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Novel Affinity Ligands And Their Use

Abstract: The present invention relates to novel affinity ligand-matrix conjugates comprising a ligand with the general formula (a) which ligand is attached to a support matrix in position (A), optionally through a spacer arm interposed between the matrix and ligand. The invention furthermore relates to these novel affinity ligand-matrix conjugates and the preparation and use thereof in the purification of proteinaceous materials such as e.g. immunoglobulins, insulins, Factor VII, or human Growth Hormone or analogues, derivatives and fragments thereof and precursors.

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Patent Information

Application #
Filing Date
19 September 1996
Publication Number
15/2012
Publication Type
INA
Invention Field
CHEMICAL
Status
Email
Parent Application

Applicants

NOVO NORDISK A/S
NOVO ALLE, 2880 BAGSVAERD
AFFINITY CHROMATOGRAPHY LTD.
307 HUNTINGDON ROAD, GIRTON, CAMBRIDGE, CB3 OJX

Inventors

1. CHRISTOPHER R. LOWE
THE LIMES, HEMPSTEAD, SAFFRON WALDEN CB10 2PW
2. KENNETH SPROULE
3 WINDNALL CLOSE, GRANTCHESTER, CAMBRIDGE, CB3 9NN
3. RONGXIU LI
31 CLIVEDON CLOSE, CAMBRIDGE, CB4 3LX
4. JAMES C PEARSON
1 PYE TERRACE, CHURCH STREET, CHESTERTON, CAMBRIDGE, CB4 1DX
5. STEVEN J BURTON
TOUCHWOOD, 23 HARLTON ROAD, LITTLE EVERSDEN, CAMBRIDGE, CB3 7HB
6. DAVID J STEWART
188 SPRING ROAD, HUNTINGDON, NY 11743

Specification

Novel Affinity Ligands And Their Use The present invention relates to novel affinity ligands, their preparation and attachment to matrices which may consist of solid, semi-solid, particulate or colloidal materials, or soluble polymers. The invention furthermore relates to these novel affinity ligand-matrix conjugates and the preparation and use thereof in the purification of proteinaceous materials such as e.g. immunoglobulins, insulins, Factor VII, or Human Growth Hormone or analogues, derivatives and fragments thereof and precursors. BACKGROUND OF THE INVENTION Modern protein purification principles very much rely upon chromatographic separation techniques such as gel permeation chromatography (GPC), ionexchange chromatography (IEC), hydrophobic interaction chromatography (HIC), reversed phase high pressure chromatography (RP-HPLC), and affinity chromatography (AC). These techniques are easily adapted to laboratory scale purification of peptides and proteins meant for research and scientific experiments, resulting in pure and biologically active substances. In most cases little or no interest is paid to process economy, process validation, or cleaning in place procedures, as the material will only rarely be used for clinical experiments and because labour costs far exceed the costs of equipment and matrices. However, large scale industrial downstream processing must take into consideration factors such as economy, robustness of matrices and cleaning in place with e.g. NaOH, urea, or ethanol. Today the demand for inexpensive and robust matrices stable in 1M NaOH, 7M urea, or 80% v/v ethanol is met by a number of commercial suppliers within the field of GPC, IEC, HIC, and RP- HPLC. A combination of these principles has for many years resulted in almost pure protein bulk substances although use of extreme buffers and many purification steps have resulted in poor recoveries, increased costs and questionable stability of the bulk preparations. It has long been realised that the principle of affinity chromatography could also be applied to large scale operations. Unfortunately, adsorbents created with natural biological ligands such as monoclonal or polyclonal antibodies tend to be expensive to produce because the ligands themselves often require extensive purification, are biologically and chemically labile and tend to be difficult to immobilise with retention of their biological activity. Therefore, there has been a long term need to replace the expensive chemically and biologically labile monoclonal or polyclonal antibodies with less expensive and more robust ligands mimicking the specificity of antibodies. Affinity chromatography occupies a unique place in separation technology as the protein to be purified adsorbs selectively and reversibly to the complementary binding substance such as an antibody molecule. Purification factors of several thousandfold are often observed with high recoveries, in contrast to the conventional purification methods offering factors from 5-50 times. The high purification factors obtained in affinity chromatography dramatically reduces the number of purification steps in the downstream process. Further, the very low non specific binding observed in affinity chromatography, makes it possible to purify a given protein from complex biological mixtures, to separate incorrectly folded forms from native molecules, and to recover the protein specifically from even large volumes of tissue extracts or fermentation cultures. The affinity sorbent comprises a solid, usually permeable, support matrix, to which a suitable ligand is covalently attached, contained in a conventional chromatographic column. A crude sample, containing the complementary biopolymer is passed over the support matrix, under conditions which promote specific binding interactions with the immobilised ligand. The column is washed with buffer to remove unretarded molecules followed by an elution step in which the protein is eluted in its pure form. A typical affinity adsorbent is based on a solid support, a spacer arm and a ligand. The solid support can be made of bead-formed agarose with an open pore structure. The spacer arm may encourage protein binding by making the ligand more accessible. The length and nature of the spacer arm can be determined by a person skilled in the art. The ligand should exhibit specific and reversible binding to the protein to be purified even after immobilization. In addition to antibodies, a number of compounds including enzymatic co-factors, amino acids, peptides, proteins, concanavalin A, Lectin, thiols, and dyes have been used as affinity ligands. Affinity chromatography has been used in many applications. A comprehensive list is given in e.g. "Affinity Chromatography A Practical Approach" from IRL Press, 1985, and "Affinity Chromatography, Principles and Methods" from Pharmacia Fine Chemicals 1979. Conventional substrate or substrate analogue affinity ligands, especially dyes, have been used for large scale purification of specific enzymes or groups of enzymes. (Scawen M.D. and Atkinson T. 1987, Reactive Dyes in Protein and Enzyme Technology, Ed. Clonis Y.D. etah, Macmillan Press, pp. 51-85). Dye affinity chromatography has over the years gained much interest because of the relative low price of such matrices, their robustness and their ability to withstand NaOH, urea and ethanol. Some of the more widely used ligands in this type of affinity chromatography have been a variety of reactive triazine-based textile dyes immobilised to agarose and other supports. The use of affinity chromatography on immobilized dyes has been reviewed (Lowe C.R. and Pearson J.C. 1984, Methods in Enzymology 104, pp. 97-113). Selective interactions with the NAD+-binding site of horse liver alcohol dehydrogenase were shown with dye analogues of Cibacron Blue F3G-A (Lowe C.R. et a/ 1986; Journal of Chromatography 376, pp. 121-130). The selective purification approach was further illustrated with the computer aided design of a novel affinity adsorbent mimicking the phenyl-arginine dipeptide substrate for the purification of porcine pancreatic kallikrein (Burton N.P. and Lowe C.R. 1992, Journal of Molecular Recognition 5, pp. 55-58). US Patent No. 4,562,252 discloses a particular ligand structure consisting of two m-aminophenyl boronic acid groups attached to a triazine ring for glycoprotein separation. However, despite the rapid progress in affinity technology over the past few years, the need is still to develop a technology by which a specific nimetic ligand can be identified for a protein in order to produce an nexpensive and stable affinity column capable of repeated large scale aurification of the said protein, e.g. in the separation and purification of aroteinaceous materials, such as immunoglobulins, insulins, Factor VII, or human Growth Hormone or analogues, derivatives and fragments thereof and recursors, whether derived from natural or recombinant sources. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel affinity ligands, their preparation and attachment to matrices, and the use of these novel affinity ligand-matrices in the purification of proteinaceous materials. The current invention is based on the notion that the selectivity of hydrophobic ligands may be increased by increasing the complexity and spatial geometry of the hydrophobic component, and the incorporation of various functional groups capable of partaking in electrostatic and hydrogen bonding interactions thereby promoting selective interactions with protein binding sites. This work led to the discovery of a generic group of novel affinity ligands, which have been unexpectedly found to be generally applicable to the isolation and purification of proteins by affinity chromatography. In contrast to the above mentioned selective approach where enzyme substrates, analogues thereof or substrate mimetics were used as ligands, the ligands defined in this application are directed towards any surface of the protein molecule, making the principle applicable for any protein. The ligands are designed by computer modelling techniques and/or by screening of mimetic ligand libraries. Further, the current invention has the advantage that the structure of the protein binding site architecture is not required for design and development of the ligand, and consequently the materials and techniques described herein have a significantly greater utility. A feature of the present invention is the provision of a general tool for protein resolution, isolation and purification. A family of subtly different chemical structures has been synthesised, which have the ability to interact with different proteins. A particularly effective ligand structure for a given protein is identified by screening a range of ligands provided by the invention for suitable binding properties. By way of example, affinity ligands of high selectivity and specificity which are currently available for the separation and purification of immunoglobulins are often proteinaceous materials derived from either bacterial or recombinant sources and include materials such as Protein A, Protein G and Protein L. Immobilisation of these, and similar, proteins often results in a significant loss of biological activity. Continual and repeated use of immobilised proteins as affinity media leads to a further diminution of biological activity. Furthermore, the inherent nature of these biological macromolecules imposes strict limitations with respect to the use of buffer salts, organic solvents and pH levels in affinity chromatography and related techniques. Novel affinity ligands provided by this invention can be used in place of protein A and Protein G and are significantly more flexible in their use, are more robust, less expensive to produce and offer equivalent levels of purification. Another example is the use of novel affinity matrices provided by this invention in biotechnology. The present invention relates to affinity ligand matrix conjugates comprising a ligand with the general formula (a): wherein R1 represents a hydrogen atom, an alkyl group containing from 1 to 6 carbon atoms, a hydroxyalkyl group containing from 1 to 6 carbon atoms, a cyclohexyl group, an amino group, a phenyl group, naphthyl group, henylpyrazole, indazole, benzthiazole group, benzoxazole group, or a benzimidazoie group, each of which benzene, naphthalene, phenylpyrazole, ndazole, benzthiazole, benzoxazole or benzimidazoie ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl groups containing from 1 to 6 carbon atoms, alkoxy groups containing from 1 to 6 carbon atoms, acyloxy or acylamino groups containing from 1 to 6 carbon atoms, amino groups, hydroxyl groups, carboxylic acid groups, sulphonic acid groups, carbamoyl groups, sulphamoyl groups, alkylsulphonyl groups containing from 1 to 6 carbon atoms or halogen atoms; Y represents an oxygen atom, a sulphur atom or a group N-R2; Z represents an oxygen atom, a sulphur atom or a group N-R3; R2 and R3 each independently represent a hydrogen atom, an alkyl group containing from 1 to 6 carbon atoms; a hydroxyalkyl group containing from 1 to 6 carbon atoms, a benzyl group or a p-phenylethyl group; R4, RS and R6 each independently represent a hydrogen atom, a hydroxyl group, an alkyl group containing from 1 to 6 carbon atoms, an alkoxy group containing from 1 to 6 carbon atoms, an amino group, an acyloxy or acylamino group containing from 1 to 6 carbon atoms, a carboxylic acid group, a sulphonic acid group, a carbamoyl or sulphamoyl group, an alkylsulphonyl group containing from 1 to 6 carbon atoms or a halogen atom; one of the symbols X represents a nitrogen atom and the other symbol X represents a nitrogen atom or a carbon atom carrying a chlorine atom or a cyano group; Q represents a benzene, naphthalene, benzthiazole, benzoxazole 1- phenylpyrazole, indazole or benzimidazoie ring; n is an integer between 0 and 6; p is an integer between 0 and 20; and which ligand is attached to a support matrix in position A, optionally through a spacer arm interposed between the matrix and ligand. The optional spacer arm is preferably represented by the general formula wherein T represents an oxygen atom, a sulphur atom or a group N-R7; wherein R7 represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms; V represents an an oxygen atom, a sulphur atom, a -COO- group, a CONH group or an NHCO group or a -P03H- group , an NH-arylene-S02-CH2- CH2 group or an N-R8 group; wherein R8 represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms; L represents an optionally substituted hydrocarbon linkage containing from 2 to 20 carbon atoms; and m is 0 or 1. The support matrix may be any compound or material, particulate or non particulate, soluble or insoluble, porous or non porous which may be used in conjunction with affinity ligands to form a affinity ligand matrix conjugate and which provides a convenient means of separating the affinity ligands from solutes in a contacting solution. The present invention provides novel affinity ligand-matrix conjugates, which affinity ligand-matrix conjugates may be used in the separation and purification of proteinaceous materials, such as immunoglobulins, insulins, Factor VII, or human Growth Hormone or analogues, derivatives and fragments thereof and precursors, whether derived from natural or recombinant sources. In a preferred embodiment, the invention provides novel affinity ligand matrix conjugates which are represented by the General Formula (I): vherein R,, Y, Z, R2, R3, R4, R6, R6, X, Q, n and p have the meanings specified ibove, T represents an oxygen atom, a sulphur atom or a group N-R7; V represents an oxygen atom, a sulphur atom, a -COO- group, a CONH jroup or an NHCO group or a -P03H- group , an NH-arylene-S02-CH2-CH2 group sr an N-R8 group; R7 and R8 each independently represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms; L represents an optionally substituted hydrocarbon linkage containing from 2 to 20 carbon atoms; m is 0 or 1; and M represents the residue of a support matrix. The term "alkyl group containing from 1 to 6 carbon atoms" as used herein, alone or in combination, refers to a straight or branched, saturated hydrocarbon chain having 1 to 6 carbon atoms such as e.g. methyl, ethyl, n- propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 2- methylbutyl, 3-methylbutyl, n-hexyl, 4-methylpentyl, neopentyl, n-hexyl and 2,2-dimethylpropyl. The term "hydroxyalkyl group containing from 1 to 6 carbon atoms" as used herein, alone or in combination, refers to a straight or branched, saturated hydrocarbon chain having 1 to 6 carbon atoms substituted with one or more hydroxy groups, preferably one hydroxy group, such as e.g. hydroxymethyl, 2- hydroxyethyl, 3-hydroxypropyl, 2-hydroxypropyl, 4-hydroxybutyl, 5- hydroxypentyl, 6-hydroxyhexyl. The term " alkoxy group containing from 1 to 6 carbon atoms" as used herein, alone or in combination, refers to a straight or branched monovalent substituent comprising an alkyl group containing from 1 to 6 carbon atoms linked through an ether oxygen having its free valence bond from the ether oxygen and having 1 to 6 carbon atoms e.g. methoxy, ethoxy, propoxy, isopropoxy, butoxy, pentoxy. Term "halogen" means fluorine, chlorine, bromine or iodine. The term "acyloxy or acylamino containing from 1 to 6 carbon atoms " as used herein refers to a monovalent substituent comprising an alkyl group containing from 1 to 5 carbon atoms linked through a carbonyloxy or oxycarbonyl group such as a methylcarbonyloxy, ethylcarbonyloxy, methyloxycarbonyl or ethyloxycarbonyl group or linked through a carbonylamino or aminocarbonyl group such as a methylcarbonylamino, ethylcarbonylamino, methylaminocarbonyl or ethylaminocarbonyl group. The term "alkylsulfonyl containing from 1 to 6 carbon atoms" as used herein refers to a monovalent substituent comprising a alkyl group containing from 1 to 6 carbon atoms linked through a sulfonyl group such as e.g. methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, isopropylsulfonyl, n- butylsulfonyl, sec-butylsulfonyl, isobutylsulfonyl, tert-butylsulfonyl, n- pentylsulfonyl, 2-methylbutylsulfonyl, 3-methylbutylsulfonyl, n-hexylsulfonyl, 4-methylpentylsulfonyl, neopentylsulfonyl, n-hexylsulfonyl and 2,2- dimethylpropylsulfonyl. The term "one or more substituents independently selected from" shall more preferably refer to from 1 -3 substituents. The term shall further preferably refer to 1 -2 substituents and most preferably refer to one substituent. In the present specification, whenever the term insulin is used in a plural or a generic sense, it is intended to encompass both naturally occurring insulins and insulin analogues and derivatives thereof and precursors. By the term insulin is thus also meant insulin from any species, e.g. human insulin. By the term "insulin analogue" as used herein is meant human insulin with one or several amino acid substitutions, one or several amino acid deletions, one or several amino acid additions or combinations hereof. The term "insulin derivative" means insulin chemically modified in one or several residues. By "insulin precursor" as used herein is meant any molecule which by enzymatic or chemical conversion results in formation of insulin, insulin fragments, e.g. des-Thr(B30)-insulin, insulin analogues, or insulin derivatives. The term "optionally substituted hydrocarbon linkage containing from 2 to 20 carbon atoms" as used herein refers to one or more linear or branched alkyl chains, optionally substituted with for example hydroxy or alkoxy groups containing from 1 to 6 carbonatoms, and optionally linked together by amino, ether, thioether, ester, amide or sulphonamide bonds providing a chain containing from 2 to 20 carbon atoms. The construction is preferably flexible. The construction of such optionally substituted hydrocarbon linkages is for example described in Lowe, C. R. and Dean, P.D.G, 1974, Affinity Chromatography, John Wiley & Sons, London, which hereby are incorporated by reference. In a preferred embodiment, these conjugates are represented by the General Formula (I): wherein R, represents a hydrogen atom, an alkyl group containing from 1 to 6 carbon atoms, a hydroxyalkyl group containing from 1 to 6 carbon atoms, a cyclohexyl group, an amino group, a phenyl group or a naphthyl group, which may be substituted on the benzene or naphthalene ring by alkyl groups containing from 1 to 6 carbon atoms, alkoxy groups containing from 1 to 6 carbon atoms, acyloxy or acylamino groups containing from 1 to 6 carbon atoms, amino groups, hydroxyl groups, carboxylic acid groups, sulphonic acid groups, carbamoyl groups, sulphamoyl groups, alkylsulphonyl groups or halogen atoms; T represents an oxygen atom, a sulphur atom or a group N-R7; Y represents an oxygen atom, a sulphur atom or a group N-R2; Z represents an oxygen atom, a sulphur atom or a group N-R3; R2 and R3 each independently represent a hydrogen atom, an alkyl group containing from 1 to 6 carbon atoms; a hydroxyalkyl group containing from 1 to 6 carbon atoms, a benzyl group or a p-phenylethyl group; R4, R5 and R6 each independently represent a hydrogen atom, a hydroxy! group, an alkyl group containing from 1 to 6 carbon atoms, an alkoxy group containing from 1 to 6 carbon atoms, an amino group, an acyloxy or acylamino group containing from 1 to 6 carbon atoms, a carboxylic acid group, a sulphonic acid group, a carbamoyl or sulphamoyl group, an alkylsulphonyl group or a halogen atom; one of the symbols X represents a nitrogen atom and the other symbol X represents a nitrogen atom or a carbon atom carrying a chlorine atom or a cyano group; V represents an oxygen atom, a sulphur atom, a -COO- group, a CONH group or an NHCO group or a -P03H- group , an NH-arylene-S02-CH2-CH2 group or an N-Rs group; R7 and R8 each independently represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms; L represents an optionally substituted hydrocarbon linkage containing from 2 to 20 carbon atoms; Q represents a benzene or naphthalene ring; n is an integer between 0 and 6; p is an integer between 0 and 20; m is 0 or 1; and M represents the residue of a support matrix which may be any compound or material, particulate or non particulate, soluble or insoluble, porous or non-porous which may be used in conjunction with affinity ligands to form a novel affinity ligand-matrix conjugate of General Formula (I) and which provides a convenient means of separating the affinity ligands from solutes in a contacting solution. It will be appreciated that this invention relates, inter alia, to the use of compounds which are pyridines, diazines or triazines carrying a T-[L-V]0.1-M substituent, or the precursor thereof, and other substituents linked to the ring via a heteroatom. Such substituents may include any non-interfering group comprising 0 to 10 or 20 C atoms. In a preferred embodiment of the invention, R, represents a phenyl or naphthyl group each of which is optionally substituted on the benzene or naphthalene ring with one or more independently selected from the group consisting of hydroxyl groups or carboxylic acid groups. In another preferred embodiment of the invention, R2 represents a hydrogen atom. In another preferred embodiment of the invention, R3 represents a hydrogen atom. In another preferred embodiment of the invention, R4 represents a hydrogen atom, a hydroxyl group, a carboxylic acid group, or an amino group. In another preferred embodiment of the invention, R5 represents a hydrogen atom, a hydroxyl group, a carboxylic acid group, or an amino group. In another preferred embodiment of the invention, R6 represents a hydrogen atom, a hydroxyl group, a carboxylic acid group, or an amino group. In another preferred embodiment of the invention, R7 represents a hydrogen atom. In another preferred embodiment of the invention, T represents an oxygen atom or an NH group. In another preferred embodiment of the invention, Y represents N-R2 wherein R2 is as defined above. In another preferred embodiment of the invention, Z represents N-R3 wherein R3 is as defined above. In another preferred embodiment of the invention, both X represents a nitrogen atom. In another preferred embodiment of the invention, Q represents a benzene or naphthalene ring. In another preferred embodiment of the invention, n represents 0 or 2. In another preferred embodiment of the invention, p represents 0 or 2. In another preferred embodiment of the invention, m represents 0 or 1. In another preferred embodiment of the invention, L represents an ethyl, propyl, hydroxypropyl, butyl, pentyl, hexyl, octyl or decyl group and V and m are as defined above. In another preferred embodiment of the invention, V represents an oxygen atom, a -COO- group, a -P03H- group, or an N-R8 group; and more preferred an oxygen atom or an NH group and L and m are as defined above. In another preferred embodiment of the invention, m represents 1 and L and V are as defined above. The term "integer between x and y" may include the values x (including zero) and y. The invention also provides methods for the manufacture of the novel affinity ligand-matrix conjugates according to the invention which comprises reacting, in any order, a halogenoheterocyclic compound of General Formula (II): wherein the symbols X have the meaning hereinbefore specified and W represents a halogen atom with (i) a compound of General Formula (III): R1-(CH2)P-Y-H (III) wherein the symbols R1f Y and p have the meanings hereinbefore specified and H is hydrogen, (ii) a compound of General Formula (IV) hereinbefore specified; the compound of General Formula (VII) is then reacted further with a support matrix whose residue is represented by M using activating and coupling procedures well known to those skilled in the art. As examples of halogenoheterocyclic compounds of General Formula (II) there may be mentioned 5-chloro-2,4,6-trifluoropyrimidine, 5-cyano-2,4,6- trichloropyrimidine, cyanuric fluoride, cyanuric bromide and, above all, cyanuric chloride. As examples of compounds of General Formula (III) there may be mentioned amines such as ammonia, methylamine, ethylamine, propylamine, isopropylamine, diisopropylamine, isobutylamine, amylamine, hexylamine, ethanolamine, diethanolamine, aniline, N-methylaniline, N-ethylaniline, N- isopropylaniline, 1,4-diaminobutane, 1,6-diaminohexane, N-tert-butylaniline, p- toluidine, p-butylaniline, 2,4-dimethylaniline, p-anisidine, p-ethoxyaniline, p- aminoacetanilide, p-aminophenol, p-chloroaniline, orthanilic acid, metanilic acid, sulphanilic acid, 4-methylaniline-2-sulphonic acid, 4-methoxyaniline-2-sulphonic acid, aniline-2,5-disulphonic acid, N-methylmetanilic acid, o-, m- and p- aminobenzoic acid, p-aminobenzamide, p-aminobenzenesulphonamide, 1- amino-2-, 3-, 4-, 5-, 6-, 7- and 8-naphthol, 2-amino-3-, 4-, 5-, 6-, 7- and 8- naphthol, 5-, 6- and 7-amino-1-naphthol-3-sulphonic acid, N-benzylaniline, benzylamine, 4-methylbenzylamine, 4-hydroxybenzylamine, 4- methoxybenzylamine, 4-acetoxybenzylamine, 4-acetylaminobenzylamine, N- methylbenzylamine, p-phenylethylamine, N-butyl-benzylamine, N-benzyl-(3- phenylethylamine, N-(P-hydroxyethyl)-benzylamine, N-tert-butyl-benzylamine, N-benzyltyramine and tyramine; phenols such as phenol, o-, m- and p-cresol, catechol, resorcinol, hydroquinone, p-chlorophenol, 1-naphthol and 2-naphthol, 1-naphthol-4-sulphonic acid, 2-naphthol-6-sulphonic acid and 2-hydroxy-3- naphthoic acid; thiols such as ethylthiol, thioglycollic acid, thiophenol and thio- p-cresol, and aromatic heterocycles such as 5-amino-1 -phenylpyrazole, 6- aminoindazole, 2-aminobenzimidazole, 2-aminobenzthiazole, and 2-amino-5- chlorobenzoxazole. As examples of compounds of General Formula (IV) there may be mentioned amines such as aniline, N-methylaniline, N-ethylaniline, N-perfluorodecalin which provide affinity-ligand matrix conjugates for use in the formation of affinity emulsions. For the avoidance of doubt, a support matrix is defined herein as any compound or material whether particulate or non- particulate, soluble or insoluble, porous or non-porous which may be used to form a novel affinity ligand-matrix conjugate according to the invention and which provides a convenient means of separating the affinity ligand from solutes in a contacting solution. Also included within the definition of support matrices whose residue is represented by M are support matrices such as agarose, cellulose, dextran, starch, alginate, carrageenan, synthetic polymers, silica, glass and metal oxides which have been, or are, modified by treatment with an activating agent prior to, or during, attachment of the ligand. In a preferred embodiment of the invention M represents optionally activated agarose, silica, cellulose, glass, toyopearl, hydroxyethylmethacrylate, polyacrylamide, styrenedivinylbenzene, Hyper D, perfluorocarbons. Preferably M represents optionally tresyl activated, sulphonylchloride activated, tosyl activated, vinylsulphone activated or epoxy activated agarose. Preferred affinity ligand matrix conjugates according to the invention are there may be mentioned such diverse compounds as cyanogen bromide, cyanuric chloride, epichlorohydrin, divinyl sulphone, p-toluenesulphonyl chloride, 1,1'-carbonyldiimidazole, sodium meta-periodate, 2-fluoro-1- methylpyridiniumtoluene-4-sulphonate, glycidoxypropyltrimethoxysilane and 2,2,2-trifluoroethanesulphonyl chloride. As indicated above, the procedures by which such activating steps are carried out are well known to those skilled in the art. Similarly, a wide variety of condensing agents may be used to attach the compounds of General Formulae (VI) to support matrices such as agarose, cellulose, dextran, starch, alginate, carrageenan, silica or glass. Again these compounds, and their method of use are well known to those skilled in the art and, again, since the nub of the present invention lies in the nature of the ligand and not in the mode of attachment, any of these condensing agents will serve in the preparation of the new matrix-ligand conjugates of the invention. As non-limiting examples of such condensing agents, there may be mentioned N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, dicyclohexyl carbodiimide and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. As examples of linking units of General Formula (VI) which may be used to produce compounds of General Formula (VII) there may be mentioned diamines such as ethylene diamine, N,N'-dimethylethylene diamine, N- ethylethylene diamine, N-(P-hydroxyethyl)-ethylene diamine, propylene diamine, N-methylpropylene diamine, N-(J3-hydroxyethyl)-propylene diamine, 1,4- diaminobutane, 1,5-diaminopentane, 1,6-diaminohexane, 1,7-diaminoheptane, 1,8-diaminooctane, 1,9-diaminononane, 1,10-diaminodecane, 1,12- diaminododecane, piperazine, 3-hydroxy-1,5-diaminopentane, m- and p- phenylene diamine, m- and p-aminobenzylamine; amino alcohols such as ethanolamine, N-methylethanolamine, N-propylethanolamine, diethanolamine, 3- hydroxypropylamine, 2,3-dihydroxypropylamine, isopropanolamine, 5- aminopentan-1-ol and 6-aminohexan-1-ol; aminophenols such as o-, m- and p- aminophenol, aminocarboxylic acids such as glycine, N-methylglycine, 3- and 4- aminobutyric acid, 3-aminoisobutyric acid, 5-aminovaleric acid, 6- aminocaproic acid, 7-aminoheptanoic acid, m- and p-aminobenzoic acid; aminophosphonic acids such as m-aminobenzenephosphonic acid and p- aminobenzylphosphonic acid; and aminoarylene vinylsulphone precursors such as aniline-3-p-sulphatoethylsulphone and aniline-4-p-sulphatoethylsulphone. The reaction of halogenoheterocyclic compounds of General Formula (II) with compounds of General Formulae (III), (IV) and (V) or (VI) may be carried out in an organic solvent which is not miscible with water; or in an organic solvent which is miscible with water, or in a mixture of water and a water miscible organic solvent. Examples of suitable organic solvents which are not miscible with water are toluene, xylene or chlorobenzene; Examples of suitable organic solvents which are miscible with water are acetone, methyl ethyl ketone or dioxan. The first reaction of the halogenoheterocyclic compound may be carried out at temperatures between 0°C and 25°C, ideally between 0°C and 5°C; the second reaction may be carried out at temperatures between 20°C and 50°C, ideally between 30°C and 45°C and the third reaction at temperatures between 20°C and 100°C. During such reactions, the inorganic acid such as hydrochloric acid or hydrofluoric acid which is produced is neutralised by the use of an acid binding agent such as sodium hydroxide, sodium carbonate, sodium bicarbonate, calcium hydroxide or calcium carbonate. Additionally, compounds of General Formula (VII) may be reacted with a reactive polymerisable monomer to form a polymerisable compound of General Formula (VIII): wherein Rv R4, R5, R6, Q, L, T, V, X, Y, Z, m, n and p have the meanings hereinbefore specified; R9 represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms; R10 represents a carbonyl group, a methylene group, a -NH-CHr group or a -S-CH2- group. Examples of reactive polymerisable monomers include acryloyl chloride, methacryloyl chloride, allyl bromide, allylamine or 3,4-epoxybutene. Polymerisable compounds of General Formula (VIII) may be polymerised, optionally in the presence of other polymerisable monomers, to form affinity ligand matrix conjugates of General Formula (I). Such polymerisation procedures are well known to those skilled in the art. The invention further covers the use of all such affinity ligand-support matrices in the separation, isolation, purification, quantification, identification and characterisation of proteinaceous materials, such as immunoglobulins, insulins, Factor VII, or Human Growth Hormone or analogues, derivatives and fragments thereof and precursors. Immunoglobulins are a family of proteins, often abbreviated as lg, which share a common structure. Immunoglobulins are also known collectively as antibodies and either word may be used to describe this group of proteins. Immunoglobulins exist in a number of different forms, for example, the most significant antibody types being lgA, lgD, lgE, lgG, lgM and lgY and various sub¬classes thereof. Immunoglobulins may occur in body fluids, such as plasma, ascities, saliva, milk or egg yolk or may be produced using genetic engineering methodologies. Immunoglobulins may be altered by a variety of techniques to confer desirable properties upon them. Such procedures are well known to those skilled in the art and the resulting modified antibodies are also subject to the Claims of this invention. As non-limiting Examples of antibody modification techniques, antibody fragments, labelled antibodies, antibody conjugates or antibody-fusion proteins may be obtained through chemical modification, by treatment with one or more enzymes or by a combination of both techniques. There exists a considerable number of chemical modification reagents and enzymes which have found use in antibody modification and these compounds and their use are well known to those skilled in the art. A further way of obtaining modified or novel antibodies is to produce them using genetic engineering methodologies. Such methodologies and their use are well known to those skilled in the art and may be used to produce, for example, antibody Fragments or antibody-fusion products. Modified or novel antibodies derived by genetic engineering methodologies are also subject to the Claims of this invention. A valuable group of affinity ligand-support matrices is represented by the General Formula (IX): wherein R,, R4, R5, R6, M, Q, n and p have the meanings hereinbefore specified and j is an iriteger between 2 and 20. An especially valuable group of affinity ligand-support matrices is represented by the general Formula (X): wherein j and M have the meanings hereinbefore specified. Typically, reaction of compounds of General Formula (XI) with 3-propoxy-(1,2-epoxy)-agarose at temperatures between 10°C and 30°C in the presence, of an acid binding agent produces novel affinity ligand-matrix conjugates which are of outstanding value in the purification of proteinaceous materials. These new affinity ligand-matrix conjugates possess high affinity for the immunoglobulin group of proteins. This unique property makes them of exceptional value in the separation, isolation, purification quantification, identification and characterisation of proteins of this class. In another embodiment the invention relates to novel affinity ligands of General Formula (XII): wherein R1# R4, Rs, R6, Q, X, Y, Z, n and p have the meanings specified above and Halogen represents a fluorine, chlorine, bromine or iodine atom. Furthermore, the invention relates to a method of attaching novel affinity iigands of General Formula (XII) as defined above to a matrix of General Formula (V) as defined above by reacting the novel affinity ligands with the matrix at temperatures between -20°C and 121°C, optionally in the presence of an acid binding agent. In another embodiment the invention relates to novel affinity iigands of General Formula (XIII): wherein R„ R4, R5, R6, Q, X, Y, Z, m, n and p have the meanings specified above and j is an integer between 2 and 20. Furthermore the invention relates to a method of preparing above novel affinity ligands of General Formula (XIII) by reacting a compound of above General Formula (XII) with an alkylene diamine of General Formula H2N-(CH2)r NH2 at temperatures between 0°C and 100°C in the presence of an acid binding agent. In another embodiment the invention relates to novel affinity ligands of General Formula (XIV): wherein R1# R4, R5, Q, X, Y, Z, m, n and p have the meanings specified above, q is 0 or 1 and j is an integer between 2 and 20. Furhermore the invention relates to a method of preparing novel affinity ligands of above General Formula (XIV) by reacting a compound of above General Formula (XII) with an amino hydroxy compound of General Formula H2N-{CH2)j-(CO)q-OH at temperatures between 0°C and 100°C, optionally in the presence of an acid binding agent. In another embodiment the invention relates to novel affinity ligands of above General Formula (VIII) wherein RV R4, R6, R6, Q, n and p have the meanings specified in anyone of the preceding claims. 36. Novel affinity ligands of General Formula (XVI): wherein R1# R4/ R5/ R6/ Q, n and p have the meanings specified in anyone of the preceding claims and j is an integer between 2 arid 20. 37. Affinity ligands according to anyone of claims 28, 30, 32, 34, 35 or 36 wherein R, represents a phenyl or naphthyl group each of which is optionally substituted on the benzene or naphthalene ring with one or more independently selected from hydroxyl groups and carboxylic acid groups. 38. Affinity ligands according to anyone of claims 28, 30, 32, 34, 35 or 36 wherein R4 represents a hydrogen atom, a hydroxyl group, a carboxylic acid group or an amino group. 39. Affinity ligands according to anyone of claims 28, 30, 32, 34, 35 or 36 wherein R6 represents a hydrogen atom, a hydroxyl group, a carboxylic acid group or an amino group. 40. Affinity ligands according to anyone of claims 28, 30, 32, 34, 35 or 36 wherein R6 represents a hydrogen atom, a hydroxyl group, a carboxylic acid group or an amino group. 41. Affinity ligands according to anyone of claims 28, 30, 32, 34, 35 or 36 wherein Q represents a benzene or naphthalene ring. 42. Affinity ligands according to anyone of claims 28, 30, 32, or 34 wherein X represents a nitrogen atom. 43. Affinity iigands according to anyone of claims 28, 30, 32, 34 or 35 wherein Y represents a -NH- group. 44. Affinity Iigands according to anyone of claims 28, 30, 32, 34 or 35 wherein Z represents a -NH- group. 45. Affinity Iigands according to anyone of claims 28, 30, 32, 34, 35 or 36 wherein n is 0 or 2. 46. Affinity Iigands according to anyone of claims 28, 30, 32, 34, 35 or 36 wherein p is 0 or 2. 47. Affinity Iigands according to anyone of claims 30, 32, or 36 wherein j is 2, 4 or 6. 48. Affinity Iigands according to claim 34 wherein L is an ethyl, butyl, or hexyl group. 49. Affinity Iigands according to claim 34 wherein T represents a -NH- group. 50. Affinity Iigands according to claim 34 wherein V represents a -NH- group. 51. Affinity Iigands according to claim 34 wherein m is 1. 52. Novel affinity Iigands of General Formula (XI): wherein is an integer between 2 and 20. 53. Affinity ligands according to anyone of claims 28 to 51 selected among the following: 54. The use of affinity Iigands according to anyone of the preceding claims for the preparation of affinity ligand matrix conjugates 55. A method of attaching the novel affinity Iigands of General Formulaen(VII) as defined in claim 27 and (XIII) as defined in Claim 30, (XVI) as defined in Claim 36 and (XI) as defined in Claim 52 to carbohydrate or organic polymer matrices by reacting the carbohydrate or organic polymer matrix with an activating agent followed by reaction of the activated matrix with the novel affinity ligand, optionally in the presence of an acid binding agent. 56. A method of attaching the novel affinity Iigands of General Formulae (XIV) as defined in Claim 32 to carbohydrate or organic polymer matrices by condensation with the matrix. 57.A method of attaching the novel affinity Iigands of General Formulae(VII) as defined in Claim 27 and (XIII) as defined in Claim 30, (XVI) as defined in Claim 36 and (XI) as defined in Claim 52 to metal oxide, glass or silica matrices, optionally coated with an organic polymer by reacting the optionally metal oxide, glass or silica matrix with an activating agent followed by reaction of the activated matrix with the novel affinity ligand, optionally in the presence of an acid binding agent. 58. A method of attaching the novel affinity ligands of General Formulae (XIV) as defined in Claim 32 to metal oxide, glass or silica matrices, optionally coated with an organic polymer by condensation with the matrix. 59. A method of attaching novel affinity ligands of General Formula (XV) as defined in Claim 35 and (XII) as defined in Claim 28 to a matrix of General Formula (V) as defined in Claim 26 by reacting the novel affinity ligands with the matrix at temperatures between -20°C and 121°C, optionally in the presence of an acid binding agent. 60. Affinity ligand-matrix conjugates, prepared as claimed in Claims 26, 27, 29, 54, 55, 56, 57 and 58. 61. The use of affinity ligand-matrix conjugates, according to anyone of the preceding claims to affinity ligand-matrix conjugates for the separation, isolation, purification, characterisation, identification or quantification of proteins. 62. . The use according to claim 61 wherein the proteinaceous material is lgG, lgM, lgA, insulins, Factor VII,or Human Growth Hormone or analogues, derivatives and fragments thereof and precursors. 63. A process for the separation or purification of proteinaceous materials comprising carrying out affinity chromatography using as the biospecific ligand a ligand of general formula (a) as defined above. 64. The use according to claim 61 wherein the proteinaceous material is immunoglobulins or subclasses, fragments, precursors or derivatives thereof, whether derived from natural or recombinant sources. 65. The use according to claim 61 wherein the proteinaceous material is immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA) or subclasses, fragments, precursors or derivatives thereof, whether derived from natural or recombinant sources. 66. The use according to claim 61 wherein the proteinaceous material is insulins or insulin analogues, derivatives and fragments thereof and precursors, whether derived from natural or recombinant sources. 67. The use according to claim 66 wherein the affinity ligand-matrix conjugates comprises a ligand selected among the following: which ligand is attached to a support matrix in position (A), optionally through a spacerarm represented by the general formula (b) as specified above. 68. The use according to claim 67 wherein the ligand is 11a. 69. The use according to claim 67 or 68 wherein the support matrix is optionally activated agarose, cellulose, silica or glass. 70. The separation, isolation, purification, characterisation, identification or quantification of immunoglobulins by any process whereby the said immunoglobulins are applied to affinity ligand-matrix conjugates, as defined in anyone of above affinity ligand-matrix conjugate claims at a pH in the range 5.0 to 12.0 and subsequently removed, eluted or desorbed by reducing the pH to 4.9 or lower. 71. The separation, isolation, purification, characterisation, identification or quantification of insulins or insulin analogues or derivatives thereof and precursors by any process whereby the said insulins, insulin derivatives, analogues, and precursors are applied to affinity ligand-matrix conjugates, as defined in anyone of above affinity ligand-matrix conjugate claims at a pH in the range 4,0 to 9,0 and subsequently removed, eluted or desorbed by reducing the pH to 3,99 or lower or to 9,01 or higher. 72. Affinity ligand-natrix conjugates, substantially as hereinabove desoribed and exemplified.

Documents

Application Documents

# Name Date
1 1654-MAS-1996 FORM-6.pdf 2012-03-02
2 1654-MAS-1996 FORM-4.pdf 2012-03-02
3 1654-MAS-1996 FORM-1.pdf 2012-03-02
4 1654-MAS-1996 DESCRIPTION (COMPLETE).pdf 2012-03-02
5 1654-MAS-1996 CORRESPONDENCE OTHERS.pdf 2012-03-02
6 1654-MAS-1996 CLAIMS.pdf 2012-03-02
7 1654-MAS-1996 ABSTRACT.pdf 2012-03-02
8 1654-MAS-1996FORM-13.pdf 2012-03-06
9 1654-MAS-1996 FORM-19.pdf 2012-03-06
10 1654-MAS-1996 CORRESPONDENCE OTHERS 09-11-2012.pdf 2012-11-09
11 1654-MAS-1996 CORRESPONDENCE OTHERS 22-03-2013.pdf 2013-03-22
12 1654-MAS-1996 CORRESPONDENCE OTHERS 03-04-2014.pdf 2014-04-03
13 1654-MAS-1996 CORRESPONDENCE OTHERS 14-07-2014.pdf 2014-07-14
14 1654-MAS-1996_EXAMREPORT.pdf 2016-07-02