Patent Description:
Isolation of highly pure biomolecules is a requirement of a number of fields including biotechnology applications and the manufacture of biopharmaceutical products.

Chromatography is used extensively in industry for purification of biopharmaceuticals including therapeutic proteins, nucleic acids, and cells etc. The downstream process for purification of biomolecules using traditional chromatography and alternative isolation methods such as the Cohn Process is inefficient and costly especially at large scale. The use of affinity chromatography in the purification of target biomolecules affords potential for far greater specificity than traditional separation methods allowing more efficient and cost-effective downstream bioprocesses.

<CIT> discloses a process for preparing a functionalised polymeric chromatography medium, including a functionalisation in which:.

<CIT> discloses triazine-based detoxification agents.

The article <NPL> discloses scaffolds with ligands specific for the purification of DNA polymerase which are bound to activated substrates.

The performance of affinity adsorbents on repeated use in terms of maintained selectivity and capacity is dependent on cleaning the solid phase matrix to restore it ready for subsequent use by means of a clean in place (CIP) step in the chromatography process. Sanitisation of the matrix is also necessary to avoid contamination from bacteria, viruses, and endotoxin. The conditions typically used for this cleaning and sanitisation is aqueous NaOH (<NUM> to <NUM> NaOH) which means exposure of the matrix to solutions in excess of pH <NUM>. NaOH is favoured for this purpose as it is cheap, non-toxic, freely available, and relatively easy to dispose of.

However, high pH conditions used in CIP and sanitisation steps are detrimental to the stability of some affinity ligands, notably protein-based ligands. High pH can affect the tertiary structure of the affinity ligand and in some cases cleavage of the ligand by hydrolysis which may diminish the performance of the adsorbent. This degradation can limit the re-use potential of an adsorbent and therefore increase the cost of bioprocesses and in turn the products manufactured with them.

Affinity ligands which have improved caustic stability are known in the art. The modification of asparagine residues in protein ligands to improve alkali stability is discussed in <CIT>. Patent <CIT> also discusses the mutation of asparagine residues to amino acids other than glutamine or aspartic acid in immunoglobulin binding protein ligands to improve caustic stability. The use of functional protein fragments such as domain C from Staphylococcus protein A to provide adsorbents with improved stability to high pH is captured in <CIT>. Use of peptide ligands with improved caustic stability compared to protein-based ligands for immunoglobulin purification is described in <CIT>.

The availability of highly stable, cost effective affinity chromatography adsorbents with low toxicological risk is therefore of particular interest in bioprocessing to achieve affordable therapeutics.

Further, it is desirable for activated adsorbents used for covalent attachment of affinity ligands to possess a high density of chemically reactive groups. High activation densities on solid supports afford increased reaction rates for ligand coupling which in turn allows reduction in ligand concentration required to drive the coupling reaction, manufacturing process time and reduction in reaction temperatures. This is particularly important for costly ligands and fragile ligands that require mild immobilisation reaction conditions to avoid degradation. Another advantage to high activation density is achievement of higher immobilised ligand concentrations. This affords the possibility of increasing the binding capacity for target biomolecules. Triazine adsorbents disclosed in the prior art, for example <CIT>, describe the use of activating agents such as epichlorohydrin for epoxide activation. This type of activation chemistry, whist offering caustic stability, is limited in terms of the possible density of chemically reactive groups achievable on beaded agarose supports.

The present invention arises from the inventors' attempts to overcome the problems associated with the prior art.

Therefore, in accordance with a first aspect of the invention, there is provided a method of producing an activated substrate according to claim <NUM>.

The activated substrate, also referred to herein as the activated base matrix, may be understood to be activated in that it is configured to further react with a molecule comprising a ligand to provide a scaffold for isolation of a biomolecule. Advantageously, therefore, the method provides an activated substrate, which can be further reacted with a molecule comprising a ligand to provide a scaffold for isolation of a biomolecule.

Activation of a substrate as described herein may achieve a high surface concentration of chemically active groups which may be conveniently converted to a high concentration of more highly reactive groups
The substrate may be a solid support. The solid support may be selected from the group consisting of a controlled pore glass, a magnetic controlled pore glass, a silica-containing particle, a polymer, and a controlled pore glass grafted with a polymer. The solid support may comprise a polymer. The polymer may be a natural polymer or a synthetic polymer. The polymer may be a polysaccharide, a polymethacrylate, a polymer of styrene, a copolymer of styrene and divinylbenzene, a copolymer of styrene and divinylbenzene grafted with polyethyleneglycol or a copolymer of dimethylacrylamide and N,N,-bisacryloylethylenediamine. The polysaccharide may be agarose, cellulose, hemicellulose, dextran, carrageenan or chitin.

The substrate may be a fibre, a fibre mat, a membrane, a solid bead, a porous bead, a monolith or a solid gel.

Preferably, the substrate comprises a nucleophilic moiety. The nucleophilic moiety may be an -OH, SH or -NH<NUM> group. Preferably, the nucleophilic moiety is a hydroxyl moiety.

The method preferably comprises modifying the substrate to form a base matrix comprising a leaving group. The leaving group may be a halogen.

Modifying the substrate to form a base matrix comprising a leaving group comprises:.

The method comprises modifying the substrate to form a base matrix comprising an optionally substituted heterocyclic ring, which may be a <NUM> membered ring.

The optionally substituted heterocyclic ring may be an optionally substituted epoxide. Modifying the substrate to form a base matrix comprising an optionally substituted heterocyclic ring comprises contacting the substrate with an electrophile, wherein the electrophile comprises an optionally substituted heterocyclic ring, to thereby provide a base matrix comprising an optionally substituted heterocyclic ring.

Accordingly, the method comprises contacting the substrate with an electrophile.

The electrophile may comprise <NUM> to <NUM> carbon atoms, more preferably the electrophile comprises <NUM> to <NUM> carbon atoms or <NUM> to <NUM> carbon atoms, and most preferably <NUM> to <NUM> carbon atoms, or <NUM> to <NUM> carbon atoms.

The electrophile may comprise a C<NUM>-<NUM> unsaturated hydrocarbon chain, more preferably a C<NUM>-<NUM> unsaturated hydrocarbon chain, and most preferably a C<NUM>-<NUM> unsaturated hydrocarbon chain. The unsaturated hydrocarbon chain may be an allyl group.

The electrophile may comprise an ether group.

The electrophile is a compound of formula (I):.

R<NUM>-L<NUM>-X<NUM>-L<NUM>-R<NUM>     (I).

The term "alkyl", as used herein, unless otherwise specified, refers to a saturated straight or branched hydrocarbon. An alkyl group can be unsubstituted or substituted with one or more of halogen, OH, SH, COOH, NH<NUM> or oxo.

"Alkenyl" refers to olefinically unsaturated hydrocarbon groups which can be unbranched or branched. An alkenyl group can be unsubstituted or substituted with one or more of halogen, OH, SH, COOH, NH<NUM>, oxo or an optionally substituted C<NUM>-<NUM> alkynyl.

"Alkynyl" refers to acetylenically unsaturated hydrocarbon groups which can be unbranched or branched. An alkenyl group can be unsubstituted or substituted with one or more of halogen, OH, SH, COOH, NH<NUM>, oxo or an optionally substituted C<NUM>-<NUM> alkenyl.

The term "alkylene", as used herein, unless otherwise specified, refers to a bivalent saturated straight or branched hydrocarbon. An alkylene group can be unsubstituted or substituted with one or more of halogen, OH, SH, NH<NUM>, COOH or oxo.

The term "alkenylene", as used herein, unless otherwise specified, refers to a bivalent olefinically unsaturated straight or branched hydrocarbon. An alkenylene group can be unsubstituted or substituted with one or more of halogen, OH, SH, COOH, NH<NUM>, oxo or an optionally substituted C<NUM>-<NUM> alkynyl.

The term "alkynylene", as used herein, unless otherwise specified, refers to a bivalent acetylenically unsaturated straight or branched hydrocarbon. An alkynylene group can be unsubstituted or substituted with one or more of halogen, OH, SH, COOH, NH<NUM>, oxo or an optionally substituted C<NUM>-<NUM> alkenyl.

A "heterocyclic ring" may refer to <NUM> membered monocyclic rings in which at least one ring atom is a heteroatom. The or each heteroatom may be independently selected from the group consisting of oxygen, sulfur and nitrogen. A heterocyclic ring may be saturated or partially saturated. A heterocyclic group can be unsubstituted or substituted with one or more of halogen, OH, SH, COOH, NH<NUM>, oxo, optionally substituted C<NUM>-C<NUM> alkyl, optionally substituted C<NUM>-C<NUM> alkenyl or optionally substituted C<NUM>-C<NUM> alkynyl.

"Heteroaryl" and "heteroaromatic ring" refers to a monocyclic or fused, e.g. bicyclic, aromatic <NUM> to <NUM> membered ring system in which at least one ring atom is a heteroatom. The or each heteroatom may be independently selected from the group consisting of oxygen, sulfur and nitrogen.

"Aryl", "arylene" and "aromatic ring" refers to a monocyclic or fused, e.g. bicyclic, aromatic <NUM>-<NUM> membered aromatic ring system.

An optionally substituted aromatic or heteroaromatic group as described anywhere herein can be unsubstituted or substituted with one or more of halogen, OH, SH, COOH, NH<NUM>, oxo, optionally substituted C<NUM>-C<NUM> alkyl, optionally substituted C<NUM>-C<NUM> alkenyl or optionally substituted C<NUM>-C<NUM> alkynyl.

A "heteroatom" may be O, NR<NUM> or S, wherein R<NUM> is H, an optionally substituted C<NUM>-C<NUM> alkyl, an optionally substituted C<NUM>-C<NUM> alkenyl or an optionally substituted C<NUM>-C<NUM> alkynyl.

A "halogen" may be fluorine, chlorine, bromine or iodine.

R<NUM> may be an optionally substituted <NUM> membered heterocyclic ring.

Accordingly, in a preferred embodiment, the compound of formula (I) is a compound of formula (Ia):
<CHM>.

Preferably, R<NUM>, R<NUM> and R<NUM> are independently H, a C<NUM>-C<NUM> alkyl, a C<NUM>-C<NUM> alkenyl or a C<NUM>-C<NUM> alkynyl. Preferably, R<NUM>, R<NUM> and R<NUM> are each H.

Accordingly, in a more preferred embodiment, the compound of formula (I) is a compound of formula (Iai):
<CHM>.

In embodiments where R<NUM> is a leaving group, the leaving group may be a halogen.

L<NUM> may be absent, a C<NUM>-<NUM> alkylene, a C<NUM>-<NUM> alkenylene or a C<NUM>-<NUM> alkynylene. Preferably, L<NUM> is absent or a C<NUM>-<NUM> alkylene, more preferably is absent, -CH<NUM>- or -CH<NUM>CH<NUM>-, and most preferably is -CH<NUM>-.

L<NUM> may be absent, a C<NUM>-<NUM> alkylene, a C<NUM>-<NUM> alkenylene or a C<NUM>-<NUM> alkynylene. Preferably, L<NUM> is absent or a C<NUM>-<NUM> alkylene, more preferably is absent, -CH<NUM>- or -CH<NUM>CH<NUM>-, and most preferably is absent or -CH<NUM>-.

In one embodiment, R<NUM> may be an optionally substituted C<NUM>-C<NUM> alkenyl or an optionally substituted C<NUM>-C<NUM> alkynyl. More preferably, R<NUM> is a C<NUM>-C<NUM> alkenyl or an optionally substituted C<NUM>-C<NUM> alkynyl. Most preferably, R<NUM> is -CH<NUM>CHCH<NUM>.

Accordingly, the electrophile may by allyl glycidyl ether (AGE).

The substrate and the electrophile may be contacted in a weight ratio of between <NUM>:<NUM> and <NUM>:<NUM>, between <NUM>:<NUM> and <NUM>:<NUM>, between <NUM>:<NUM> and <NUM>:<NUM>, more preferably between <NUM>:<NUM> and <NUM>:<NUM>, between <NUM>:<NUM> and <NUM>:<NUM> or between <NUM>:<NUM> and <NUM>:<NUM>, and most preferably between <NUM>:<NUM> and <NUM>:<NUM>, between <NUM>:<NUM> and <NUM>:<NUM> or between <NUM>:<NUM> and <NUM>:<NUM>.

The substrate and the electrophile may be contacted in a weight ratio sufficient to provide a base matrix comprising between <NUM> and <NUM>µg allyl groups or epoxide groups per gram, more preferably between <NUM> and <NUM>µg allyl groups or epoxide groups per gram or between <NUM> and <NUM>µg allyl groups or epoxide groups per gram, and most preferably between <NUM> and <NUM>µg allyl groups or epoxide groups per gram or between <NUM> and <NUM>µg allyl groups or epoxide groups per gram.

The substrate and the electrophile may be contacted in a solvent. The solvent may comprise water and/or an alcohol. The alcohol may be ethanol, propan-<NUM>-ol and/or propan-<NUM>-ol. In some embodiments, the solvent is water.

The substrate and the electrophile may be contacted under alkaline conditions. Accordingly, the substrate and the electrophile may be contacted in the presence of a base. The base may be sodium hydroxide (NaOH). The base may be present at a concentration between <NUM> and <NUM>, more preferably between <NUM> and <NUM>, between <NUM> and <NUM> or between <NUM> and <NUM>, and most preferably between <NUM> and <NUM>, between <NUM> and <NUM> or between <NUM> and <NUM>.

The substrate and the electrophile may be contacted in the presence of an emulsifying agent. The emulsifying agent could be sodium sulphate.

The weight ratio of the substrate to the emulsifying agent may be between <NUM>,<NUM>:<NUM> and <NUM>:<NUM>,<NUM>, between <NUM>,<NUM>:<NUM> and <NUM>:<NUM> or between <NUM>,<NUM>:<NUM> and <NUM>:<NUM>, more preferably is between <NUM>:<NUM> and <NUM>:<NUM>, between <NUM>:<NUM> and <NUM>:<NUM>, between <NUM>:<NUM> and <NUM>:<NUM> or between <NUM>:<NUM> and <NUM>:<NUM>, and most preferably between <NUM>:<NUM> and <NUM>:<NUM> or between <NUM>:<NUM> and <NUM>:<NUM>.

The substrate and the electrophile may be contacted in the presence of a reducing agent. The reducing agent may be sodium borohydride.

The weight ratio of the substrate to the reducing agent may be between <NUM>,<NUM>:<NUM> and <NUM>:<NUM>, between <NUM>,<NUM>:<NUM> and <NUM>:<NUM> or between <NUM>,<NUM>:<NUM> and <NUM>:<NUM>, more preferably is between <NUM>:<NUM> and <NUM>:<NUM>, between <NUM>:<NUM> and <NUM>:<NUM>, between <NUM>:<NUM> and <NUM>:<NUM> or between <NUM>:<NUM> and <NUM>:<NUM>, and most preferably between <NUM>:<NUM> and <NUM>:<NUM> or between <NUM>:<NUM> and <NUM>:<NUM>.

The substrate and the electrophile may be contacted at a temperature between <NUM> and <NUM>, more preferably between <NUM> and <NUM>, between <NUM> and <NUM> or between <NUM> and <NUM>, and most preferably between <NUM> and <NUM> or between <NUM> and <NUM>.

The substrate and the electrophile may be contacted for at least <NUM> minute, at least <NUM> minutes, at least <NUM> hour or at least <NUM> hours, and more preferably for at least <NUM> hours, at least <NUM> hours or at least <NUM> hours. The substrate and the electrophile may be contacted for between <NUM> minute and <NUM> hours, between <NUM> hour and <NUM> hours or between <NUM> and <NUM> hours, more preferably between <NUM> and <NUM> hours, between <NUM> and <NUM> hours, between <NUM> and <NUM> hours or between <NUM> and <NUM> hours.

In some embodiments, the method comprises contacting a base matrix comprising an unsaturated hydrocarbon chain with a halogenating matrix.

Prior to contacting the halogenating agent, the base matrix comprising an unsaturated hydrocarbon chain may be washed with a solvent. The solvent may be water and/or an alcohol. The alcohol may be ethanol.

The halogenating agent may be a fluorinating agent, a chlorinating agent or a brominating agent. Preferably, the halogenating agent is a brominating agent.

The halogenating agent may be N-bromosuccinimide, N-chlorosuccinimide, bromine or chlorine. Preferably, the halogenating agent is N-bromosuccinimide.

The weight ratio of the substrate to the halogenating agent may be between <NUM>,<NUM>:<NUM> and <NUM>:<NUM> or between <NUM>,<NUM>:<NUM> and <NUM>:<NUM>, more preferably is between <NUM>,<NUM>:<NUM> and <NUM>:<NUM>, between <NUM>:<NUM> and <NUM>:<NUM> or between <NUM>:<NUM> and <NUM>:<NUM>, and most preferably between <NUM>:<NUM> and <NUM>:<NUM> or between <NUM>:<NUM> and <NUM>:<NUM>.

The base matrix comprising an unsaturated hydrocarbon chain may be contacted with the halogenating agent at a temperature between -<NUM> and <NUM>, more preferably between <NUM> and <NUM>, between <NUM> and <NUM> or between <NUM> and <NUM>, and most preferably between <NUM> and <NUM>.

The base matrix comprising an unsaturated hydrocarbon chain may be contacted with the halogenating agent in a solvent. The solvent may be water.

The base matrix comprising an unsaturated hydrocarbon chain may be contacted with the halogenating agent under acidic conditions. The base matrix comprising an unsaturated hydrocarbon chain may be contacted at a pH between <NUM> and <NUM>, more preferably between <NUM> and <NUM> or between <NUM> and <NUM>, and most preferably between <NUM> and <NUM>.

The base matrix comprising an unsaturated hydrocarbon chain may be contacted for at least <NUM> minute, at least <NUM> minutes, at least <NUM> minutes or at least <NUM> minutes, and more preferably for at least <NUM> minutes, at least <NUM> minutes or at least <NUM> hour. The base matrix comprising an unsaturated hydrocarbon chain may be contacted for between <NUM> minute and <NUM> hours, between <NUM> minutes and <NUM> hours or between <NUM> minutes and <NUM> hours, more preferably between <NUM> minutes and <NUM> hours, between <NUM> minutes and <NUM> hours or between <NUM> and <NUM> minutes.

Prior to contacting the aminating agent, the halogenated base matrix may be washed with a solvent. The solvent may be water.

The aminating agent may be NH<NUM>R<NUM> or
<CHM>
wherein R<NUM> and R<NUM> are independently H, an optionally substituted C<NUM>-<NUM> alkyl, an optionally substituted C<NUM>-<NUM> alkenyl or an optionally substituted C<NUM>-<NUM> alkynyl and L<NUM> is an optionally substituted C<NUM>-<NUM> alkylene, an optionally substituted C<NUM>-<NUM> alkenylene or an optionally substituted C<NUM>-<NUM> alkynylene, where the backbone of the alkylene, alkenylene or alkynlene is optionally interrupted by one or more heteroatoms.

Preferably, R<NUM> and R<NUM> are both H.

Preferably, L<NUM> is a C<NUM>-<NUM> alkylene, a C<NUM>-<NUM> alkenylene or a C<NUM>-<NUM> alkynylene. More preferably, L<NUM> is a C<NUM>-<NUM> alkylene, most preferably is-CH<NUM>- or -CH<NUM>CH<NUM>-.

Preferably, the aminating agent is ammonia.

The halogenated base matrix and the aminating agent may be contacted in a solvent. The solvent may be water.

The halogenated base matrix and the aminating agent may be contacted in a weight ratio of between <NUM>:<NUM> and <NUM>:<NUM>, between <NUM>:<NUM> and <NUM>:<NUM>, between <NUM>:<NUM> and <NUM>:<NUM>, more preferably between <NUM>:<NUM> and <NUM>:<NUM>, between <NUM>:<NUM> and <NUM>:<NUM> or between <NUM>:<NUM> and <NUM>:<NUM>, and most preferably between <NUM>:<NUM> and <NUM>:<NUM>, between <NUM>:<NUM> and <NUM>:<NUM> or between <NUM>:<NUM> and <NUM>:<NUM>.

The halogenated base matrix and the aminating agent may be contacted at a temperature between <NUM> and <NUM>, more preferably between <NUM> and <NUM>, between <NUM> and <NUM> or between <NUM> and <NUM>, and most preferably between <NUM> and <NUM> or between <NUM> and <NUM>.

The halogenated base matrix and the aminating agent may be contacted for at least <NUM> minute, at least <NUM> minutes, at least <NUM> hour or at least <NUM> hours, and more preferably for at least <NUM> hours, at least <NUM> hours, at least <NUM> hours or at least <NUM> hours. The halogenated base matrix and the aminating agent may be contacted for between <NUM> minute and <NUM> hours, between <NUM> hour and <NUM> hours or between <NUM> and <NUM> hours, more preferably between <NUM> and <NUM> hours, between <NUM> and <NUM> hours, between <NUM> and <NUM> hours or between <NUM> and <NUM> hours.

Prior to contacting the heteroaromatic compound, the aminated base matrix may be washed with a solvent. The solvent may be water and/or a pH neutral solution. The aminated base matrix is preferably washed with water and then a pH neutral solution. The pH neutral solution may be an aqueous solution. The pH neutral solution may comprise alkali metal ions, alkaline earth metal ions, phosphate ions, sulphate ions and/or halogen ions. The pH neutral solution may comprise sodium phosphate, potassium phosphate and/or sodium chloride.

The heteroaromatic compound is a heteroaromatic ring which is substituted with at least two halogens, and optionally one or more further substituents. The heteroaromatic ring may be a <NUM> to <NUM> membered heteroaromatic ring, more preferably a <NUM> or <NUM> membered heteroaromatic ring, and most preferably a <NUM> membered heteroaromatic ring.

The heteroaromatic ring may be a pyridine ring, a pyridazine ring, a pyrimidine ring, a pyrazine ring, a <NUM>,<NUM>,<NUM>-triazine ring or a <NUM>,<NUM>,<NUM>-triazine ring. Preferably, the heteroaromatic group comprises a <NUM>,<NUM>,<NUM>-triazine ring or a <NUM>,<NUM>,<NUM>-triazine ring and most preferably a <NUM>,<NUM>,<NUM>-triazine ring.

The heteroaromatic ring is substituted with at least two halogen atoms, and may be substituted with <NUM>, <NUM>, <NUM> or <NUM> halogen atoms. Preferably, the heteroaromatic ring is substituted with <NUM> or <NUM> halogen atoms, most preferably <NUM> halogen atoms.

The or each halogen may be fluorine, chlorine, bromine or iodine. Preferably, the or each halogen is chlorine or bromine. In one embodiment, each halogen is chlorine.

The heteroaromatic ring may be substituted with one or more further substituents. The or each further substituent may be independently selected from the group consisting of OH, SH, COOH, NH<NUM>, optionally substituted C<NUM>-C<NUM> alkyl, optionally substituted C<NUM>-C<NUM> alkenyl and optionally substituted C<NUM>-C<NUM> alkynyl.

The heteroaromatic compound may be dichloro-triazine or cyanuric chloride, and is preferably cyanuric chloride.

The aminated base matrix and the heteroaromatic compound may be contacted in a solvent. The solvent may be water and/or acetone.

The aminated base matrix and the heteroaromatic compound may be contacted in the presence of a buffer. The buffer may be potassium phosphate. The buffer may be present at a concentration of between <NUM> and <NUM>, more preferably between <NUM> and <NUM> or between <NUM> and <NUM>, and most preferably between <NUM> and <NUM> or between <NUM> and <NUM>.

The weight ratio of the aminated base matrix to the heteroaromatic compound may be between <NUM>,<NUM>:<NUM> and <NUM>:<NUM> or between <NUM>:<NUM> and <NUM>:<NUM>, more preferably is between <NUM>:<NUM> and <NUM>:<NUM>, between <NUM>:<NUM> and <NUM>:<NUM>, between <NUM>:<NUM> and <NUM>:<NUM> or between <NUM>:<NUM> and <NUM>:<NUM>, and most preferably between <NUM>:<NUM> and <NUM>:<NUM> or between <NUM>:<NUM> and <NUM>:<NUM>.

The aminated base matrix and the heteroaromatic compound may be contacted at a temperature between -<NUM> and <NUM>, more preferably between -<NUM> and <NUM>, between - <NUM> and <NUM> or between -<NUM> and <NUM>, and most preferably between -<NUM> and <NUM>, between <NUM> and <NUM> or between <NUM> and <NUM>.

The aminated base matrix and the heteroaromatic compound may be contacted for at least <NUM> minute, at least <NUM> minutes, at least <NUM> minutes or at least <NUM> minutes, and more preferably for at least <NUM> minutes, at least <NUM> minutes or at least <NUM> hour. The aminated base matrix and the heteroaromatic compound may be contacted for between <NUM> minute and <NUM> hours, between <NUM> minutes and <NUM> hours or between <NUM> minutes and <NUM> hours, more preferably between <NUM> minutes and <NUM> hours, between <NUM> minutes and <NUM> hours or between <NUM> and <NUM> minutes.

The activated substrate may be washed with a solvent. The solvent may be water and/or acetone. The activated substrate is preferably washed with a water and acetone solution and then water.

In accordance with a second aspect, there is provided an activated substrate of formula (II):
<CHM>.

It may be appreciated that the circle in formula (II) represents a substrate. The substrate may be as defined in relation to the first aspect.

L<NUM> may be an optionally substituted C<NUM>-<NUM> alkylene, an optionally substituted C<NUM>-<NUM> alkenylene or an optionally substituted C<NUM>-<NUM> alkynylene, where the backbone of the alkylene, alkenylene or alkynlene is optionally interrupted by one or more heteroatoms. Preferably, L<NUM> is an optionally substituted C<NUM>-<NUM> alkylene, an optionally substituted C<NUM>-<NUM> alkenylene or an optionally substituted C<NUM>-<NUM> alkynylene, where the backbone of the alkylene, alkenylene or alkynlene is optionally interrupted by one or more heteroatoms. More preferably, L<NUM> is an optionally substituted C<NUM>-<NUM> alkylene, an optionally substituted C<NUM>-<NUM> alkenylene or an optionally substituted C<NUM>-<NUM> alkynylene, where the backbone of the alkylene, alkenylene or alkynlene is optionally interrupted by one or more heteroatoms. Most preferably, L<NUM> is an optionally substituted C<NUM> alkylene, an optionally substituted C<NUM> alkenylene or an optionally substituted C<NUM> alkynylene, where the backbone of the alkylene, alkenylene or alkynlene is optionally interrupted by one or more heteroatoms. The alkylene, alkenylene or alkynlene may be substituted with one or more hydroxyl groups. The alkylene, alkenylene or alkynlene may be substituted with between <NUM> and <NUM> hydroxyl groups, between <NUM> and <NUM> hydroxyl groups or between <NUM> and <NUM> hydroxyl groups. Preferably the alkylene, alkenylene or alkynlene is substituted with two hydroxyl groups.

Preferably, the backbone of the alkylene, alkenylene or alkynlene is interrupted with at least one heteroatom. The backbone of the alkylene, alkenylene or alkynlene may be interrupted with between <NUM> and <NUM> heteroatoms, between <NUM> and <NUM> heteroatoms or between <NUM> and <NUM> heteroatoms. Preferably, the backbone of the alkylene, alkenylene or alkynlene is interrupted with two heteroatoms. The one or more heteroatoms may each be O, NR<NUM> or S, wherein R<NUM> is H, an optionally substituted C<NUM>-C<NUM> alkyl, an optionally substituted C<NUM>-C<NUM> alkenyl or an optionally substituted C<NUM>-C<NUM> alkynyl. Preferably, the or each heteroatom is oxygen. Preferably, the backbone is interrupted with at least two oxygen atoms.

Accordingly, L<NUM> may be
<CHM>
R<NUM> may be H, an optionally substituted C<NUM>-<NUM> alkyl, an optionally substituted C<NUM>-<NUM> alkenyl or an optionally substituted C<NUM>-<NUM> alkynyl. Preferably, R<NUM> is H, an optionally substituted C<NUM>-<NUM> alkyl, an optionally substituted C<NUM>-<NUM> alkenyl or an optionally substituted C<NUM>-<NUM> alkynyl. More preferably, R<NUM> is H, an optionally substituted C<NUM>-<NUM> alkyl, an optionally substituted C<NUM>-<NUM> alkenyl or an optionally substituted C<NUM>-<NUM> alkynyl. Most preferably, R<NUM> is H.

R<NUM> may be a <NUM> to <NUM> membered heteroaryl group substituted with at least one halogen, and optionally substituted with further substituents, more preferably a <NUM> or <NUM> membered heteroaryl group substituted with at least one halogen, and optionally substituted with further substituents, and most preferably a <NUM> membered heteroaryl group substituted with at least one halogen, and optionally substituted with further substituents.

The heteroaryl group which is substituted with at least one halogen, and optionally one or more further substituents, may be a pyridinyl, a pyridazinyl, a pyrimidinyl, a pyrazinyl, a <NUM>,<NUM>,<NUM>-triazinyl or a <NUM>,<NUM>,<NUM>-triazinyl. Preferably, the heteroaryl group is a <NUM>,<NUM>,<NUM>-triazinyl or a <NUM>,<NUM>,<NUM>-triazinyl substituted with at least one halogen, and optionally one or more further substituents and most preferably <NUM>,<NUM>,<NUM>-triazinyl substituted with at least one halogen, and optionally one or more further substituents.

The heteroaryl group is substituted with at least one halogen atom, and may be substituted with <NUM>, <NUM>, <NUM> or <NUM> halogen atoms. Preferably, the heteroaryl group is substituted with <NUM> or <NUM> halogen atoms, most preferably <NUM> halogen atoms.

The heteroaryl group may be substituted with one or more further substituents. The one or more further substituents may be OH, SH, COOH, NH<NUM>, optionally substituted C<NUM>-C<NUM> alkyl, optionally substituted C<NUM>-C<NUM> alkenyl or optionally substituted C<NUM>-C<NUM> alkynyl.

Accordingly, R<NUM> may be
<CHM>
wherein R<NUM> is a halogen and R<NUM> is a halogen, H, OH, SH, COOH, NH<NUM>, optionally substituted C<NUM>-C<NUM> alkyl, optionally substituted C<NUM>-C<NUM> alkenyl or optionally substituted C<NUM>-C<NUM> alkynyl. Preferably, R<NUM> is a halogen or H.

Accordingly, in a preferred embodiment, the compound of formula (II) is a compound of formula (IIa):
<CHM>
In accordance with a third aspect, there is provided a method of producing a scaffold for isolation of a biomolecule, the method comprising:.

It will be understood that the ligand may be selected according to the biomolecule to be isolated.

Advantageously, the method provides an alkaline-stable structure which can be used as an affinity medium or ligand for bioprocessing, where sanitisation with caustic solutions is highly desirable.

The biomolecule is selected from the group consisting of an amino acid, a peptide, an affimer, a protein, an enzyme, a lipopolysaccharide, an antibody or a fragment thereof, a nucleic acid, a virus, a bacterium, and a cell. The antibody may be an isoagglutinin. The virus may be adeno-associated virus (AAV) or Lentivirus. The cell may be an animal cell.

The molecule for forming the ligand attached to the activated substrate may be a compound of formula (IV):.

More preferably, L<NUM> is an optionally substituted C<NUM>-<NUM> alkylene, an optionally substituted C<NUM>-<NUM> alkenylene or an optionally substituted C<NUM>-<NUM> alkynylene, where the backbone of the alkylene, alkenylene or alkynlene is optionally interrupted by one or more heteroatoms. Most preferably, L<NUM> is an optionally substituted C<NUM>-<NUM> alkylene, an optionally substituted C<NUM>-<NUM> alkenylene or an optionally substituted C<NUM>-<NUM> alkynylene, where the backbone of the alkylene, alkenylene or alkynlene is optionally interrupted by one or more heteroatoms.

L<NUM> may be an alkylene, an alkenylene or an alkynylene where the alkylene, alkenylene or alkynylene is substituted with one or more oxo groups. Preferably, the alkylene, alkenylene or alkynylene is substituted with an oxo group.

L<NUM> may be an alkylene, an alkenylene or an alkynylene wherein the backbone of the alkylene, alkenylene or alkynylene is interrupted by one or more heteroatoms selected from NR<NUM>, O or S, wherein R<NUM> is H, an optionally substituted C<NUM>-<NUM> alkyl, an optionally substituted C<NUM>-<NUM> alkenyl or an optionally substituted C<NUM>-<NUM> alkenyl. More preferably, L<NUM> is an alkylene, an alkenylene or an alkynylene wherein the backbone of the alkylene, alkenylene or alkynylene is interrupted by an NH group.

The ligand may comprise an optionally derivatized saccharide molecule, an optionally derivatized amino acid, an optionally derivatized peptide, an optionally derivatized affimer or an optionally derivatized protein. The optionally derivatized saccharide molecule may be an optionally derivatives polysaccharide molecule.

The ligand may be derivatised with one or more functional groups. The one or more functional groups may replace a hydrogen or hydroxyl group in the ligand. In embodiments where the ligand is a polysaccharide, one or more of the saccharide monomers may be derivatized. The one or more functional groups may be an optionally substituted C<NUM>-<NUM> alkyl, an optionally substituted C<NUM>-<NUM> alkenyl, an optionally substituted C<NUM>-<NUM> alkenyl, OR<NUM>, SR<NUM>, C(O)R<NUM>, NR<NUM>R<NUM>, NR<NUM>C(O)R<NUM> or SO<NUM>R<NUM>, wherein R<NUM> and R<NUM> are independently H, an optionally substituted C<NUM>-<NUM> alkyl, an optionally substituted C<NUM>-<NUM> alkenyl or an optionally substituted C<NUM>-<NUM> alkenyl.

The saccharide molecule may comprise between <NUM> and <NUM> saccharide monomers, more preferably between <NUM> and <NUM> saccharide monomers or between <NUM> and <NUM> saccharide monomers, and most preferably between <NUM> and <NUM> saccharide monomers.

The or each optionally derivatized saccharide monomer may be selected from the group consisting of optionally derivatized glucose; optionally derivatized glucosamine; optionally derivatized galactose; optionally derivatized fructose; and optionally derivatized xylose, or a stereoisomer thereof.

In some embodiments, X<NUM> is
<CHM>
or
<CHM>.

The compound of Formula (IV) may be an antigen-A trisaccharide ligand, which has Formula (IVa):
<CHM>.

The compound of Formula (IV) may be an antigen-B trisaccharide ligand, which has Formula (IVb):
<CHM>.

The ligand may have an affinity for isoagglutinins.

The ligand may, in use, be cationic, preferably protonated.

An exemplary ligand which may be cationic in use is a ligand formed from a compound of formula (IVc):.

H<NUM>N-L<NUM>-N(Ak-NH<NUM>)<NUM>     (IVc).

wherein Ak in each occurrence is a C<NUM>-<NUM> alkylene where, in the case of a C<NUM>-<NUM> alkylene, the backbone of the alkylene is optionally interrupted by one or more heteroatoms, e. g one or more O atoms.

A preferred compound of formula (IVc) is formula (IVd):
<CHM>.

The ligand formed from the compound of formula (IVd) may have an affinity to lipopolysaccharide and albumin.

X<NUM> of formula (IV) may be a boronate group. A ligand comprising a boronate group may be formed from a compound of formula (IVe):
<CHM>.

The ligand may have an affinity to glycosylated proteins.

X<NUM> of the compound of Formula (IV) may be a naphthol ligand. An exemplary compound of formula (IV) comprising a naphthol ligand has Formula (IVf):
<CHM>.

The ligand may have an affinity to insulin.

The method may comprise contacting the activated substrate and the molecule comprising the ligand in a solvent. The solvent may be water.

The activated substrate and the molecule comprising the ligand may be contacted in a weight ratio of between <NUM>,<NUM>,<NUM>:<NUM> and <NUM>:<NUM> or between <NUM>,<NUM>:<NUM> and <NUM>:<NUM>, more preferably between <NUM>,<NUM>:<NUM> and <NUM>:<NUM> or between <NUM>,<NUM>:<NUM> and <NUM>:<NUM>, and most preferably between <NUM>,<NUM>:<NUM> and <NUM>:<NUM>, between <NUM>,<NUM>:<NUM> and <NUM>:<NUM> or between <NUM>,<NUM>:<NUM> and <NUM>,<NUM>:<NUM>.

Alternatively, or additionally, the ligand may be present at a concentration of between <NUM>µg/ml and <NUM>/ml, between <NUM>µg/ml and <NUM>/ml or between <NUM>µg/ml and <NUM>/ml, more preferably between <NUM>µg/ml and <NUM>/ml, between <NUM>µg/ml and <NUM>/ml, between <NUM>µg/ml and <NUM>/ml or between <NUM>µg/ml and <NUM>/ml, most preferably between <NUM> and <NUM>/ml.

Alternatively or additionally, the ligand may be present at a concentration at least <NUM>µmol per gram of scaffold, optionally a concentration of at least <NUM>µmol/g, optionally up to <NUM> or <NUM>µmol / g.

The activated substrate and the molecule comprising the ligand may be contacted under alkaline conditions. The activated substrate and the molecule comprising the ligand may be contacted in solution at a pH between <NUM> and <NUM> at <NUM>, more preferably at a pH between <NUM> and <NUM> at <NUM>, and most preferably at a pH of between <NUM> and <NUM> at <NUM>.

The activated substrate and the molecule comprising the ligand may be contacted at a temperature between -<NUM> and <NUM>, more preferably between <NUM> and <NUM>, between <NUM> and <NUM> or between <NUM> and <NUM>, and most preferably between <NUM> and <NUM>.

The activated substrate and the ligand molecule comprising the may be contacted for at least <NUM> minute, at least <NUM> minutes, at least <NUM> minutes or at least <NUM> minutes, and more preferably at least <NUM> hour, at least <NUM> hours, at least <NUM> hours or at least <NUM> hour. The activated substrate and the molecule comprising the ligand may be contacted for between <NUM> minute and <NUM> hours, between <NUM> minutes and <NUM> hours or between <NUM> and <NUM> hours, more preferably between <NUM> and <NUM> hours, between <NUM> and <NUM> hours or between <NUM> to <NUM> hours.

The method may subsequently comprise contacting the scaffold with an alcohol, a hydroxide, ammonia, an amine or a thiol. The alcohol, hydroxide, ammonia, amine or thiol may be HOR<NUM>, -OH, HNR<NUM>R<NUM> or HSR<NUM>, wherein R<NUM> and R<NUM> are independently H, optionally substituted C<NUM>-C<NUM> alkyl, optionally substituted C<NUM>-C<NUM> alkenyl or optionally substituted C<NUM>-C<NUM> alkynyl. More preferably, R<NUM> and R<NUM> are independently H, optionally substituted C<NUM>-C<NUM> alkyl, optionally substituted C<NUM>-C<NUM> alkenyl or optionally substituted C<NUM>-C<NUM> alkynyl. Most preferably, R<NUM> and R<NUM> are independently H, optionally substituted C<NUM>-C<NUM> alkyl, optionally substituted C<NUM>-C<NUM> alkenyl or optionally substituted C<NUM>-C<NUM> alkynyl. The alkyl, alkenyl or alkynyl may be optionally substituted with -OH, NH<NUM> or SH.

The alcohol, hydroxide, ammonia, amine or thiol may be selected from the group consisting of <NUM>-aminoethanol, methylamine, ammonia, sodium hydroxide, glycine, alanine, dimethylamine, tris(hydroxymethyl)aminomethane or <NUM>-mercaptoethanol. Advantageously, this step removes residual halogen sites after ligand coupling.

The scaffold and the alcohol, hydroxide, ammonia, amine or thiol may be contacted in a weight ratio of between <NUM>:<NUM> and <NUM>,<NUM>:<NUM> or between <NUM>:<NUM> and <NUM>:<NUM>, more preferably between <NUM>:<NUM> and <NUM>:<NUM> or between <NUM>:<NUM> and <NUM>:<NUM>, and most preferably between <NUM>:<NUM> and <NUM>:<NUM>, between <NUM>:<NUM> and <NUM>:<NUM> or between <NUM>:<NUM> and <NUM>:<NUM>.

The scaffold may be washed with a solvent. The solvent may be water.

In accordance with a fourth aspect, there is provided a scaffold for isolation of a biomolecule, wherein the scaffold is represented by formula (III):
<CHM>.

The group comprising the ligand and biomolecule are as defined in relation to the third aspect.

In particular, the group comprising the ligand may have formula -L<NUM>-L<NUM>-X<NUM>, wherein L<NUM> and X<NUM> are as defined in the fourth aspect and L<NUM> is O, S or NH.

R<NUM> may be a <NUM> to <NUM> membered heteroaryl group substituted with at least one group comprising the ligand specific for a biomolecule, and optionally substituted with one or more further substituents, more preferably a <NUM> or <NUM> membered heteroaryl group substituted with at least one group comprising the ligand specific for a biomolecule, and optionally substituted with one or more further substituents, and most preferably a <NUM> membered heteroaryl group substituted with at least one group comprising the ligand specific for a biomolecule, and optionally substituted with one or more further substituents.

The heteroaryl group which is substituted with at least one group comprising the ligand specific for a biomolecule, and optionally substituted with one or more further substituents, may be a pyridinyl, a pyridazinyl, a pyrimidinyl, a pyrazinyl, a <NUM>,<NUM>,<NUM>-triazinyl or a <NUM>,<NUM>,<NUM>-triazinyl. Preferably, the heteroaryl group is a <NUM>,<NUM>,<NUM>-triazinyl or a <NUM>,<NUM>,<NUM>-triazinyl substituted with at least one group comprising the ligand specific for a biomolecule, and optionally substituted with one or more further substituents and most preferably a <NUM>,<NUM>,<NUM>-triazinyl substituted with at least one group comprising the ligand specific for a biomolecule, and optionally substituted with one or more further substituents.

The heteroaryl group is substituted with at least one group comprising the ligand, and may be substituted with <NUM>, <NUM>, <NUM> or <NUM> groups comprising the ligand. Preferably, the heteroaryl group is substituted with <NUM> or <NUM> group comprising the ligand, most preferably <NUM> group comprising the ligand.

The heteroaryl group may be substituted with one or more further substituents. The one or more further substituents may be a halogen, OR<NUM>, SR<NUM>, COOR<NUM>, NR<NUM>R<NUM>, optionally substituted C<NUM>-C<NUM> alkyl, optionally substituted C<NUM>-C<NUM> alkenyl or optionally substituted C<NUM>-C<NUM> alkynyl, wherein R<NUM> and R<NUM> are independently H optionally substituted C<NUM>-C<NUM> alkyl, optionally substituted C<NUM>-C<NUM> alkenyl or optionally substituted C<NUM>-C<NUM> alkynyl.

Accordingly, R<NUM> may be
<CHM>
wherein R<NUM> is a group comprising a ligand specific for a biomolecule and R<NUM> is a group comprising a ligand specific for a biomolecule, a halogen, H, OR<NUM>, COOR<NUM>, NR<NUM>R<NUM>, SR<NUM>, optionally substituted C<NUM>-C<NUM> alkyl, optionally substituted C<NUM>-C<NUM> alkenyl or optionally substituted C<NUM>-C<NUM> alkynyl, wherein R<NUM> and R<NUM> are independently H optionally substituted C<NUM>-C<NUM> alkyl, optionally substituted C<NUM>-C<NUM> alkenyl or optionally substituted C<NUM>-C<NUM> alkynyl. Preferably, R<NUM> and R<NUM> are independently H, optionally substituted C<NUM>-C<NUM> alkyl, optionally substituted C<NUM>-C<NUM> alkenyl or optionally substituted C<NUM>-C<NUM> alkynyl. More preferably, R<NUM> and R<NUM> are independently H, optionally substituted C<NUM>-C<NUM> alkyl, optionally substituted C<NUM>-C<NUM> alkenyl or optionally substituted C<NUM>-C<NUM> alkynyl. Most preferably, R<NUM> and R<NUM> are independently H, optionally substituted C<NUM>-C<NUM> alkyl, optionally substituted C<NUM>-C<NUM> alkenyl or optionally substituted C<NUM>-C<NUM> alkynyl. The alkyl, alkenyl or alkynyl may be optionally substituted with -OH, NH<NUM> or SH.

In a most preferred embodiment, the scaffold is represented by formula (IIIa):
<CHM>.

Preferably, R<NUM> is a group comprising a ligand specific for a biomolecule. Preferably, R<NUM> is the same as R<NUM>.

In accordance with a fifth aspect, there is provided use of the scaffold of the fourth aspect to isolate a biomolecule.

In accordance with an sixth aspect, there is provided a method of isolating a biomolecule on a scaffold, the method comprising contacting a scaffold with a biomolecule, wherein the scaffold is as defined in the fourth aspect.

It may be appreciated that the use of the fifth aspect is in affinity chromatography.

The method of the sixth aspect is preferably a method of conducting affinity chromatography.

The biomolecule is as defined in relation to the third aspect.

The method may comprise contacting the scaffold for isolation of a biomolecule with a solution comprising the biomolecule.

The biomolecule may be present in human intravenous immunoglobulin (IVIG) intermediate product. Accordingly, the method may comprise contacting the scaffold for isolation of a biomolecule with human IVIG intermediate product.

In accordance with a seventh aspect, there is provided a method of cleaning a scaffold, the method comprising contacting a scaffold with a caustic substance, wherein the scaffold is as defined in either the fifth or sixth aspect.

Advantageously, the scaffold may be cleaned to avoid contamination from bacteria, viruses, and endotoxin without loss of performance.

The caustic substance may be or comprise an alkaline solution.

The alkaline solution may have a pH of at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM> or at least <NUM> at <NUM>. The alkaline solution may have a pH of between <NUM> and <NUM>, between <NUM> and <NUM>, between <NUM> and <NUM>, between <NUM> and <NUM>, between <NUM> and <NUM> or between <NUM> and <NUM> at <NUM>.

The alkaline solution may comprise an alkali metal hydroxide or an alkaline earth metal hydroxide. Preferably, the alkaline solution comprises an alkali metal hydroxide. The alkaline solution may comprise lithium hydroxide, sodium hydroxide or potassium hydroxide. In some embodiments, the alkaline solution comprises sodium hydroxide. The alkaline solution may comprise the alkali metal hydroxide or the alkaline earth metal hydroxide at a concentration of between <NUM> and <NUM>, between <NUM> and <NUM>, between <NUM> and <NUM>, between <NUM> and <NUM>, between <NUM> and <NUM> or between, between <NUM> and <NUM>.

The method of the seventh aspect may be performed subsequent to the method of the sixth aspect. The scaffold may then be used in a further method of isolating a biomolecule. Accordingly, the method of the sixth aspect may be repeated after the method of the seventh aspect.

The methods of the sixth and seventh aspect may be cycled, one after the other. The methods of the sixth and seventh aspect may be repeated at least <NUM> times, at least <NUM> times at least <NUM> times or at least <NUM> times, more preferably at least <NUM> times, at least <NUM> times or at least <NUM> times, and most preferably at least <NUM> or <NUM> times.

All of the features described herein (including accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. However, the invention is defined by the claims.

For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which:-.

A synthetic route for producing a dihalogentriazine scaffold is shown in <FIG>. The steps are described in more detail below.

Approximately <NUM> of a solid support, such as beaded agarose, is slurried in water with up to <NUM>, optionally approximately <NUM> of sodium sulphate, <NUM> NaOH, and <NUM> of sodium borohydride. The slurry is heated to <NUM> to <NUM> before adding allyl glycidyl ether (up to <NUM>, optionally <NUM>). The slurry is left to react for no more than <NUM> hours, after which the drained gel is washed with ethanol and water. The resulting material is an activated base matrix that contains up to about <NUM>µmol allyl groups per g adsorbent.

The allyl-activated base matrix is slurried at room temperature with an acidic solution of pH around <NUM>, followed by the addition of N-bromosuccinimide, and incubation for at least one hour. The resulting brominated base matrix is then washed with water and left to settle.

The brominated base matrix is resuspended in water followed by the addition of <NUM> of an ammonia solution, and heating to no more than <NUM> under stirring for less than <NUM> hours. At the end of the reaction period, the reaction is drained and washed with water, then a pH-neutral solution, followed by settling.

The aminated base matrix resulting from the previous step is then slurried in an aqueous solution of potassium phosphate (<NUM>), followed by settling and re-suspension in the same solution, with the addition of <NUM> of acetone, under stirring at around <NUM>. Approximately <NUM> molar equivalents of cyanuric chloride with respect to the precursor activation density dissolved in acetone is added to the slurried aminated base matrix, followed by incubation under refrigeration for approximately one hour, after which it is drained, washed with aqueous acetone solutions of decreasing concentration, then a final wash in water, after which the slurry is settled under gravity. The final product of this reaction is the DCT-activated base matrix.

A DCT-activated base matrix is suspended in water and set to stir at room temperature. A solution of A-antigen trisaccharide ligand comprising a flexible linker in water is added to the DCT gel slurry maintained under high pH at room temperature for <NUM> to <NUM> hours. The A-antigen trisaccharide ligand comprising the flexible linker is a compound of formula (IVa), as described herein. After reaction, the derivatised base matrix is blocked using mercaptoethanol and thoroughly washed with water before being allowed to settle under gravity.

This reaction results in a chromatographic scaffold material containing an affinity ligand which binds A-isoagglutinins (Scaffold Example <NUM>).

A DCT-activated base matrix is suspended in water and set to stir at room temperature. A solution of B-antigen trisaccharide ligand comprising a flexible linker in water is added to the DCT gel slurry maintained under high pH conditions at room temperature for <NUM> to <NUM> hours. The B-antigen trisaccharide ligand comprising the flexible linker is a compound of formula (IVb), as described herein. After reaction, the derivatised base matrix is blocked using mercaptoethanol, thoroughly washed with water then settled under gravity.

This reaction results in a chromatographic scaffold material containing an affinity ligand which binds B-isoagglutinins (Scaffold Example <NUM>).

A column containing the antigen A ligand (Scaffold Example <NUM>) is packed, followed by the application of an IVIG solution containing isoagglutinins at a flow rate of <NUM>/min.

The flow-through from the loading process is collected. The load and flow-through samples are analysed for of A-isoagglutinins using a standard agglutination assay.

Human red blood cells are washed with PBS buffer by adding <NUM> red blood cells into a <NUM> centrifuge tube and centrifuging at <NUM> rpm for <NUM> minutes. The supernatant from the centrifuged cells is removed before adding PBS buffer to the cell pellet to make the suspension up to a <NUM> volume and the cells mixed by inverting the tube. This PBS wash is repeated three times.

Freeze dried papain is reconstituted with PBS buffer (<NUM>) before adding <NUM>µL to the cells after centrifugation and removal of the supernatant. These suspensions are then made up to <NUM> volume with PBS buffer before incubation with mixing for <NUM> minutes at <NUM>.

At the end of this incubation period, the suspension is centrifuged for <NUM> minutes at <NUM> rpm. The supernatant is removed from the centrifuged cells, then PBS buffer added to make the suspension up to a <NUM> volume. The cells are mixed by inverting the tube. This PBS wash is repeated three times.

The spun cells are mixed with inversion made up to <NUM> volume with <NUM>/mL BSA solution.

A sample of the feedstock from step <NUM> is diluted <NUM>/<NUM>, <NUM>/<NUM>, <NUM>/<NUM>, <NUM>/<NUM>, <NUM>/<NUM>, <NUM>/<NUM>, <NUM>/<NUM> and <NUM>/<NUM> with a solution of <NUM>/mL BSA solution.

A sample of the non-bound from step <NUM> is diluted <NUM>/<NUM>, <NUM>/<NUM>, <NUM>/<NUM>, <NUM>/<NUM>, <NUM>/<NUM>, <NUM>/<NUM>, <NUM>/<NUM> and <NUM>/<NUM> with a solution of <NUM>/mL BSA solution.

The feedstock and non-bound dilutions described above (<NUM>µL) are transferred to a <NUM> well v-bottomed plate along with the blank <NUM>/mL BSA solution.

The red blood cells prepared in stage <NUM>. <NUM> are mixed by inversion before transferring to a pipette reservoir. The blood cell suspension (<NUM>µL) is transferred to the plate using a multichannel pipette. The plate is agitated to mix the solutions for <NUM> seconds before centrifuging the plate at <NUM> rpm for <NUM> minutes.

The plate is placed onto a stand at a <NUM>° angle for <NUM> minutes. Each well of the plate is studied to determine the level of clearance of isoagglutinin from the red blood cells. This is achieved by assessing the sample dilution required to prevent agglutination, indicated by streaming of the red blood cells in the plate wells (<FIG>).

As shown in <FIG>, a dilution of greater than <NUM>/<NUM> for the load sample causes the red blood cells to stream. For the non-bound solution sample, a dilution of greater than <NUM>/<NUM> gives streaming of the blood cells. It can be concluded from this assay that the A-antigen adsorbent (Scaffold Example <NUM>) gives a <NUM>/<NUM> to <NUM>/<NUM> clearance of isoagglutinin from the feed.

A column containing the antigen B ligand (Scaffold Example <NUM>) is packed followed by the application of an IVIG solution containing isoagglutinins at a flow rate of <NUM>/min. The flow-through from the loading process is collected. The load and flow-through samples are analysed for the content of B-isoagglutinins using a standard agglutination assay.

To make the type-B red blood cells up in PBS buffer, the cells are washed with PBS buffer by adding <NUM> red blood cells into a <NUM> centrifuge tube and centrifuging at <NUM> rpm for <NUM> minutes. The supernatant from the centrifuged cells is removed before adding PBS buffer to the cell pellet to make the suspension up to a <NUM> volume, and the cells are mixed by inverting the tube. This PBS wash is repeated three times.

Freeze dried papain was reconstituted with PBS buffer (<NUM>) before adding <NUM>µL to the cells after centrifugation and removal of the supernatant. These suspensions are then made up to <NUM> volume with PBS buffer before incubation with mixing for <NUM> minutes at <NUM>.

As shown in <FIG>, a dilution of greater than <NUM>/<NUM> for the load sample causes the red blood cells to stream. For the non-bound solution sample, any dilution gives streaming of the blood cells. It can be concluded from this assay that the B-antigen adsorbent (Scaffold Example <NUM>) gives a <NUM>/<NUM> to neat clearance of isoagglutinin from the feed.

Two samples of the A-Antigen adsorbent were incubated in <NUM> NaOH at <NUM> for <NUM> week, and subsequently tested for performance using a standard agglutination assay. The samples both showed reduction in agglutinin titres of <NUM>/<NUM> to <NUM>/<NUM> before and after incubation under caustic conditions, indicating the stability of the adsorbent under those conditions.

In another experiment, a batch of the A-Antigen adsorbent was subjected to a cycling study, where a packed column of the adsorbent went through <NUM> cycles of a procedure containing a caustic-based cleaning-in-place (CIP) step using <NUM> NaOH. The isoagglutinin titre of samples taken before and after the column run both showed a reduction of agglutinin titre from <NUM>/<NUM> to <NUM>/<NUM> after the first and <NUM>st runs, demonstrating the stability of the material to caustic conditions.

A batch of the B-Antigen adsorbent was subjected to a cycling study, where a packed column of the adsorbent went through <NUM> cycles of a procedure containing a caustic-based cleaning-in-place (CIP) step using <NUM> NaOH. The isoagglutinin titre of samples taken before and after the column run both showed a reduction of agglutinin titre from <NUM>/<NUM> to <NUM>/<NUM> (neat) after the first and <NUM>st runs, demonstrating the stability of the material to caustic conditions.

A range of DCT activated substrates with activation densities ranging from <NUM>µmol/g to <NUM>µmol/g were generated using the method described in Example <NUM>.

A solution of A-antigen trisaccharide ligand comprising a flexible linker in water was added to the DCT gel slurries maintained under high pH at room temperature for <NUM> hours. The A-antigen trisaccharide ligand comprising the flexible linker is a compound of formula (IVa), as described herein. After <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> hours, samples of the reaction supernatants were collected for quantitation of unreacted ligand.

As shown in <FIG>, higher activation densities afforded by the invention allow coupling of the ligand to the target concentration within <NUM>-hours reaction time. It can be concluded that activation densities equal to or lower than <NUM>µmol/g would fail to meet the target immobilisation level within <NUM> hours. Other activation methods such as epichlorohydrin activation (Example <NUM>) are not capable of achieving activation densities higher than <NUM>µmol/g settled on a beaded agarose support.

A synthetic route for producing a n-hydroxy succinimide scaffold is shown in Scheme <NUM>. The steps are described in more detail below.

Approximately <NUM> of a solid support, such as beaded agarose, is suspended in water before heating to <NUM> to <NUM>. Sodium chloroacetate (approximately <NUM>) is added to the reaction slurry and left to react for no more than <NUM> hours, after which the drained gel is washed with water. The resulting material is an activated matrix that contains <NUM> to <NUM>µmol carboxy groups per g adsorbent.

The carboxylated support is acidified by washing with <NUM> HCl and then washed free of water with acetone. The gel is then suspended in acetone before reacting with N-hydroxysuccinimide (NHS) (<NUM> per kg of support) and N-ethyl-N'-(<NUM>-(dimethylamino)propyl)carbodiimide (EDC) (<NUM> per kg of support) at ambient temperature for at least <NUM> hours. After the reaction,_the gel is drained and washed with N,N-dimethylformamide. _The resulting material is an activated matrix that contains <NUM> to <NUM>µmol NHS groups per g adsorbent.

The bromo activated base matrix produced as described in Example <NUM> is suspended in water before adding tris <NUM>-aminoethyl amine ligand. The charge of tris <NUM>-aminoethyl ligand is calculated with respect to the precursor allyl activation density to give an excess of <NUM> molar equivalents.

After addition of the amine, the reaction is left to react at <NUM> for at least <NUM> hours. At the end of the reaction period, the gel is drained and washed with water, <NUM> HCl and <NUM> NaCl before settling under gravity.

This reaction results in a chromatographic material containing an affinity ligand which binds bovine serum albumin.

The NHS activated base matrix produced as described in Example <NUM> is suspended in N,N-dimethylformamide before adding tris <NUM>-aminoethyl ligand. The charge of tris <NUM>-aminoethyl ligand is calculated with respect to the precursor NHS activation density to give an excess of <NUM> molar equivalents.

After addition of the amine, the reaction is left to react at ambient temperature for at least <NUM> hours. At the end of the reaction period, the gel is drained and washed with water, <NUM> HCl and <NUM> NaCl before settling under gravity.

The DCT activated base matrix produced as described in Example <NUM> is suspended in water before adding tris <NUM>-aminoethyl ligand. The amount of tris <NUM>-aminoethyl ligand is calculated with respect to the precursor activation density to give an excess of <NUM> molar equivalents.

After addition of the amine, the reaction is left to react at <NUM> temperature for at least <NUM> hours. At the end of the reaction period, the gel is drained and washed with water, <NUM> HCl and <NUM> NaCl before settling under gravity.

A sample of the tren adsorbent produced as described in Example <NUM> (bromo attachment, Comparative Scaffold <NUM>), <NUM> (NHS attachment, Comparative Scaffold <NUM>) and <NUM> (DCT attached product of the invention, Scaffold Example <NUM>) were subjected to a cycling study, where packed columns of the adsorbents went through <NUM> cycles of a procedure containing a caustic-based cleaning-in-place (CIP) step using <NUM> NaOH. On the first, <NUM>th and <NUM>st cycle, the column was loaded to <NUM>% breakthrough with bovine serum albumin (BSA) and the binding capacity was calculated.

As shown in <FIG>, the BSA binding capacity of the Scaffold Example <NUM> adsorbent at <NUM>% break through (approximately <NUM>/mL) did not change significantly from cycle <NUM> to <NUM>, demonstrating stability of the Scaffold Example <NUM> to caustic conditions. In contrast, binding capacity of Scaffold Example <NUM> fell significantly under the same conditions.

The bromo attached Tren adsorbent of Comparative Scaffold <NUM> also showed good caustic stability however, as described in Example <NUM> this required much stronger reaction conditions compared to the triazine attachment process for Scaffold Example <NUM>, including higher temperature and a three-fold excess of amine (Tren ligand).

A solution of <NUM> of Mimetic Blue SA ligand available from Astrea Bioseparations (referred to herein as "Blue ligand") per kg of adsorbent is prepared in water with adjustment to pH <NUM> with NaOH. The bromo activated base matrix produced as described in Example <NUM> is suspended in the Blue ligand solution and the reaction is left to react at <NUM> for at least <NUM> hours. At the end of the reaction period, any residual reactive sites on the gel are blocked by addition of ethanolamine followed by reaction at <NUM> for at least <NUM> hours. After the blocking reaction, the gel is drained and washed with water. This reaction results in a chromatographic material containing an affinity ligand at approximately <NUM>µmol ligand per g of adsorbent which binds human serum albumin.

A solution of <NUM> of Blue ligand per kg of adsorbent is prepared in water with adjustment to pH <NUM> with NaOH. The NHS activated base matrix produced as described in Example <NUM> is suspended in the Blue ligand solution and the reaction is left to react at ambient temperature for at least <NUM> hours. At the end of the reaction period, any residual reactive sites on the gel are blocked by addition of ethanolamine followed by reaction at room temperature for at least <NUM> hours. After the blocking reaction, the gel is drained and washed with water. This reaction results in a chromatographic material containing an affinity ligand at approximately <NUM>µmol ligand per g of adsorbent which binds human serum albumin.

A solution of <NUM> of Blue ligand per kg of adsorbent is prepared in water with adjustment to pH <NUM> with NaOH. The DCT activated base matrix produced as described in Example <NUM> is suspended in the Blue ligand solution and the reaction is left to react at ambient temperature for at least <NUM> hours. At the end of the reaction period, any residual reactive sites on the gel are blocked by addition of ethanolamine followed by reaction at <NUM> for at least <NUM> hours. After the blocking reaction, the gel is drained and washed with water. This reaction results in a chromatographic material containing an affinity ligand at approximately <NUM>µmol ligand per g of adsorbent which binds human serum albumin.

A sample of the Blue chromophore_ligand scaffolds produced as described in Example <NUM> (bromo attachment, Comparative Scaffold <NUM>), <NUM> (NHS attachment, Comparative Scaffold <NUM>) and <NUM> (DCT attached product of the invention, Scaffold Example <NUM>) were subjected to a cycling study, where a packed column of the adsorbents went through <NUM> cycles of a procedure containing a caustic-based cleaning-in-place (CIP) step using <NUM> NaOH. On the first, <NUM>th and <NUM>st cycle, the columns were loaded to <NUM>% breakthrough with human serum albumin (HSA) and the binding capacity was calculated.

As shown in <FIG>, the HSA binding capacity of the DCT coupled adsorbent (Scaffold Example <NUM>) and bromo coupled adsorbent (Comparative Scaffold <NUM>) at <NUM>% break through did not change significantly from cycle <NUM> to <NUM>, demonstrating stability of the adsorbents to caustic conditions. The NHS coupled comparative example (Comparative Scaffold <NUM>) showed slight decrease in binding capacity by the <NUM>st cycle.

Despite being activated to the same density and having the same excess of ligand in the immobilisation reaction, the triazine adsorbent showed higher binding capacity than the bromo attached adsorbent. Without wishing to be bound by any theory, this is afforded by the higher reactivity of the chlorotriazine reactive group leading to increased ligand density of the adsorbent and demonstrating an advantage in efficiency of the synthesis process.

In another experiment, a sample of the Blue chromophore ligand adsorbents produced as described in Example <NUM> (NHS coupled Comparative Scaffold <NUM>) and <NUM> (DCT attached product of the invention Scaffold Example <NUM>), were incubated in <NUM> NaOH at <NUM> for three days. Approximately every <NUM> hours, a supernatant sample was collected for quantitation of ligand leachate concentration by HPLC.

As shown in <FIG>, this analysis showed negligible levels of ligand leachate in the incubation supernatant of the triazine coupled Scaffold Example <NUM> demonstrating the stability of the product to caustic conditions. Conversely, the NHS coupled Comparative Scaffold <NUM> showed significant levels of Blue ligand leachate increasing over the course of the incubation, indicating a lack of caustic stability.

The two adsorbents were tested for HSA binding capacity at <NUM>% breakthrough before the stability study and after incubation in <NUM> NaOH at <NUM> for <NUM> hours.

As shown in <FIG>, the triazine coupled Scaffold Example <NUM> showed negligible change in binding capacity after exposure to <NUM> NaOH at <NUM> for <NUM> hours. This demonstrates the caustic stability of the product. For the NHS coupled Comparative Scaffold <NUM>, binding capacity significantly dropped over the <NUM>-hour incubation period demonstrating the poor caustic stability of this attachment method.

Approximately <NUM> of a beaded agarose solid support, is suspended in <NUM> of water before addition of <NUM> of <NUM> NaOH. Epichlorohydrin is added to the reaction slurry at <NUM> per kg of base matrix. The reaction is left to stir at <NUM> for at least <NUM> hours before adding an additional <NUM> portion of epichlorohydrin and <NUM> of <NUM> NaOH. The reaction mixture is left to react for <NUM> hours before draining and washing with water. The resulting material is an activated matrix that contains up to <NUM>µmol epoxide groups per g adsorbent, demonstrating the limited activation density of epichlorohydrin activation method on beaded agarose support.

The inventors have shown that their functionalised solid support can be used to selectively isolate isoagglutinin. However, it will be appreciated that the scaffold could be functionalised with alternative ligands to enable the isolation of other biomolecules.

The inventors have also shown that the functionalised solid support is compatible with cleaning and sanitisation using sodium hydroxide, thereby preventing contamination thereof. The functionalised solid support is stable under these conditions, allowing repeated use thereof. This will significantly reduce the cost of isolating biomolecules, and in turn any products manufactured therewith.

In addition, the inventors have shown that their activated solid support can be functionalised under mild reaction conditions (low temperature, low molar excess of amine and short reaction times) by virtue of the high activation densities achievable by this invention and the high reactivity of the dichlorotriazine attachment chemistry. It will also be appreciated by those skilled in the art that this high potential activation density enables higher ligand densities which in turn can provide increased binding capacity of target biomolecules.

Claim 1:
A method of producing an activated substrate, the method comprising:
(a) modifying a substrate comprising a base matrix to form a base matrix comprising a leaving group, wherein modifying the substrate comprises:
(ai) contacting the substrate with an electrophile to form a base matrix comprising an unsaturated hydrocarbon chain, wherein the electrophile is a compound of formula (I):

        R<NUM>-L<NUM>-X<NUM>-L<NUM>-R<NUM>     (I)

, wherein R<NUM> is an optionally substituted <NUM> to <NUM> membered heterocyclic ring;
R<NUM> is an optionally substituted C<NUM>-C<NUM> alkenyl or an optionally substituted C<NUM>-C<NUM> alkynyl;
L<NUM> and L<NUM> are each independently absent, an optionally substituted C<NUM>-<NUM> alkylene, an optionally substituted C<NUM>-<NUM> alkenylene or an optionally substituted C<NUM>-<NUM> alkynylene, where the backbone of the alkylene, alkenylene or alkynlene is optionally interrupted by one or more heteroatoms;
X<NUM> is NR<NUM>, O or S; and
R<NUM> is H, an optionally substituted C<NUM>-C<NUM> alkyl, an optionally substituted C<NUM>-C<NUM> alkenyl or an optionally substituted C<NUM>-C<NUM> alkynyl;
(aii) contacting the base matrix formed in step (ai) with a halogenating agent, to provide a halogenated base matrix;
(b) contacting the base matrix formed in step (aii) with an aminating agent, to thereby provide an aminated base matrix; and
(c) contacting the aminated base matrix formed in step (b) with a heteroaromatic compound, wherein the heteroaromatic compound is a <NUM> to <NUM> membered heteroaromatic ring substituted with at least two halogens and optionally one or more further substituents, to produce an activated substrate.