₄ in H₂SO₄, without causing significant damage to the physical structure of the surface. Physical erosion of the thermoplastic (tunnels and holes inside the plastic material to increase its binding capacity, prior to modification of the surface of the plastic material) can be achieved by treatment of the plastic with more aggressive oxidizers such as trifluoroacetic acid applied at higher concentrations and higher temperatures.

Preferably the oxidizing agent is KMnO in H₂SO4. This results in gentle but effective oxidation without damage to the physical structure of the plastic [25]. The types of the functional groups that are present on the surface of the matrix depend on the type of the reaction that is employed to generate them. In most cases, carboxyl or hydroxyl groups are produced. Aldehyde and keto groups can also be generated as side products of the reaction. Carboxyl or hydroxyl functions can be substituted by more stable and potentially reactive functions, for instance, amines. Amino groups can be chemically introduced directly onto the thermoplastic surface or attached via spacer molecules (linkers).

Although surface amination is a very useful technique for modifying the surface of the solid support for most DNA RNA based applications, it confers a strong positive charge to the matrix at neutral pH. This leads to non-specific binding of any negatively-charged molecules from the solution to the matrix. If desired, the amine functions can largely be converted to neutral at physiological pH to amide bonds by the consecutive reaction with a linker (or ligand).

Preferably, the matrix is modified to provide a surface which is essentially free of amine groups. The matrix is also preferably modified in order to provide a surface which presents a plurality of carboxyl groups.

After the surface of the matrix has been functionalized, the surface may or may not be reacted with one or more linkers or spacers. The function of such entities is (i) to facilitate the attachment of a desired ligand to the surface of the matrix and/or (ii) if desired, to allow the ligand to be placed at a certain distance away from the surface of the matrix. Advantageously, the unmodified surface remains hydrophobic and chemically inert thus significantly reducing the non-specific background binding. Linker technology helps to preserve to a large extent the native conformation of any immobilized proteins, and also any proteins which are purified on such matrices. Utilization of a non-cleavable linker on the matrix allows permanent covalent coupling of the protein to the matrix thus radically reducing leaching of any immobilized molecules from the matrix. In the context of the present invention, the terms "linker", "spacer" and "handle" may be used interchangeably. Preferably, a linker is bound to the surface of the matrix. Most preferably, the linker is bound to the surface of the matrix immediately after the surface has been modified.

The selection of an appropriate linker will be dependent on the surface functionalization of the matrix and the ligand intended to be bound to the matrix. Numerous such linkers are known in the art. In particular, reactions which may be employed for coupling polypeptide or DNA/RNA molecules to certain linkers or directly to solid supports are well known in the art. Conveniently, functional groups can be incorporated into a ligand (e.g. peptide or DNA/RNA) during its chemical synthesis. Potential functional groups include ethers, esters, thiols, dialkylamides, hydrazides, diamines and many others. Appropriate linkers will be those that contain groups which are capable of reacting with one or more of the aforementioned functional groups. For example, a linker which utilizes the formation of thioether bond between the ligand and the linker could have the thiol group on one (ligand) end and bromoacetyl group on the other (linker).

In contrast to chemically-synthesized molecules, the conjugation of naturally-occurring biological molecules (for example, enzymes and antibodies) requires care to be taken to ensure that the activity of the biological molecule is preserved, at least to a significant extent, once it is bound to the matrix. This restricts the choice of linker strategies for the following reasons: Non-denaturing (i.e. physiological or mild) conditions must be used to link the protein to the linker; not all linkers can be used under such conditions. The biological activity of a protein might be dependent on the accessibility (to a substrate) of a particular functional group; such groups must therefore not be used to link the protein to the matrix. Furthermore, many of the potential functional groups may be modified post- translationally (e.g. by phosphorylation, acetylation, etc.) and therefore will not be accessible for the linking reaction.

Preferred reactions for conjugation of biologically active molecules and linkers include:

1) Amino-linkage, or formation of an amide bond between a linker and a ligand (e.g. protein) via reaction between ester function at the linker's end and the protein's primary and/or secondary amines). Such reactions are generally reliable and the activity of the immobilized protein is very rarely affected. Furthermore, the reaction can be performed at neutral pH (for primary amines) rising to pH 8.0 -8.3 (for secondary amines). Furthermore, the reaction requires no free amines in the reaction mixture. 2) Thio-linkage, or formation of the covalent bond between a thiol present on the matrix and another thiol originating from the protein. In this reaction, the conjugation reaction is reversible, i.e. the ligand can be removed back into fluid phase after reduction with 2- mercapto ethanol or DTT. This can be very convenient for studying interactions between proteins, for example. The reaction requires some special condition for conjugation, i.e. ~ the absence of II-valent metals in the solution; and the protein must have SH-groups reduced prior to conjugation.

3) Carboxylic linkage, or formation of the covalent bond between the functional group on the matrix and carboxy-terminus of the protein. This type of reaction is less efficient and reliable because many proteins have C-termini which are naturally modified (i.e. blocked). Furthermore, naturally-occurring cyclic polypeptides have no C termini and in some cases, modification of the C-terminal end significantly affects the functional activity of the protein. Additionally, this reaction requires special pH and ionic conditions, which can result in protein denaturation.

Preferably, an amino-linker is used. This reaction utilizes the carboxylic function of the linker and primary or/and secondary amines of the ligand, resulting in formation of an amide bond between the linker and ligand in high yield. The irreversible linkage created by this reaction is very stable at physiological conditions.

In one embodiment of the invention, the matrix is an affinity matrix or an enzyme-bound matrix. In this embodiment, post-sintering, the matrix is provided with a surface which is non-aminated or essentially non-aminated. In this method, after oxidation (and preferably immediately after oxidation), a spacer is generated in a reaction between a carboxyl function on the matrix and 6-aminohexanoic acid. This reaction produces a linker with the anchoring carboxylic function. Importantly, this approach does not involve generation of unbound amines on the surface, which significantly reduces the non-specific background binding to the modified surface.

Thus in one embodiment of the invention there is provided a process for modifying the surface of a matrix which comprises a sintered thermoplastic polymer comprising the steps

(i) oxidizing the surface of the matrix; and (ii) reacting a carboxyl group on the surface of the matrix with 6-aminohexanoic acid. Preferably, the oxidizing step (i) does not result in the production of amine groups.

The linker is preferably one which is long enough to prevent any steric hindrance between the support and the molecules which bind to the ligand. Linkers may also be introduced to create a large enough distance between ligand attachment sites thus providing non- restricted access of the ligands to reagents and also preventing aggregation of the ligands on the surface of the polymer.

There are different approaches where the length of the linker (and, alternatively its presence) may or may not be important.

For example, in solid-phase synthesis technology, the linkers (or handles) are made for handling an anchor part of the synthesized molecule. They must contain a cleavable part so that the linker may be cleaved to release the newly -synthesized molecule when the synthesis is complete. The length of the linker in this case would be determined by the length of the cleavable and anchor parts of the linker and depends on the type of cleavage reaction used to cleave the linker. The length of a linker can vary significantly and very often includes aromatic rings. This approach can be applied to both peptide and DNA synthesis techniques.

In conjugation of biologically active molecules, the length of the linker will determine the distance between the ligand and solid support. It has been shown that this length may significantly affect the functional activity of a biological molecule which is attached via the linker or, in case of an antigen, availability of antigenic determinants to be recognized by the antibody in a fluidic system and preservation of their stereochemical properties. In practice, the effective length of the linker may be determined experimentally according to its capability to retain the native biological functions of the ligand molecule (e.g. its enzymatic activity). However, spacers with longer hydrocarbon bonds (especially if aromatic rings are included) may affect the hydrophobic properties of the matrix itself.

Thus use of such matrices, for example, for affinity chromatography might be seriously affected because of probable non-affinity (hydrophobic) binding of the hydrophobic molecules from the fluid to the solid phase.

Preferably, the linker will comprise from 3 to 11 carbon atoms, most preferably 3, 4, 5, 6,

7 or 8 carbon atoms.

The linker may either be a cleavable linker or a non-cleavable linker. In the context of the present invention, the term "cleavable linker" is intended to mean a linker that is cleavable under conditions which do not affect the activity of the material which is bound via the linker to the matrix. Examples of non-cleavable linkers which can be used in the context of the present invention include 6-aminohexanoic acid, others known in the art [11, 26, 27] and those of Table 1.

Table 1.

Examples of non-cleavable linkers (spacers) that can be used with SPE (classified according to the ligand functions).

Linker Ligand Spacer length (in carbons)

Thiopropyl SH equivalent to 13

Epoxy NH₂, OH, SH equivalent to 11

Aminopropyl COOH 3

Aminohexyl COOH 6

Carboxylhexyl NH₂ 6

N-Hydroxysuccinimidylhexyl NH₂ 8

Adipic acid dihydrazide Aldehydes, COOH 11

Hydrazide Aldehydes, COOH Preferred materials which may be attached to the matrix, optionally via a linker, include biological material such as proteins, polypeptides, peptides, peptide mimetics, enzymes, antibodies (e.g. monoclonal, polyclonal, hybrid), ribozymes, RNA, DNA, PNA (peptide nucleic acid) and whole cells or cell fractions. Particularly preferred is the use of Protein A for the purification of antibodies .

In other embodiments, it is preferred to immobilize an enzyme, such as restriction endonuclease or a phosphatase, which can be used for manipulation of a nucleic acid, e.g. DNA. In such embodiments, the matrix may be in the form of a column, to which the nucleic acid in solution may be added. With such columns, the usual requirement to extract the DNA with phenol/chloroform post-reaction with the enzyme may be obviated.

The matrix of the invention will in general be porous, i.e. pores or spaces will be present within the matrix through which liquids may pass. In certain embodiments of the invention, however, it may be advantageous for one, two or more sides of the matrix to be impermeable to fluids. For example, the matrix may be cylindrical in shape, wherein the sides of the cylinder are impermeable and the ends of the cylinder are permeable. The cylinder will therefore allow the passage of fluids from one end to the other without any leakage of the fluid out of the sides.

The invention also provides a process for making a sintered thermoplastic polymer matrix of the invention, comprising treating the surface of a sintered thermoplastic polymer matrix by one or more of gas plasma amination, gamma irradiation and chemical oxidation. Preferably, the process additionally comprises sintering a plurality of thermoplastic polymer particles to produce said sintered thermoplastic polymer matrix; and/or additionally comprises immobilizing a ligand on the surface of the matrix, optionally via a linker. Most preferably, the ligand or linker is immobilized on the matrix immediately after the surface treatment of the matrix.

In most embodiments of the invention, it will be preferable to modify the surface of the particles after the sintering process. In some case, however, the modification of the surface may be carried out before the sintering process. The matrix of the invention may take any convenient physical form, for example sheets, filters, membranes, cylinders, fibres, tubes, microtitre plates and columns. In one preferred embodiment, the matrix may be produced in a mould having a pre-defined shape. The matrix may also be provided in a rolled-up form of a sheet, from which a desired length of matrix is capable of being cut. The matrix will be of a size that it suitable for its purpose.

Preferably, the matrix is in the form of a disc or a column.

The matrix may be provided as a separate entity or it may form an integral part of another entity. For example, the matrix may be incorporated into separation devices such as columns, centrifuge vials, microtitre plates, cartridges or syringes, and, depending on the sample and the downstream processes to be operated, one or more of such devices may be provided in a serial or parallel manner. Such devices may be handled manually, semi- automatically or in fully-automated fashion.

The matrices of the invention may be used to produce high purity antibodies for experimental uses, e.g. for use on Western blots and in situ immuno-staining. The matrices of the invention may also be used for the large-scale purification of antibodies or recombinant-derived antibody fragments for diagnostic or therapeutic applications.

These uses are generally based on two alternative approaches and they exploit the advantages of low non-specific adsorption combined with the chemical stability of the matrix: (i) Protein A immobilized on a matrix of the invention may be used for the purification of mono- or polyclonal antibodies to very high specificities; and (ii) specific peptides immobilized to a matrix of the invention may be used to provide purification based on antigen specificity.

Thus one embodiment of the invention provides the use of a matrix of the invention to produce an antibody, preferably a mono- or polyclonal antibody or recombinant-derived antibody fragments. In one embodiment, protein A is immobilized on the matrix, preferably via a non-cleavable linker. In another embodiment, a peptide is immobilized on the matrix. The invention also provides a kit comprising a matrix of the invention together with a container of Protein A or a peptide or other protein (e.g. an enzyme). The invention also provides a method of purifying an antibody from a composition which comprises the said antibody, comprising:

(i) immobilizing a ligand which selectively binds the said antibody on a matrix of the invention, optionally via a linker;

(ii) passing the composition over the matrix; and (iii) eluting the desired antibody from the matrix.

Preferably, the ligand is Protein A. The invention also relates to an antibody which has been purified by such a method.

The invention further provides a method of purifying a polypeptide from a composition comprising that polypeptide, comprising:

(i) immobilizing a ligand which selectively binds the said polypeptide on a matrix of the invention, optionally via a linker;

(ii) passing the composition over the matrix; and (iii) eluting the desired polypeptide from the matrix.

Preferably, the ligand is an antibody. The invention also relates to a polypeptide which has been purified by such a method.

The broad range over which the matrices of the invention are stable may make the matrices suitable as substrates for engineered enzymes with kinetic properties which are optimized for process conditions (high temperature, high or low pH, etc.) under which other media (especially polysaccharides) are unstable. Examples of such systems include the immobilization of glucose/xylose isomerase in the production of high fructose corn steep liquor.

Thus a further embodiment of the invention provides a use of a matrix of the invention in bioconversion, wherein an enzyme is bound to the matrix. The matrices of the invention may also be used as biosensors. In such embodiments, entities such as proteins (for example, enzymes or antibodies) may be immobilized on a matrix of the invention where they may be used to detect specific analytes. In a further embodiment of the invention, the entities may be immobilized on a surface comprising a matrix of the invention (for example, a sheet or membrane) which is capable of being subdivided into a plurality of physically discrete pieces. Preferably, the physically discrete pieces are of uniform size and carry essentially the same amount of immobilized entity. Each piece may then be assembled into a sensor. The sensor may, for example, be based on solution phase enzyme reagents.

Matrices of the invention may also be incorporated into micro-fluidics devices, wherein one or more proteins, preferably one or more enzymes, are immobilized on the matrix.

Thus in a further embodiment of the invention, there is provided a sensor, preferably a biosensor, which comprises a matrix of the invention.

In yet a further embodiment of the invention, there is provided a micro -fluidics device which comprises a matrix of the invention.

The matrices of the invention may also be used to purify nucleic acids, for example, oligonucleotides, DNA and RNA. In one embodiment, oligodT is immobilized on the matrix of the invention and this is used to purify polyA RNA. In other embodiments, nucleic acid molecules having a specific sequence are immobilized on the matrix and the matrix is used to purify nucleic acid molecules comprising a complementary nucleic acid sequence.

Thus the invention provides the use of a matrix of the invention for the purification of nucleic acid molecules.

In some cases, the matrix of the invention to which an entity (such as a protein) has been immobilized may be stored in a dried form and then reactivated prior to use.

The following abbreviations are used in the subsequent sections: B ORIS Brother Of the Regulator of Imprinted Sites

BSA bovine serum albumin

B S binding solution (20mM KH₂PO₄ pH 7.0, 0.15M NaCl, 2mM EDTA, 0.1 % Tween 20)

CTCF CCCTC binding factor

DMSO dimethyl sulfoxide

IgG Immunoglobulin G

IgY Immunoglobulin Y Mini SPE discs SPE discs with the diameter 6.5mm (thickness 4.75 mm)

Maxi SPE discs SPE discs with the diameter 13mm (thickness 4.75 mm)

PBS phosphate buffered saline (0.01M phosphate buffer, 0.0027M potassium chloride and 0.137M sodium chloride, pH 7.4)

RT room temperature SDS sodium dodecyl sulfate

SPE sintered polyethylene.

TEA triethanolamine

TE buffer Tris EDTA (lOmM Tris-HCl, lmM EDTA pH 8.0)

LEGENDS TO THE FIGURES

Figure 1: Sintered polyethylene discs and columns of the invention. Figure 1A - 13mm and 6.5mm sintered polyethylene discs; Figure IB - 15ml column and 0.5ml icro- centrifuge tube; Figure 1C - Attachment of BSA to different matrices. Lane a: sintered polypropylene; Lane b: sintered polyethylene oxidized with K₂Cr O₇ for 3 hours; Lane c: sintered polyethylene oxidized with K₂MnO₄ for 1 hour; Lane d: sintered polyethylene oxidized with K₂MnO₄ for 3 hours; and Lane e: sintered polyethylene functionalized with S-H linkers. Figure 2: SDS-PAGE gel stained with Coomassie Blue showing crude chicken immune serum (Lane 1); purified anti-BORIS antibodies (Lane 2); and protein markers (Lane 3). Figure 3 : Western blots of lysates from five human cell lines, blotted with anti -BORIS antibodies before purification (Figure 3 A) and after purification (Figure 3B). Figure 4: Comparison of prior art method of antibody purification on Sepharose 4B and the method of the invention using sintered polyethylene. Lane 1 - Western blot of total cell lysates using antibodies purified with Sepharose 4B; Lane 2 - Western blot of total cell lysates using antibodies purified with SPE-affmity column.

Figure 5: Purification of antibodies using a sintered polyethylene column. Figure 5 A - Western blot of human cell lines with antibodies before adsorption; Figure 5B - Western blot of human cells lines with antibodies after adsorption with a SPE column. Figure 6: Immobilization of Protein A onto Sepharose 4B (Lane 1) and onto sintered polyethylene (Lane 2).

Figure 7: Western blot showing the leaching of Protein A from Sepharose 4B beads (Lane 2) and from sintered polyethylene (Lane 3). Lane 1 shows Protein A (control lane). Figure 8: Activity of calf intestinal alkaline phosphatase (CIP) after immobilization on a sintered polyethylene (SPE) column. Lane 1 - CLP-SPE disc; Lane 2 - BSA-SPE disc

(control).

Figure 9: Activity of RNAse A after immobilization on sintered polyethylene. M- DNA molecular markers - 1 kb DNA ladder (NE biolabs); S- supercoiled 4kb pCi plasmid (Promega); R - relaxed 4 kb pCi plasmid (Promega).

EXAMPLES

Method 1: Preparation of discs from flat Porvair sheets

SPE discs were cut from flat sheets of sintered polyethylene (Vyon Type F, Porvair Pic, UK). The diameters of the discs were 6.5mm and 13mm (Figure 1 A), thickness 4.75 mm and pore size 8-79 μm (mean 27 μm). These diameters were selected to fit in a 15 ml chromatography column or a 0.5ml micro-centrifuge tube (Figure IB).

The two formats of SPE discs, 13mm and 6.5mm, are shown in Figure 1A. The 13mm disc can be incorporated into a 15 ml column for large-scale experiments (maxi-format) or in the 0.5ml microcentrifuge tube (mini -format) for analytical small-scale experiments (Figure IB, left and right, respectively).

Method 2: Preparation of functionalized SPE with incorporated spacer

Step 1 : Matrix oxidation

Discs from Example 1 were soaked and saturated with 10ml of 3M H₂SO under vacuum for 5min. The discs were then washed twice with 20ml of 3M H₂SO₄ and removed. KMnO₄ (5 mg) was dissolved in 3M H₂S0 (100ml) and a 20ml portion was added to the discs and the reaction was heated up to +70°C with periodical agitation. After 1 hr, a fresh portion of 20ml of the KMnO in H ₂SO₄ was added and reaction was continued for another lhr, and after that the next portion of 20 ml of KMnO₄ in H₂SO₄ was added and reaction continued for the following 1 hr. After 3hrs of reaction, the products of oxidation were filtered off and the discs were washed 5 times with 5ml 10M HCl to remove the manganese oxide completely. The discs were then washed three times with DMSO.

Step 2. Linker formation

After washing with DMSO, the discs were saturated with the solution of carbodiimide (0.21g) in DMSO (20ml). The reaction was heated by microwaving to approximately 50°C and then left at RT for 30 min with agitation, then a new portion of carbodiimide was added and the reaction continued for another 30 min. The discs were then transferred into 20 ml solution of 50% DMSO with 6-aminohexanoic acid

(1.3 lg) and left overnight at room temperature with agitating. After the reaction was completed the filters were washed with 0.5 M HCl (5x) and 0.5M NaOH (5x), the discs were immersed in 0.1M NaOH (20ml) and NaCNBH (0.3 lg) was added together with 6-aminohexanoic acid (1.3 lg) and the reaction carried out for 3hr at RT. The discs were then washed in 0.5M HCl (5X) and 0.5M NaOH (5X) and finally in 50% methanol. After linker formation the discs can be stored in 50% methanol until needed. Method 3: Protein coupling

Activation of linker function for protein/peptide conjugation

The linker was converted into the acidic form by reaction with 0.5M HCl for 5 min, then subjected to reaction with 0.25M l-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDACH) in 20% DMSO for 20min at +40°C, the reactants removed, then new portion of 0.25M EDACH in 20% DMSO was added, reaction continued another 20min at +40°C. After this step the protein or peptide ligands were immediately applied to activated SPE.

Protein solution

For protein coupling, 4mg total protein solution was used in 0.25M triethanolamine pH 8.5, 0.5-lM NaCl depending on protein solubility. Protein was re-applied on the column several times by passing the protein solution through the column and then left for 30 min for conjugation. After the reaction was completed, any remaining active groups were then blocked with 1M ethanolamine, pH8.0 for lhr at room temperature. Finally, the filter was washed once with 0.1M citric acid to remove noncovalently bound protein (this step is optional).

After this step the columns can be assembled in various formats, two examples are shown in Figure IB.

Method 4: Determination of the amount of protein immobilized on SPE

The amount of coupled protein was calculated by assaying the unbound protein concentration after the coupling reaction. The difference between the loaded and unbound protein gives the amount of protein coupled to the matrix. A "Bio-Rad" protein detection kit was used to measure protein concentration according to the manufacture's recommendation. The protein immobilized on the SPE was visualized by staining with Ponseau. Filters were immersed in the 2% Ponseau solution for 15 min and washed twice with 10% acetic acid.

Method 5: Western blol/SDS-PAGE analysis

SDS-PAGE (SDS Polyacrylamide Gel Electrophoresis) used in this study was a modified Laemmli system [28]. The polyacrylamide separating gel (375 mM Tris- HCl, pH 8.8 and 0.1% (w/v) SDS was polymerized in the presence of 0.05% (v/v)

TEMED and 0.5 % (v/v) Ammonium Persulfate (APS). The stacking gel was composed of 135 mM Tris-HCl pH 6.8 with 0.1% (w/v) SDS polymerized in the presence of 0.2 % (v/v) TEMED and 0.25 % (w/v) APS.

Total cell lysates (protein samples) were prepared from 5x10⁵ cells. Cells were collected, washed with PBS and lysed in the UREA-lysis buffer (lOOmM Tris HCl pH 6.8, 7M Urea, 4% SDS, 10% 2 mercaptoethanol), and then heated at 95°C for 5 min in a heating block, loaded onto the gel and resolved at 50 mA/120 V.

To perform a western blotting assay, we used protocols described previously [29] with some modifications. After electrophoresis, proteins were transferred onto PVDF membranes (Immobilon P, Millipore Inc.) using a semi dry electroblotting apparatus (Amersham-Pharmacia Biotech. USA) in a blotting buffer (10 mM CAPS pH 11, 2.5% (v/v) Methanol) for 2 hr at 50 mA. Membranes were then blocked with blocking buffer [lx TBS, 0.5% (w/v) Non-Fat Milk (Marvel), 0.1% (v/v) Tween 20] for 30 min at room temperature and washed 3 times (10 min each) with washing buffer [0.05%(v/v) Tween 20, lx TBS]. After the third wash, the membranes were incubated for 1 hr with the primary antibodies followed by washing (3 times, 10 min each) with the washing buffer. The membranes were then submitted to 1 hr incubation with the secondary peroxidase-conjugated antibody, followed by washing as previously described. Each membrane were then incubated with a mixture of 800 μl Solution 1 & 800 μl Solution 2 (ECL, Amersham, UK) and then exposed to the autoradiography film (KODAK, Japan) for 0.1 - 30 minutes. The films were then developed using an automated X-zograph (UK) developing machine or manually.

Method 6: SDS/PAGE staining with the Coomassie Brilliant Blue

The SDS-PAGE gel containing protein was stained with the Coomassie-staining solution (0.025% (w/v) Coomassie Brilliant Blue G-250 ; 40% (v/v) methanol and 7% (v/v) acetic acid) for 2 hrs followed by 4 hours/overnight soaking in sufficient amount of de-staining solution (5% (v/v) methanol, 7% (v/v) acetic acid).

Method 7: Small scale isolation of plasmid DNA (Alkaline lysis method)

A standard method of plasmid isolation was used for the preparation of the crude plasmid + RNA fraction to test the activity of RNAse immobilized on SPE [29] with some modifications. A single colony of bacteria transformed with the plasmid pCi (Promega) was inoculated in 1.5 mL of LB broth containing antibiotic ampicillin, and incubated overnight at 37°C with vigorous shaking. The overnight culture was transferred into a sterile micro centrifuge tube and centrifuged at maximum speed for 30 seconds. The supernatant was discarded and the pellet was re-suspended in 100 μl of the ice-cold solution I (50mM Glucose, 25 mM Tris-Cl pH 8.0, 10 mM EDTA) with vigorous vortexing. 200 μl of Alkaline lysis solution II (0.2 N NaOH, 1% SDS) was added to the suspension and this was mixed by inverting the tube 5 -8 times until the suspension became clear and viscous. The tube was then left on ice for 10 minutes. 150 μl of ice cold solution III (3 M Potassium acetate, pH 5.5) was then added to the viscous suspension. The solution was mixed thoroughly by inverting the tube 8- 10 times, then incubated on ice for 5 minutes. The lysate was then centrifuged at maximum speed for 5 minutes and the supernatant was transferred to a new sterile tube. An equal amount of Phenol/Chloroform (1 :1 , v/v) was added to the plasmid suspension and this was mixed thoroughly by vortexing to separate the organic and the aqueous phase. The emulsion was then centrifuged at 13,000 rpm for 2 minutes at +4°C, and the aqueous layer containing plasmid

DNA was transferred to a new sterile microfuge tube. The extraction was repeated with chloroform and the aqueous phase was collected after centrifugation. The plasmid DNA was then precipitated from the suspension by adding 2 volumes of ice-cold 100 % (v/v) ethanol and 0.1 volumes of 3M Sodium acetate (pH 5.5) followed by incubation on ice for 30 minutes. The precipitated nucleic acids were collected by centrifugation 13,000 rpm at +4°C for 10 minutes. The supernatant was then carefully aspirated, 750 μl of cold 70% (v/v) ethanol was added to the tube and mixed by inverting the tube or by gentle vortexing. The plasmid was recovered by centrifugation at 13,000 rpm, +4°C for 10 minutes. The supernatant was removed and the pellet left to dry at room temperature for 10 minutes. The plasmid DNA was resuspended in 500 μl of sterile TE buffer (10 mM Tris-HCl, ImM EDTA, pH 8.0) and used immediately or stored at -20°C.

Method 8: Agarose gel electrophoresis of plasmid DNA

Gel electrophoresis of the plasmid DNA was performed as previously described [29] with some modifications. A total of 10 μl of plasmid DNA was added to 2 μl of 6x loading buffer (0.25% Bromophenol blue, 0.25% Xylene cyanol FF, 30% glycerol) in a clean microfuge tube. The mixture was then loaded into a slot (5 mm in length x 2.5 mm in width) cast in a 1 % agarose gel (5 mm thick) in lxTAE buffer (0.04M Tris -acetate, 0.00 IM EDTA). After the Bromophenol blue dye had migrated two -thirds to three-fourths the length of the gel, the electrophoresis was stopped and the gel was soaked in Ethidium bromide solution (0.5 μg/mL in H O) for 20 minutes. The gel was then examined and photographed under UV illumination.

Example 1: Conjugation of BSA to sintered polyethylene

The effects of different type of sintered polymer, oxidation conditions and linkers were investigated on protein binding to the filter.

The sintered polymers were activated and functionalized as described above in the Methods. BSA (4mg total protein per 0.8 ml (10x8mm) vol/ SPE disc) was diluted in 0.25M triethanolamine pH 8.3, 0.5M NaCl. Protein was re-applied on the filter several times by passing the protein solution through the column and then left for 30 min for conjugation. After the reaction was completed, the remaining active groups were blocked with 0. IM ethanolamine, pH 8.0 for lhr at room temperature. Finally, the filter was washed once with 0. IM citric acid to remove non -covalently bound protein.

The immobilized protein was visualized with Ponseau as described in the above Methods. The results are shown in Figure lC. From these experiments, the followed could be concluded: Both sintered polypropylene and sintered polyethylene can be used as a matrices for protein binding, although sintered polyethylene has a higher binding capacity for BSA. With regard to oxidizing agents, both K₂Cr₂O₇ and K₂MnO₄ could be used, although K₂Cr₂O₇ was found to be less efficient. Oxidation for three hours produced matrices with higher binding capacities. An S-H linker can be efficiently used for functionalization of the SPE.

Example 2: Conjugation of BORIS peptides to sintered polyethylene

The SPE discs were activated as described in the above Methods.

Synthetic peptides having sequences derived from the BORIS protein were designed and prepared as previously described [5]. BORIS is a novel protein with features of an oncogene [30].

Peptide solution (10 μmol per 0.8 ml of the disc volume) in 0.25M triethanolamine (TEA) pH 8.3 was applied using a pumped flow through system at a flow rate 2ml/min for 3 hr at room temperature. At the end of the incubation, the two bed volume of a 0. IM ethanolamine solution, pH 8.5, was added to neutralize any non- reacted functions. The discs were then washed through with 6M Guanidine/0. IM NaH₂PO , pH 4.0 solution to remove unbound material.

Example 3: Purification of the anti-BORIS antibodies using the sintered polyethylene column with immobilized anti-BORIS peptides

A polyclonal chicken antisera was raised against synthetic BORIS peptides. The antibodies were produced by Aves Labs (Tigard, OR) and quality tested. The antibodies contained significant amount of non-specific material cross-reacting with various cellular proteins (see figure 3 A). This antisera was purified as follows:

(NIΪ4)2S04 was added to the chicken immune serum to 33% saturation and the protein was recovered by centrifugation at 14,000 rpm at +4C°. The pellet was then dissolved (at approximately 5mg/ml) in binding solution (BS) (20mM KH₂PO₄ pH 7.0, 0.15M NaCl, 2mM EDTA, 0.1% Tween 20). The serum protein solution was applied onto the SPE column with the immobilized BORIS peptide prepared as described in Example 2 and assembled in a 15 ml format (Figure IB) for 3 hrs at +4°C, by using a pump circulation system. The column loaded with the serum proteins was then pumped-washed with ~50ml of BS and then with 10 ml of with BS + 0.4 NaCl. The bound material, I mmunoglobulin Y (IgYs) was eluted with 6M Guanidine/ 0.1M NaH₂PO₄pH 4.0 solution. The IgYs were then re-natured by dialysis against IL of PBS overnight at +4°C.

The quality of the purified antibodies was tested by SDS-PAGE with Coomassie staining as shown in Figure 2. In this Figure, Lane 1 shows the chicken immune serum prior to purification and Lane 2 shows the pure active component of IgY (indicated by an arrow) after purification.

The original and purified antibodies were then used in a Western blot assay against total cellular lysates prepared from five human cell lines. The results are shown in Figure 3. Panel A is a Western blot using antibodies before purification; Panel B is a Western blot of the same lysates using antibodies after purification through the SPE column. Lanes 1-5 correspond to extracts from five different cell lines tested in this experiment. A specific band recognized by the antibodies is shown by a double-headed arrow. Lane 1- K562

(chronic myelogenous leukemia cells; ATCC registered number: CCL-10); Lane 2 - HeLa (cervix epithelial adenocarcinoma cells; ATCC registered number: CCL-2); Lane 3 - LNCaP (prostate metastatic carcinoma cells; ATCC registered number: CRL-1740); Lane 4 - MCF7 (mammary gland adenocarcinoma cells; ATCC registered number: HTB-22); Lane 5 - 293T (embryonic kidney cells; ATCC registered number: CRL-11268). Example 4: Purification of anti-CTCF antibodies using the SPE column with immobilized CTCF N-terminal domain

Rabbit polyclonal anti-CTCF antibodies were generated against the CTCF-N terminal domain. CTCF is a conserved, ubiquitous and multifunctional 11 Zn finger (ZF) factor with features of a tumour suppressor [31]. The CTCF-N terminal domain, which was used for immunization and also for coupling to SPE and to Sepharose 4B, was produced in the bacterial expression system as previously described [3].

To affinity-purify the anti-CTCF antibodies (NH )₂SO₄ was added to the immune serum to

33%) saturation and the protein was precipitated by centrifugation at 14,000 rpm at +4 °C. The pellet was then dissolved (at approximately 5mg/ml) in binding solution (BS) (20mM KH₂PO₄ ρH 7.0, 0.15M NaCl, 2mM EDTA, 0.1% Tween 20). The protein solution was applied onto the SPE column (15 ml format, as shown in Figure IB and Example 1 ), with the immobilized CTCF-N terminal domain for 3 hrs at +4°C, by using a pump circulation system. The column loaded with the serum proteins was then washed with -50 ml of BS and then with 10ml of BS + 0.4 NaCl. The bound material (IgGs) was eluted with 6M Guanidine/0. IM NaH₂P0 pH 4.0 solution. The IgGs were then re-natured by dialysis against IL PBS overnight at +4°C.

The coupling of the CTCF-N terminal domain to Sepharose 4B was performed as previously reported [3].

The quality of these antibodies was tested as shown in Figure 4. K562 cells (chronic myelogenous leukemia; ATCC registered number: CCL-10) were lysed and total cell lysates were resolved by SDS gel followed by Western analysis with the primary anti- CTCF antibodies purified by the two methods. The antibodies purified by standard method cross-react with several bands (indicated by asterisks; Lane 1). Antibodies purified using the SPE-affinity column show no background bands and also higher specific activity of the purified antibody (stronger 130 kDa CTCF band; Lane 2). Example 5: Conjugation of total cell protein to SPE for pre- adsorption of antibodies

A SPE-total cell protein column was employed for pre-absorption of the commercial secondary antibodies. A mixture of ~ 5xl0⁷ cells of each type of K562 (chronic myelogenous leukemia cells, ATCC registered number: CCL-10), HBLIOO (cell line derived from normal breast epithelium [32]) and HMT 3552 (breast epithelial cell line derived form fibrocystic immortalized cells [33]) was used for extraction of total cell protein.

The cells were collected by centrifugation, washed with lxPBS lysed in 50mM Tris/HCl, 0.5% Triton X-100, 0.1% SDS, IM NaCl, 1 mM EDTA, 50mM DTT. The suspension was heated at 96°C for 5 min and cooled on ice. Then PEG 8000 was added to 6% final concentration, the extract was incubated for 15min longer and spun at 14,000 for lOmin. The supernatant was transferred into new tube and acetone was added to 50% final concentration. The protein extract was cooled for lOmin on ice and the protein was collected at 14,000 rpm for 10 min.

The pellet was then washed twice with 50% acetone/20mM HCl, air-dried and dissolved in 0.25M triethanolamine (TEA), 7M urea at approximate concentration 2.5mg/ml.

Three activated maxi discs (13mm) were assembled into 15ml columns and used for coupling of the protein as described in Example 1. The estimated amount of conjugated protein was 4mg/ml.

Goat anti-rabbit peroxidase conjugated secondary antibody was purchased from Cappel. Western blot analysis demonstrated that these antibodies had non-specific activities, cross- reacting with the cellular proteins (Figure 5A). These antibodies were diluted at the ratio 1 :4 with solution PBS + 0.1% Tween 20 (total volume 500 μl) and processed through a SPE/cell protein column with recycling, using the pumped flow through system. The pre- absorbed antibodies were collected, concentrated and tested as shown in Figure 5B. The immortalized human prostate epithelial cell line BPH-l (Lane 1) and its 8 tumorigenic sub lines BPH 1-CAFTD-Ol through 08 (Lanes 2-9, respectively) [34] were lysed and total cell lysates were resolved by SDS gel. This was followed by the Western analysis with the primary anti-CTCF antibodies purified through SPE column as described in Example 4 (see also Figure 4, Lane 2) and then probed with the secondary anti-rabbit peroxidase- conjugated IgG antibody (Cappel) before (Figure 5 A) and after (Figure 5B) pre- absorption.

It can be seen that, prior to pre -absorption, the secondary antibody cross-reacts with other cellular proteins to generate spurious background signals (indicated by asterisks), whereas the secondary antibody pre-absorbed through the SPE-column show no background bands.

Example 6: Immobilization of Protein A onto SPE: assessment of the effectiveness of binding of rabbit IgG.

S. aureus Protein A was immobilized on SPE and its effectiveness in binding serum immunoglobulins compared with that of commercially-available Protein A immobilized on Sepharose 4B.

Protein A [35] and Protein A- Sepharose 4B Fast flow (ProtA/sepharose) were purchased from Sigma.

Coupling of Protein A to SPE was performed as described in Example 1. Both ProtA sepharose and mini SPE disc were then incubated with shaking for 3 hrs at +4°C with the rabbit pre-immune serum diluted at the ratio 1 :4 with solution 50mM Tris/HCl pH

7.0, 0.25M NaCl, 0.5% Tween 20, 2mM EDTA (Solution 1) followed by washing with Solution 1 and elution of the bound IgG with 2x SDS-Loading Buffer (0.125M Tris- HCL;4% SDS, 20% glycerol, 0.2M dithiothreitol, 0.02% Bromophenol Blue pH 6.8). These materials were analyzed by SDS-PAGE/ Western as shown in Figure 6. The Western blot was carried out using goat anti -rabbit IgG heavy chain specific peroxidase-conjugated antibodies. The efficiency of binding of IgG was identical in the case of binding to ProtA/sepharose (Lane 1) and mini SPE disc (Lane 2).

Example 7: Immobilization of Protein A onto SPE: evaluation of the leaching of Protein A form from ProtA/SPE matrix

The stability of ProtA/SPE and ProtA/Sepharose 4B were compared under the denaturing and hydrolytic conditions during the elution of IgG.

50 μl of ProtA/sepharose 4B beads and a single ProtA/SPE-disc were incubated with IM glycine/HCl pH 2.5 for 10 min. To detect Protein A leaching from the matrix, the flow through fluids were collected and precipitated with acetone as follows. Ice-cold acetone was added to 50% (final concentration) to lOOμl of the flow through and the tube was placed at -20°C for 30min. The precipitated proteins were then collected by centrifugation at 14,000 rpm for 15 min. The pellet was washed with a fresh portion of 50% ice-cold acetone/ 20mM HCl, and the mixture was centrifuged at 14,000 rpm for 15 min. The supernatant was aspirated and the pellet was dried under vacuum for approximately 5 min. The resulting pellet was then dissolved in 20 μl SDS-Loading Buffer of SDS-LYSIS Buffer and analyzed on the SDS-PAGE, followed by Western blotting assay with the primary rabbit anti-Protein A (Sigma) antibody and the secondary anti-rabbit IgG antibody conjugated with peroxidase as described in the above Methods. The results are shown in Figure 7.

Lane 1 represents 2 μg of Protein A (Sigma) loaded as control. It can be seen that Protein

A represents a heterogeneous population of polypeptides of different sizes. Under the experimental conditions used, there is significant leaching of Protein A from the ProtA/sepharose 4B beads (Lane 2). However, no leaching of Protein A is detected from the ProtA/SPE-disc (Lane 3). Example 8: Assessment of the stability of proteins bound to SPE by analyzing the activity of the enzyme calf intestinal alkaline phosphatase (CIP) after immobilization onto SPE

The enzyme calf intestinal alkaline phosphatase (CEP) was purchased from New England

Biolabs. For conjugation with SPE, 10 nmol of protein was used and the processed as described in Example 1 (protein conjugation).

To test if the enzyme was still active, the CIP assay using NTB (Sigma) was performed. NTB, or 5-Bromo-4-Chloro-3-Indolyl phosphate/ Nitro Blue Tetrazolium, is supplied as a ready-made substrate solution and in the presence of CIP develops a blue color. For this reaction, the SPE disc with immobilized CEP and the SPE disc with immobilized BSA, serving as control, were immersed in the NBT- substrate for 15 min at room temperature, and then washed with distilled water 3 times for 5 min. Figure 8 shows intense colour development on the disc comprising SPE-CEP (Lane 1) compared with no colour development on the disc comprising SPE-BSA (Lane 2).

Example 9: Assessment of the quality of the proteins bound to SPE by analyzing the activity of the enzyme enzyme Ribonuclease A (RNAse A) after immobilization onto SPE

50nmol enzyme RNAse A (Boehringer Mannheim) was conjugated to mini-SPE as described in Example 1. To test the activity of the SPE-immobilized RNAse A, a crude extract containing bacterial plasmid and RNA was prepared as described in the Methods. The resulting plasmid DNA+RNA sample was dissolved in 500 μl of a TE buffer (10 mM

Tris-HCl, ImM EDTA, pH 8.0), loaded onto an SPE/RNAse column and incubated at room temperature for lhr. The reactions were collected and processed through a 1% agarose gel.

As can be seen in Figure 9, the immobilized RNAse A efficiently degrades RNA in the analyzed sample (Lane 2) compared to BSA SPE matrix (Lane 1) . REFERENCES

1. Chernukhin IV, Klenova EM ( 2000 ) A method of immobilization on the solid support of complex and simple enzymes retaining their activity. Analytical Biochemistry, 280, 178 - 181.

2. Klenova EM, Chernukhin IV, El-Kady Al, Lee RE, Pugacheva EM, Goodwin GH, Delgado D, Filippova GN, Leon J, Neiman PE, Lobanenkov W (2001 ). Functional CK II phosphorylation sites in the C-terminal region of the multivalent pluripotent transcriptional factor, CTCF. Mol. Cell Biology, 21, 6, 2221-2234. 3. Chernukhin IV, Shamsuddin S, Robinson AF, Came A, Paul A, El-Kady A,

Lobanenkov VV, Klenova EM (2000) Physical and functional interaction between two pluripotent proteins, the Y-box DNA/RNA binding factor, YB-1, and the multivalent Zinc- finger factor, CTCF. JBiol Chemistry, 275, 38, 29915-29921

4. Chernukhin TV, Shamsuddin S, Chiang CM., Wu S.Y, Lobanenkov W, Klenova EM. (2003) The multivalent DNA-binding factor, CTCF, interacts with the RNA Polymerase II large subunit in vivo and in vitro, (manuscript submitted to EMBO J)

5. Loukinov DI, Pugacheva E, Vatolin S, Pack SD, Moon H, Chernukhin I, Mannan P, Larsson E, Kanduri C , Vostrov A, Cui H, Niemitz EL, Rasko JEL, Docquier FM, Kistler M, Breen J, Zhuang Z, Quitschke W, Renkawitz R, Klenova EM, Feinberg AP, Ohlsson R, Morse III HC, Lobanenkov VV (2002) BORIS, a novel male germline-specific protein associated with epigenetic reprogramming events, shares the same 11 Zn-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma. Proc.Natl.Acad.Sci. USA, 99,6806-6811

6. Ginjala V, Pant V, Chernukin I, Lai Y-P, Kanduri C, Mukhopadhyay R, Oberg F, Oshimura M, Feinberg AP, Lobanenkov V, Klenova E, Ohlsson R A (2002) A novel type of chromatin marking in genomic imprinting: Allele-specific poly(ADP-ribosyl)ation is essential for the chromatin insulator function of the HI 9 ICR (submitted to Nature Genetics)

7. Narayanan SR, Crane LJ. (1990) Affinity chromatography supports: a look at performance requirements. Trends Biotechnol. (1990) 8: 12-16

8. Stellwagen E. (1990) Gel filtration. Methods Enzymol. ( "Guide to protein purification", Ed. MP Deutscher) 182:317-328 9. Cutler P. (1996) Affinity chromatography. Methods Mol Biol. ( "Protein Purification protocols", Humana Press, Totowa, New Jersey, Ed. S Doonan) 59:157-168.

10. Bergbreiter DE. (1999) Alternative polymer supports for organic chemistry. Med Res Rev.19 :439-50 11. Meldal M Properties of solid supports. (1997) Methods Enzymol. 289:83-104

12. Spier RE. (1991) An overview of animal cell biotechnology: the conjoint application of science, art, and engineering. Biotechnology. 17:3-18

13. Kinoshita Y, Kuzuhara T, Kirigakubo M, Kobayashi M, Shimura K, Ikada Y. (1993) Reduction in tumour formation on porous polyethylene by collagen immobilization. Biomaterials. 14:546-50

14. Kinoshita Y, Kuzuhara T, Kirigakubo M, Kobayashi M, Shimura K, Ikada Y. (1993) Soft tissue reaction to collagen-immobilized porous polyethylene: subcutaneous implantation in rats for 20 wk. Biomaterials. 14 :209-15

15. Kinoshita Y, Kuzuhara T, Kobayashi M, Ikada Y.(1995) Reduction in tumor formation on polyethylene by collagen immobilization J. Long Term Eff. Med Implants.

5:275-84

16. Ito Y, Zheng J, Imanishi Y. (1997) Enhancement of cell growth on a porous membrane co-immobilized with cell-growth and cell adhesion factors. Biomaterials. 18: 197-202

17. Hsu WC, Green JP, Spilker MH, Rubin PA. (2000) Primary placement of a titanium motility post in a porous polyethylene orbital implant: animal model with quantitative assessment of fibrovascular ingrowth and vascular density. Ophthal. Plast. Reconstr. Surg. 16:370-379

18. Purdy EP (1997) Oculoplastic and orbital applications of porous high -density polyethylene implants. Curr. Opin. Ophthalmol. 8:57-63 19. US 5,679,539

20. US 5,122,452 21. US20020064413

22. GB 2,369,796

23. Abstract book, SYMPOSIUM Y Plasma Deposition and Treatment of Polymers November 29 - December 2, 1998 Chairs; http ./ www.mrs .org/meetings/fall98/absbook AbstractBookY.html

24. US 5,679,539 25. Zammatteo N, Girardeaux C, Delforge D, Pireaux JJ, Remade J. Amination of polystyrene microwells: application to the covalent grafting of DNA probes for hybridization assays.Anal. Biochem. 1996236(l):85-94

26. Songster MF, Barany G. (1997) Handles for solid-phase peptide synthesis. Methods Enzymol. 289:126-174

27. Ostrove, S. (1990) Affinity chromatography: general methods. Methods Enzymol. ("Guide to protein purification", Ed. MP Deutscher) 182:357-371.

28. Laemmli (1970) "Cleavage of structural proteins during the assembly of the head of bacteriophage T4". Nature 227: 680 -5) 29. Sambrook, J. and Russel. D.W. (2001). Molecular Cloning: A laboratory manual. 3^rd edition. Cold Spring Harbor Press. Cold Spring Harbor, NY 30. Klenova EM, Morse UJ HC, Ohlsson R, Lobanenkov VV (2002) The novel BORIS+CTCF gene family is uniquely involved in the epigenetics of normal biology and cancer. Seminars in Cancer Biology, 12, 399-414 31. Ohlsson R, Renkawitz R, Lobanenkov V. CTCF is a uniquely versatile transcription regulator linked to epigenetics and disease. Trends Genet. 2001, 17: 520-527

32. Gaffney EV. A cell line (HBL-100) established from human breast milk. Cell Tissue Res. 1982, 227:563-8.)

33. Briand P, Petersen OW, Van Deurs B.A new diploid non-tumorigenic human breast epithelial cell line isolated and propagated in chemically defined medium. In Vitro Cell

Dev Biol. 1987, 23:181-8

34. Hayward SW, Wang Y, Cao M, Horn YK, Zhang B, Grossfeld GD, Sudilovsky D, Cunha GR. Malignant transformation in a non-tumorigenic human prostatic epithelial cell line. Cancer Res. 2001,61:8135-42 35. Forsgren A, Sjoquist J. Protein A from Staphylococcus aureus. Reaction with rabbit gamma-globulin. J. Immunol. 1967, 99:19-24

Claims

1. A porous rigid matrix comprising sintered thermoplastic polymer particles, wherein the matrix has a modified surface which is chemically reactive.

2. A porous rigid matrix as claimed in claim 1, wherein the matrix has a modified surface which is functionalized.

3. A porous rigid matrix as claimed in claim 1 or claim 2, wherein the matrix has a modified surface which provides pendant functional groups which are suitable for attaching a ligand to the surface, optionally via a linker.

4. A matrix as claimed in any one of the preceding claims, wherein the thermoplastic polymer comprises or consists of a polyolefin or a vinyl polymer.

5. A matrix as claimed in claim 4, wherein the polyolefin comprises polyethylene and/or polypropylene.

6. A matrix as claimed in claim 4 or claim 5, wherein the thermoplastic polymer is polyethylene.

7. A matrix as claimed in any one of the preceding claims, wherein the pore sizes of the matrix are between l-1000μm.

8. A matrix as claimed in any one of the preceding claims, wherein the modified surface has been produced by gas plasma amination, gamma irradiation and/or chemical oxidation.

9. A matrix as claimed in claim 8, wherein the modified surface has been produced by chemical oxidation.

10. A matrix as claimed in claim 9, wherein the modified surface has been produced by the treatment of the surface with one or more oxidizing acids.

11. A matrix as claimed in any one of the preceding claims, wherein the modified surface has been produced by treating the matrix with a composition comprising KMnO in H₂SO _.

12. A matrix as claimed in any one of the preceding claims, wherein the modified surface provides one or more functional groups selected from carboxyl, hydroxyl, aldehyde, keto and amino groups.

13. A matrix as claimed in any one of the preceding claims, wherein one or more linkers or spacers are attached to the surface of the matrix.

14. A matrix as claimed in any one of the preceding claims, wherein the linker is a non - cleavable linker.

15. A matrix as claimed in claim 14, wherein the linker is 6-aminohexanoic acid.

16. A matrix as claimed in any one of the preceding claims, wherein a ligand is immobilized on the matrix, optionally via a linker.

17. A matrix as claimed in claim 16, wherein the ligand is selected from proteins, polypeptides, peptides, peptide mimetics, enzymes, antibodies, ribozymes, RNA, DNA, PNA, whole cells and cell fractions.

18. A matrix as claimed in claim 17, wherein the ligand is Protein A.

19. A matrix as claimed in claim 17, wherein the ligand is oligo-dT.

20. A matrix as claimed in any one of the preceding claims, wherein the matrix is in the form of a sheet, filter, membrane, cylinder, fibre, tube, microtitre plate or column.

21. A matrix as claimed in any one of the preceding claims, wherein the matrix is incorporated into a separation device.

22. A matrix as claimed in claim 21, wherein the separation device is a column, centrifuge vial, microtitre plate, cartridge or syringe.

23. A biosensor comprising a matrix as claimed in any one of claims 1 to 22.

24. An affinity column comprising a matrix as claimed in any one of claims 1 to 22.

25. A micro-fluidics device comprising a matrix as claimed in any one of claims 1 to 22.

26. Use of a matrix as claimed in any one of claims 1 to 22 in affinity chromatography.

27. Use of a matrix as claimed in any one of claims 1 to 22 for the purification of antibodies.

28. Use of a matrix as claimed in claim 27, wherein Protein A is immobilized on the matrix, preferably via a non-cleavable linker.

29. Use of a matrix as claimed in any one of claims 1 to 22, wherein an enzyme is immobilized on the matrix, optionally via a linker.

30. A method of purifying a substrate from a composition which comprises the said substrate, comprising:

(i) immobilizing a ligand which selectively binds the said substrate on a matrix as claimed in any one of claims 1 to 22, optionally via a linker; (ii) passing the composition over the matrix; and (iii) eluting the desired substrate from the matrix.

31. A substrate which has been purified by the method of claim 30.

32. A method of purifying an antibody from a composition which comprises the said antibody, comprising:

(i) immobilizing a ligand which selectively binds the said antibody on a matrix as claimed in any one of claims 1 to 22, optionally via a linker; (ii) passing the composition over the matrix; and

(iii) eluting the desired antibody from the matrix.

33. A method as claimed in claim 32 wherein the ligand is Protein A.

34. An antibody which has been purified by the method of claims 32 or 33.

35. A method of purifying a polypeptide from a composition comprising that polypeptide, comprising:

(i) immobilizing a ligand which selectively binds the said polypeptide on a matrix as claimed in any one of claims 1 to 22, optionally via a linker;

(ii) passing the composition over the matrix; and (iii) eluting the desired polypeptide from the matrix.

36. A method as claimed in claim 35, wherein the ligand is an antibody.

37. A polypeptide which has been purified by the method of claim 35 or claim 36.

38. A process for making a sintered thermoplastic polymer matrix having a modified surface, comprising treating the surface of a sintered thermoplastic polymer matrix by one or more of gas plasma amination, gamma irradiation and chemical oxidation.

39. A process as claimed in claim 38, which additionally comprises sintering a plurality of thermoplastic polymer particles to produce said sintered thermoplastic polymer matrix.

40. A process as claimed in claim 38 or claim 39, which additionally comprises immobilizing a ligand on the surface of the matrix, optionally via a linker.

41. A porous matrix for use in affinity chromatography comprising sintered polyethylene particles, wherein the surfaces of the sintered particles have been chemically oxidized to provide pendant functional groups which are suitable for attachment to a ligand.

42. A matrix as claimed in claim 41, wherein a ligand is immobilized on the surface of the matrix, optionally via linker.