New affinity ligands and their application

 

(57) Abstract:

The invention relates to novel conjugates of affinity ligand - matrix containing the ligand associated with the matrix carrier, optionally via a spacer elements located between the matrix and ligand, and new conjugates affinity ligand - matrix, to their preparation and use for the purification of protein materials, such as, for example, immunoglobulins, insulin, factor VII, or human growth factor, or its analogs, derivatives and fragments, and their predecessors. The invention allows to provide an inexpensive and stable affinity columns. 26 C. and 57 C.p. f-crystals, 12 tab.

The present invention relates to new affinis ligands, to receive them and to the binding of these ligands with matrices, which may consist of solid, semi-solid, coarse or colloidal materials, or from water soluble polymers. In addition, the present invention relates to novel conjugates of affinity ligand-matrix, to receive them and to use for the purification of protein materials, such as, for example, immunoglobulins, insulin, factor VII, or human growth hormone or analogues, derivatives, fragments, and precursors.

However, large-scale industrial processing should take into account such factors as efficiency, reliability matrices and their on-site purification using, for example, NaOH, urea or ethanol. Currently, the demand for affordable and durable matrix, which is stable in 1 M NaOH, 7 M urea, or 80% (vol./about.) ethanol, met a number of suppliers specialising in the supply of products for civil, ZIOC, H.H. and OF ghvd. Coproduct, although the use of finite buffers and multiple stages of purification leads to a small yield, increase the cost of the procedure and to the low stability of the main mass of the drug.

Long ago it became clear that the methods of affinity chromatography should be also applicable for large-scale operations. Unfortunately, the use of adsorbents formed by natural biological ligands, such as monoclonal or polyclonal antibodies, leads to higher production, because these ligands are themselves often require extensive treatment and are biologically and chemically labile, making it difficult immobilization of these molecules with retention of their biological activity. Therefore, already long overdue to replace expensive, chemically and biologically labile monoclonal or polyclonal antibodies are less expensive and more reliable ligands that mimic the specificity of the antibodies.

Affinity chromatography is a unique place in separation technology, because the purified protein selectively and reversibly adsorbed on the complementary binding substance such as an antibody molecule. In this case, the coefficients of purification, when h is s cleaning range from 5 to 50 times. High collection efficiencies obtained by affinity chromatography, allow to drastically reduce the number of stages of purification in the subsequent process. In addition, very low levels of specific binding observed in affinity chromatography, allows to distinguish this protein from complex biological mixtures; separate forms of the protein with the wrong styling from the native molecules and the specific allocation of protein even of large amounts of tissue extracts or culture for fermentation.

Affinity sorbent is a solid, generally, permeable matrix carrier, which covalently binds the corresponding ligand and which is contained in a standard chromatographic column. The crude sample containing complementary biopolymer, is passed through the matrix carrier in the enabling conditions for specific binding to the immobilized ligand. The column is washed with buffer to remove nezadelannyh molecules, and then conduct phase elution, in which elute protein in its pure form. Typical affinity adsorbent consists of a solid carrier, spacer elements "legs" (spacer elements group) and ligand. A solid carrier can be made of agar is Yu protein due to the fact, she makes the ligand more accessible. The length and nature of the spacer elements of the group can be determined by the specialist. The ligand must be able to specific and reversible binding protein designed to clean, even after immobilization. In addition to antibodies as affinity ligands has been used a number of other compounds including cofactors of enzymes, amino acids, peptides, proteins, concanavalin a, a lectin, thiols and dyes.

Affinity chromatography has been used in many applications. An exhaustive list is given, for example, Affinity Chromatography A Practical Approach", IRL Press, 1985, and Affinity Chromatography, Principles and Methods, Pharmacia Fine Chemicals, 1979.

For krupnomasshtabnoi purification of specific enzymes or groups of enzymes was used a standard substrate or substrate, similar affinity ligands, and in particular, the dyes (Scawen, M. D. & T. Atkinson 1987, Reactive Dyes in Protein and Enzyme Technology, Ed. Clonis Y. D. et al.; Macmilian Press, pp. 51-85).

Affinity chromatography-based dye for many years is for professionals special interest because of the relatively low cost of the matrices used in this chromatography, their reliability and resistance to NaOH, urea and ethanol. Some of lignie textile dyes based on triazine, immobilized on agarose, and other media. Using affinity chromatography on immobilized dyes described Lowe C. R. & Pearson, J. C. (1984, Methods in Enzymology 104, pp.97-113). Were described selective interaction NAD+binding of the center of alcohol dehydrogenase horse liver with dyes-blue analogues Cibacron Blue F3G-A (Lowe C. R. et ai., 1986; Journal of Chromatography 376, pp.121-230). In addition, the illustrated method of selective treatment, developed using computer technology in order to obtain a new affinity adsorbents, imitating phenyl-arginine-dipeptide substrate, for purification of porcine pancreatic kallikrein (Burton N. P. & Lowe, C. R., 1992, Journal of Molecular Recognition 6, pp. 55-58).

In U.S. patent N 4562252 described the structure of a specific ligand consisting of two m-aminophenylacetylene groups related to the triazine ring and used for isolation of glycoproteins.

However, despite rapid progress in the field of affine technology in recent years, remains an urgent need to develop technologies through which this protein could be identified simulating specific ligand for the preparation of low-cost and stable affinity columns, to the tion and purification of protein materials such as immunoglobulins, insulin, factor VII, human growth hormone or analogues, derivatives, fragments and precursors, derived from natural or recombinant sources.

The present invention relates to novel affinity ligands, their preparation and binding matrix; and using these new complexes affinity ligand - matrix for purification of protein materials.

The present invention is based on the assumption that the selectivity hydrophobic ligands can be improved by increasing complexity and spatial geometry of the hydrophobic component and that the incorporation of various functional groups able to participate in electrostatic and Bogorodskaya interactions, stimulates selective interaction with blockswaputaki sites. This work led to the discovery of a characteristic group of new affinity ligands, which, as it has been unexpectedly discovered, can be mainly used for separation and purification of proteins by affinity chromatography.

In contrast to the above-mentioned selective method in which the substrates of the enzymes, their analogues, or imitators of substrates used in kachestve their principle applicable for purification of any protein. These ligands were designed using computer modeling and/or screening of libraries of ligands imitators. In addition, the present invention has the advantage that the design and development of ligand does not require any particular architecture for the structure blackslatewash site, and therefore the materials and methods described in this application have significantly more value.

The hallmark of the present invention is to develop a General method of separation, isolation and purification of proteins. Was synthesized by a family of different slightly different from each other of chemical structures, which are able to interact with various proteins. The structure of the ligand, particularly effective for this protein, was identified by screening of a series of ligands with suitable for the present invention binding properties.

So, for example, affinity ligands high selectivity and specificity, which are currently used for separation and purification of immunoglobulins, are often protein materials originating either from bacterial or recombinant sources, and include the mother of the e biological activity. Prolonged and repeated use of immobilized proteins as affine environment leads to an additional reduction of biological activity. In addition, the nature of these biological macromolecules imposes strict restrictions on the use of buffer salts, organic solvents and pH levels in affinity chromatography and similar methods.

New affinity ligands of the present invention can be used instead of protein a and protein G, and are significantly more flexible, more stable, less expensive and often give equivalent levels of treatment.

Another example of application could be the use of new Affinia matrices of the present invention in biotechnology.

The present invention relates to conjugates "affinity ligand - matrix" containing the ligand of General formula (a):

< / BR>
where R1represents a hydrogen atom, an alkyl group containing 1-6 carbon atoms, hydroxyalkyl group containing 1-6 carbon atoms, tsiklogeksilnogo group, amino group, phenyl group, naftalina group, 1-phenylpyrazole group indazol group, benzothiazolyl group, benzoxazolyl group or beningo, benzoxazole or benzimidazole ring optionally substituted by one or more substituents independently selected from the group comprising alkyl groups containing 1-6 carbon atoms; alkoxygroup containing 1-6 carbon atoms; acyloxy or acylamino-group containing 1-6 carbon atoms; amino groups; hydroxyl groups; carboxyl groups; sulfonylurea group; carbamoyl group; sulfamoyl group; alkylsulfonyl group containing 1-6 carbon atoms or halogen atoms;

Y represents an oxygen atom, a sulfur atom or a group N-R2;

Z represents an oxygen atom, a sulfur atom or a group N-R3;

each of R2and R3independently represents a hydrogen atom; an alkyl group containing 1-6 carbon atoms; hydroxyalkyl group containing 1-6 carbon atoms; benzyl group or phenylethylene group;

each of R4, R5and R6independently represents a hydrogen atom; hydroxyl group; alkyl group containing 1-6 carbon atoms; alkoxy group containing 1-6 carbon atoms; the amino group; acyloxy or acylamino-group containing 1-6 carbon atoms; a carboxyl group; sulfoximine group, halogen;

one of the symbols X represents a nitrogen atom and the other symbol X represents a nitrogen atom or a carbon atom bearing a chlorine atom or a cyano;

Q represents a benzene, naphthalene, benzothiazole, benzoxazole, 1-phenylpyrazole, indazol or benzimidazole ring;

n represents an integer from 0 to 6;

p represents an integer from 0 to 20; and

where the specified ligand bound to the matrix carrier in position And not necessarily by means of spacer elements located between the matrix and ligand.

Optional spacer elements group is preferably represented by the General formula:

-T-[-L-V-]m-, (b)

where T represents an oxygen atom, a sulfur atom or a group N-7where R7represents a hydrogen atom or an alkyl group containing 1-6 carbon atoms;

V represents an oxygen atom, a sulfur atom, a group-COO-group CONH or NHCO group or group - PO3-H-, a group NH-aralen-SO2-CH2-CH2or N-R8where R8represents a hydrogen atom or an alkyl group containing 1-6 carbon atoms;

L represents an optionally substituted hydrocarbon linkage containing from 2 to 20 carbon atoms; and

m = 0 or 1.

Matronym or insoluble, porous or non-porous, which can be used for conjugation with affinity ligands with the formation of the conjugate affinity ligand - matrix, and which can be carried out the selection of affinity ligands of dissolved solids in the contacting solution.

The present invention relates to novel conjugates of affinity ligand - matrix, which can be used for isolation and purification of protein materials, such as immunoglobulins, insulin, factor VII, or human growth hormone, or their analogues, derivatives and fragments, and precursors, derived from natural or recombinant sources.

In a preferred embodiment, the present invention relates to novel conjugates of affinity ligand - matrix, which are represented by the General formula (I):

< / BR>
where R1, Y, Z, R2, R3, R4, R5, R6, X, Q, n, and p are defined above;

T represents an oxygen atom, a sulfur atom or a group N-R7;

V represents an oxygen atom, a sulfur atom, a group-COO-group, CONH, or NHCO group or group-PO3-H, group NH - aralen-SO2-CH2-CH2or a group N-R8,

each of R7and R8independently represents the atoms is a hydrocarbon bond, containing from 2 to 20 carbon atoms; and

m = 0 or 1; and

M represents the residue of a matrix carrier.

Used in the present description, the term "alkyl group containing 1-6 carbon atoms", taken alone or in combination with other groups, means a straight or branched, saturated hydrocarbon chain having 1-6 carbon atoms, such as, for example, 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.

Used in the present description, the term "hydroxyalkyl group containing 1-6 carbon atoms", taken alone or in combination with other groups, means a straight or branched, saturated hydrocarbon chain having 1-6 carbon atoms, substituted by one or more hydroxy groups, preferably one hydroxy-group, such as, for example, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxypropyl, 4-hydroxybutyl, 5-hydroxyphenyl and 6-hydroxyhexyl.

Used in the present description, the term "alkoxy group containing 1-6 carbon atoms", taken alone or in combination with other groups, oznachaet the kind related through the simple oxygen of the ether group having its svobodovaflat communication from oxygen, and containing 1-6 carbon atoms, e.g. methoxy, ethoxy, propoxy, isopropoxy, butoxy, pentox.

Used in the present description, the term "halogen" means fluorine, chlorine, bromine or iodine.

Used in the present description, the term "acyloxy or acylamino containing from 1 to 6 carbon atoms" means a monovalent Deputy, including alkyl group containing 1-5 carbon atoms connected by carbonyloxy or oxycarbonyl group, such as, methylcarbonate, ethylcarbonate-, methyloxycarbonyl or ethoxycarbonyl group, or linked through carbylamine or aminocarbonyl groups, such as methylcobalamin, ethylcarbodiimide, methylaminomethyl or ethylaminomethyl group.

Used in the present description, the term "alkylsulfonyl containing 1 to 6 carbon atoms" means a monovalent Deputy, including alkyl group containing 1-6 carbon atoms, linked by sulfonyloxy group, such as, for example, methylsulphonyl, ethylsulfonyl, n-propylsulfonyl, isopropylbenzenesulfonyl, 3-methylbutylamine, n-hexylsilane, 4-methylphenylsulfonyl, neopentylene, n-hexylsilane and 2,2-diethyproprion.

Used in the present description, the term "one or more substituents independently selected from --" preferably refers to 1 to 3 substituents. In this case, preferably, when he refers to 1 or 2 substituents, more preferably one substituent.

In the present description, the term "insulin", regardless of whether it is in the multivalued sense or in a generalized sense, is as natural insulin, and their analogues and derivatives, and to their predecessors. The term "insulin" refers to the insulin, derived from any animal species, including humans. In the context of the present description, the term "insulin analog" means the human insulin having one or more amino acid substitutions, one or more amino acid deletions, one or more amino acid insertions, or combinations of these modifications. The term "derived insulin" means insulin is chemically modified in one or more of its residues. The term "precursor of insulin" refers to any molecule, which, after its permalloy insulin or derivative of insulin.

Used in the present description, the term "optionally substituted hydrocarbon linkage containing from 2 to 20 carbon atoms" means one or more linear or branched alkyl chain, optionally substituted, for example, hydroxy - or alkoxy groups containing 1-6 carbon atoms, and optionally bonded together by amino, ether, thioester, ester, amide or sulfonamidnuyu links with the education chain containing from 2 to 20 carbon atoms. This construction is preferably flexible. The design of such optionally substituted hydrocarbon linkages are described, for example, Lowe, C. R. & Dean, P. D. G., 1974, Affinity Chromatography, John Wiley & Sons, London, which is introduced in the present description by reference.

In a preferred embodiment of the present invention, these conjugates represented by the General formula (I):

< / BR>
where represents a hydrogen atom, an alkyl group containing 1-6 carbon atoms, hydroxyalkyl group containing 1-6 carbon atoms, tsiklogeksilnogo group, amino group, phenyl group or naftalina group which may be substituted on the benzene or naftalanovoy ring alkyl groups containing 1-6 atoms plerogyra, amino groups, hydroxyl groups, carboxyl groups, sulfoximine groups, carbamaepine groups, alfamarine groups, alkylsulfonyl groups or halogen atoms;

T represents an oxygen atom, a sulfur atom or a group N - R7;

Y represents an oxygen atom, a sulfur atom or a group N-R2;

Z represents an oxygen atom, a sulfur atom or a group N-R3;

each of R2and R3independently represents a hydrogen atom, an alkyl group containing 1-6 carbon atoms, hydroxyalkyl group containing 1-6 carbon atoms, benzyl group or phenylethylene group;

each of R4, R5and R6independently represents a hydrogen atom, hydroxyl group, alkyl group containing 1-6 carbon atoms, alkoxy group containing 1-6 carbon atoms, amino group, acyloxy or acylamino-group containing 1-6 carbon atoms, carboxyl group, sulfoxyl group, carbamoyl or sulfamoyl group, alkylsulfonyl group or a halogen atom;

one of the symbols X represents a nitrogen atom, and other symbols X represent a nitrogen atom or a carbon atom bearing a chlorine atom or a cyano;

V predstavil>2-CH2or N-R8,

each of R7and R8independently represents a hydrogen atom or an alkyl group containing 1-6 carbon atoms;

L represents an optionally substituted hydrocarbon linkage containing from 2 to 20 carbon atoms; and

Q represents a benzene or naphthalene ring;

n represents an integer from 0 to 6;

p represents an integer from 0 to 20;

m = 0 or 1; and

M represents the residue of a matrix carrier, which may be any compound or material comprising or consisting of particulate, soluble or insoluble, porous or non-porous, which can be used in combination with affinity ligands with the formation of new conjugate affinity ligand - matrix of General formula (I) and which serves as a convenient means of selection of affinity ligands of dissolved solids in the contacting solution.

It should be noted that the present invention relates, inter alia, to the use of compounds that are pyridine, diazine or triazine bearing Deputy T-[L-V]0-1-M, or its predecessor, and the other substituents linked to the ring through a heteroatom. Such substituents may be dear to the embodiment of the present invention R1represents phenyl or naftalina group, each of which is optionally substituted on the benzene or naphthalene ring by one or more groups independently selected from hydroxyl groups or carboxyl groups.

In another preferred embodiment of the present invention R2represents a hydrogen atom.

In another preferred embodiment of the present invention R3represents a hydrogen atom.

In another preferred embodiment of the present invention R4represents a hydrogen atom, hydroxyl group, carboxyl group or amino group.

In another preferred embodiment of the present invention R5represents a hydrogen atom, hydroxyl group, carboxyl group or amino group.

In another preferred embodiment of the present invention R6represents a hydrogen atom, hydroxyl group, carboxyl group or amino group.

In another preferred embodiment of the present invention, a7represents a hydrogen atom.

In another preferred embodiment of the present invention T is an oxygen atom or the NH group.

akim, as specified above.

In another preferred embodiment of the present invention Z is N-R3where R3is the same as above.

In another preferred embodiment of the present invention both X represent a nitrogen atom.

In another preferred embodiment of the present invention, Q represents a benzene or naphthalene ring.

In another preferred embodiment of the present invention n is 0 or 2.

In another preferred embodiment of the present invention p is 0 or 2.

In another preferred embodiment of the present invention m is 0 or 1.

In another preferred embodiment of the present invention L is ethyl, through hydroxypropyl, boutelou, pentelow, hexeline, octillo or decile group, and V and m are as described above.

In another preferred embodiment of the present invention V is an oxygen atom, a group-COO-, group-PO3H-, or a group N-R8and more preferably an oxygen atom or the NH group, and L and m are as described above.

In another preferred embodiment, nascido x and y" may include the value of x (including zero) and y.

The present invention also relates to a method of production of new conjugates "affinity ligand - matrix of the present invention, providing for reactions, in any order, halogen-containing heterocyclic compounds of General formula (II):

< / BR>
where the symbols X have the meanings mentioned above, and W represents a halogen atom,

(i) with a compound of General formula (III):

R1-(CH2)p-Y-H, (III)

where the symbols R1, Y and p are as above, and H denotes hydrogen;

(ii) with a compound of General formula (IV):

< / BR>
where the symbols R4, R5, R6, Q, Z and n have the meanings defined above; and

(iii) or with optional derivational matrix-carrier of the General formula V:

H-T-[-L-V-]m-M (V)

where the symbols L, M, V, T and m have the meanings given above,

or linking the structural unit of General formula (VI)

H-T-L-V-H (VI)

where the symbols H, L, V and T have the meanings given above,

obtaining compounds of General formula (VII)

< / BR>
where R1, R4, R5, R6, Q, L, T, V, X, Y, Z, m, n and p are as described above; and then carrying out the reaction of compounds of General formula (VII) with a matrix carrier, the remainder of Ista.

Examples of halogenated heterocyclic compounds of General formula (II) can serve 5-chloro-2,4,6-triptorelin, 5-cyano-2,4,6-trichloropyridin, floramite cyanuric acid, bromohydrin cyanuric acid, and basically, the acid chloride cyanuric acid.

Examples of compounds of General formula (III) can serve 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, Artemyeva acid, matriliny acid, Sultanalieva acid, 4-methyl - aniline-2-acid, 4-methoxyaniline-2-acid, aniline - 2,5-disulfonate, N-methylmethanamine acid, ortho-, meta -, and para-aminobenzoic acid, p-aminobenzamide, p - aminobenzenesulfonamide, 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-acid, N-benzylaniline, benzylamine, 4 - methylbenzylamine, 4-hydroxy-benzylamine, 4-methoxybenzylamine, 4-acetoxybenzoic, 4-ACE the Il-benzylamine, N-tert - butylbenzylamine, N-benzyltoluene and tyramine; phenols such as phenol, ortho-, meta - and para-cresol, catechol, resorcinol, hydroquinone, p-chlorophenol, 1-naphthol and 2-naphthol, 1-naphthol-4 - naphthol-4-acid, 2-naphthol-6-acid and 2-hydroxy-3-naphthoic acid; thiols, such as, atitool, thioglycolate acid, thiophenol and thio-p-cresol; and aromatic heterocycles, such as, 5-amino-1-phenylpyrazol, 6-aminoindazole, 2 - aminobenzimidazole, 2-amino-benzothiazole and 2-amino-5 - chlorobenzoxazol.

Examples of compounds of General formula (IV) are amines, such as aniline, N-methylaniline, N-ethylaniline, N - isopropylaniline, N-tert-butylaniline, p-toluidine, p-butylaniline, 2,4-dimethylaniline, p-anisidine, p-ethoxyaniline, p-aminoacetanilide, p-aminophenol, p-Chloroaniline, Artemyeva acid, matriliny acid, Sultanalieva acid, 4-methylaniline-2-acid, 4-methoxyaniline-2-acid, aniline-2,5-disulfonate, N - methylmethanamine acid, ortho-, meta -, and para-aminobenzoic acid, 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-acid, p-aminobenzamide, p-aminobenzenesulfonamide, N - benzylaniline, benzylamine, 4-methylbenzylamine, 4-hydroxybenzylidene, 4-methoxybenzylamine, 4-AC is ethylamine, N-(hydroxyethyl)-benzylamine, N-tert-butylbenzylamine, N-benzyltoluene and tyramine; felony, such as phenol, ortho-, meta - and para-cresol, catechol, resorcinol, hydroquinone, p-chlorophenol, 1-naphthol and 2-naphthol, 1-naphthol-4-acid, 2-naphthol-6-acid and 2-hydroxy-3-naphthoic acid; thiols, such as thiophenol and thio-p-cresol; and aromatic heterocycles, such as, 5-amino-1 - phenylpyrazol, 6-aminoindazole, 2-aminobenzimidazole, 2 - aminobenzothiazole and 2-amino-5-chlorobenzoxazol.

As examples of the matrix-carriers, the remainder of which is represented by M, can serve as an insoluble matrix carrier, such as a natural polymer, for example, polypeptide or protein, such as, crosslinking albumin or a polysaccharide such as agarose, alginate, curagen, chitin, cellulose, dextran or starch; synthetic polymers such as polyacrylamide, polystyrene, polyacrolein, polyvinyl alcohol, polymethylacrylate, fluorocarbon; inorganic compounds such as silica, glass, diatomaceous earth, alumina, oxide of iron, or other metallic oxides, or copolymers comprising any combination of two or more natural polymers, synthetic polymers, or inorganic compounds. The concept of "matrix-norimura, such as, dextran, polyethylene glycol, polyvinyl alcohol or hydrolyzed starch, and which form the conjugates of affinity ligand - matrix used for the separation of liquids; or matrix-carriers, which contain such compounds as perpendicular, and which form the conjugates of affinity ligand - matrix used for the manufacture of affine emulsions. For clarity it should be stated that in the context of the present description, the term "matrix-bearer" means any compound or material (regardless of whether it is coarse or necropotence, soluble or insoluble, porous or non-porous), which can be used to obtain a new conjugate affinity ligand-matrix of the present invention and which provides a convenient way of separating affinity ligand from the dissolved solids in the contacting solution.

Moreover, the notion of "matrix-carriers, the remainder of which presents M" means matrix carrier such as agarose, cellulose, dextran, starch, alginate, curagen, synthetic polymers, silica, glass and metallic oxides, which have been modified or are modified by treatment with an activating ia, M is not necessarily activated agarose, silica, cellulose, glass, small, pearl, hydroxyethylmethacrylate, polyacrylamide, stradivariuses, Hyper D, fluorosurfactants.

M preferably denotes agarose, optional activated trasylol, sulphonylchloride, Totila, vinylsulfonate or epoxy.

In accordance with the present invention, preferred conjugates affinity ligand - matrix are

i)

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ii)

< / BR>
iii)

< / BR>
iv)

< / BR>
v)

< / BR>
vi)

< / BR>
vii)

< / BR>
viii)

< / BR>
where M is defined above.

There are a large number of activating agents that can be used in the universal methods of attaching ligands to the matrix-bearer. These compounds and their method of use are well known in the art, and since the basis of the present invention is the nature of the ligand bound to the matrix, not the method of binding, then any of these activating agents can be used for the manufacture of new conjugates "matrix-ligand". Non-limiting examples of such activating agents can serve such diverse compounds as CYANOGEN bromide, acid chloride, zinurova the Oia, toluene-4-sulfonate-2 - fluoro-1-methylpyridine, glycidoxypropyltrimethoxysilane and 2,2,2 - triftoratsetofenona. As shown above, the methods that perform such a stage of activation is well known to specialists.

Similarly, to attach the compounds of General formula (IV) to the matrix-carrier can be used condensing agents a wide range, such as agarose, cellulose, dextran, starch, alginate, curagen, silica or glass. In this case, these compounds and methods for their use are well known in the art, and since, as in the previous case, the basis of the present invention is the nature of the ligand, not the method of binding, then any of these activating agents can be used for the manufacture of new conjugates matrix - ligand. Non-limiting examples of such condensing agents can serve as N - etoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, dicyclohexylcarbodiimide and 1-ethyl-3- (3-dimethylaminopropyl) - carbodiimide.

As examples of connecting links of the General formula (VI), which can be used to obtain the compounds of General formula (VII) may serve as diamines, such as Ethylenediamine, N,N'-dimethylethylenediamine, N-ethylethylenediamine, 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-phenylenediamine, m - and p-aminobenzoylamino; aminoalcohols, such as ethanolamine, N-methylethanolamine, N-problemanalysen, diethanolamine, 3-hydroxypropylamino, 2,3-dihydroxypropyl, isopropanolamine, 5-aminoindan-1-ol and 6-aminohexyl-1-ol; aminophenols, such as, o-, m - and p-aminophenol; aminocarbonyl acids such as glycine, N-methylglycine, 3 - and 4-aminobutyric acid, 3-aminoadamantane acid, 5-aminosalicilova acid, 6-aminocaproic acid, 7 - aminoheptanoic acid, m - and p-aminobenzoic acid; aminophosphonate acid, such as m-aminobenzophenone acid and p-aminomethylphosphonic acid; and aminoalkylsilane predecessors, such as aniline-sulphatoethylsulphonyl and aniline-sulphatoethylsulphonyl.

The reaction of halogenated heterocyclic compounds of General formula (II) with compounds of General formula (III), (IV) and (V) or (VI) can be carried out is not miscible with water, an organic solvent; or miscible with water, an organic solvent, or a mixture of water and miscible with water organicheskogo or chlorobenzene; examples of suitable miscible with water and organic solvents are acetone, methyl ethyl ketone or dioxane. The first reaction of halogenated heterocyclic compounds can be carried out at temperatures from 0oC to 25oC, and ideally, from 0oC to 5oC; the second reaction can be carried out at temperatures from 20oC to 50oC, and ideally from the 30oC to 45oC; and the third reaction can be carried out at temperatures from 20oC to 100oC. In the course of these reactions produced inorganic acid,

such as, hydrochloric acid or hydrofluoric acid, neutralized using acid binding agent, such as sodium hydroxide, sodium carbonate, sodium bicarbonate, calcium hydroxide or calcium carbonate.

In addition, compounds of General formula (VII) can interact with reactive curable monomer with the formation of the polymerized compounds of General formula (VIII):

< / BR>
where R1, R4, R5, R6, Q, L, T, V, X, Y, Z, m, n and p are as above; R9represents a hydrogen atom or an alkyl group containing 1-6 carbon atoms; R10represents the carbonyl is kerispatih monomers are akriloilkhlorida, methacryloylamido, allylbromide, allylamine or 3,4-epoxybutene. The polymerized compounds of General formula (VIII) can be polymerized optionally in the presence of other polymerized monomers with the formation of conjugates affinity ligand - matrix of General formula (I). Such methods of polymerization are well known to specialists.

The present invention also relates to the use of all these conjugates affinity ligand - matrix-carrier for separation, extraction, purification, quantification, identification and characterization of protein materials, such as immunoglobulins, insulin, factor VII, or human growth hormone, or their analogues, derivatives and fragments, and precursors.

Immunoglobulins is a family of proteins, often denoted Ig, which have the same structure. Immunoglobulins, in General, is often referred to as antibodies, and any of these names can be used to describe this group of proteins. Immunoglobulins exist in various forms; for example, the most well-known types of antibodies are IgA, IgD, IgE, IgG, IgM and IgY and their various subclasses. The immunoglobulins may be present in physiological fluids, such cues. To obtain the desired properties of antibodies, they can be modified in various ways. These methods are well known in the art and allow you to obtain a modified antibody, which are also claimed in the claims of the present invention. As non-limiting examples of methods of modification of the antibodies can serve as methods of obtaining fragments of antibodies, labeled antibodies, conjugates of antibodies or antibody-containing hybrid proteins by chemical modification, by processing one or more enzymes, or by using a combination of the two methods. There are a large number of chemically modifying reagents and enzymes that can be used for modifying antibodies, and these compounds and their use are well known in the art. Another way of getting modified or new antibodies is their production by genetic engineering methods. These methods are well known in the art and can be used for producing, for example, fragments of antibodies or antibody - containing hybrid products. Modified or new antibodies obtained by genetic engineering methods, also stated in the formula of izopet the

< / BR>
where R1, R4, R5, R6, M, Q, n and p above, a j represents an integer from 2 to 20.

A particularly valuable group of conjugates of affinity ligand - matrix media presents General formula (X):

< / BR>
where j and M are as described above.

Usually, the reaction of compounds of General formula (XI):

< / BR>
3-propoxy-(1,2-epoxy)-agarose at a temperature of 10-30oC in the presence of an acid binding agent leads to the production of new conjugates affinity ligand - matrix, which have exceptional efficiency in the purification of protein materials. These new conjugates affinity ligand - matrix have a high affinity to proteins, a group of immunoglobulins. These unique properties of these conjugates make them extremely valuable in the separation, isolation, purification, quantification, identification and characterization of proteins of this class.

In another preferred embodiment, the present invention relates to novel affinity ligands of General formula (XII):

< / BR>
where R1, R4, R5, R6, Q, X, Y, Z, n and p are as above, and halogen (Halogen) represents an atom of fluorine, chlorine, bromine or iodine.oC to 121oC, optionally in the presence of an acid binding agent.

In another preferred embodiment, the present invention relates to novel affinity ligands of General formula (XIII):

< / BR>
where R1, R4, R5, R6, Q, X, Y, Z, m, n and p are as above; j represents an integer from 2 to 20.

In addition, the present invention relates to a method for producing the above-mentioned new affinity ligands of General formula (XIII) by reaction of compounds of the above General formula (XII) with alkylenediamines General formula H2N- (CH2)j- NH2at temperatures from 0oC to 100oC in the presence of an agent that binds acid.

In another embodiment, the present invention relates to novel affinity ligands of General formula (XIV):

< / BR>
where R1, R4, R5, Q, X, Y, Z, m, n and p are as described above; q = 0 or 1, a j represents an integer from 2 to 20.

In addition, the present invention relates to a method of obtaining new affinity ligands of the above General formulas 2N-(CH2)j- (CO)q-OH at temperatures from 0oC to 100oC, optionally in the presence of an agent that binds acid.

In another embodiment, the present invention relates to novel affinity ligands of the above General formula (VIII), where R1, R4, R5, R6, L, Q, T, V, X, Y, Z, m, n and p are as above; R9represents a hydrogen atom or an alkyl group containing 1-6 carbon atoms; R10represents a carbonyl group, a methylene group, a group - NH-CH2- or a group-S-CH2-, L preferably represents ethyl, boutelou or hexoloy group, T preferably represents a group-NH-, V preferably represents a group - NH-, a m preferably equals 1.

In another embodiment, the present invention relates to novel affinity ligands of General formula (XV):

< / BR>
where R1, R4, R5, R6, Q, n and p are as described above.

In another embodiment, the present invention relates to novel affinity ligands of General formula (XVI):

< / BR>
where R1, R4, R5, R6, Q, n and p are as described above, a j represents an integer from 2 to 20.

The II), (XIV), (VIII), (XV) and (XVI), where R1represents phenyl or naftalina group, each of which is optionally substituted on the benzene or naphthalene ring by one or more groups independently selected from hydroxyl or carboxyl groups.

In another preferred embodiment, the present invention relates to affinity ligands of General formula (XII), (XIII), (XIV), (VIII), (XV) and (XVI), where R4represents a hydrogen atom, hydroxyl group, carboxyl group or amino group.

In another preferred embodiment, the present invention relates to affinity ligands of General formula (XII), (XIII), (XIV), (VIII), (XV) and (XVI), where R5represents a hydrogen atom, hydroxyl group, carboxyl group or amino group.

In another preferred embodiment, the present invention relates to affinity ligands of General formula (XII), (XIII), (XIV), (VIII), (XV) and (XVI), where R6represents a hydrogen atom, hydroxyl group, carboxyl group or amino group.

In another preferred embodiment, the present invention relates to affinity ligands of General formula (XII), (XIII), (XIV), (VIII), (XV) and (XVI), where Q represents a benzene or naphthalene ring.

In another p), where X represents a nitrogen atom.

In another preferred embodiment, the present invention relates to affinity ligands of General formula (XII), (XIII), (XIV) and (VIII), where Y represents a group-NH-.

In another preferred embodiment, the present invention relates to affinity ligands of General formula (XII), (XIII), (XIV) and (VIII), where Z represents a group-NH-.

In another preferred embodiment, the present invention relates to affinity ligands of General formula (XII), (XIII), (XIV), (VIII), (XV) and (XVI), where n = 0 or 2.

In another preferred embodiment, the present invention relates to affinity ligands of General formula (XII), (XIII), (XIV), (VIII), (XV) and (XVI), where p = 0 or 2.

In another preferred embodiment, the present invention relates to affinity ligands of General formula (XIII), (XIV) and (XVI), where j = 2, 4 or 6.

In another preferred embodiment, the present invention relates to affinity ligands of the above General formula (XI), where j is an integer from 2 to 20.

Preferred affinity ligands of the present invention are:

< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
In addition, the present invention relates to Sposi or matrix of organic polymer by reaction of the specified carbohydrate matrix, or a matrix of an organic polymer with an activating agent and subsequent reaction of the activated matrix with the new affinity ligand, optionally in the presence of an agent that binds acid. The present invention also relates to a method for linking the new affinity ligands of General formula (XIV), as defined above, with a carbohydrate matrix, or matrix of organic polymer by the condensation reaction with the specified matrix. The present invention also relates to a method for linking the new affinity ligands of General formula (VII), (XIII), (XVI) and (XI) as defined above, with a matrix based on oxide of metal, glass or silicon dioxide, and optionally coated with an organic polymer, by reaction of optionally covered with a matrix based on oxide of metal, glass or silicon dioxide with an activating agent, and the subsequent reaction of the activated matrix with the new affinity ligand, optionally in the presence of an agent that binds acid. In another embodiment, the present invention relates to a method of linking the new affinity ligands of General formula (XIV), as defined above, with a matrix of metal oxide, glass or silicon dioxide, optionally coated with an organic polymer by condensation of these ligands with the specified matrix. In another embodiment, the present invention relates to a method of linking the new am new reaction affinity ligands to the matrix at a temperature of from -20oC to 121oC, optionally in the presence of an agent that binds acid. The present invention also applies to all conjugates of affinity ligand - matrix obtained by the above described methods.

In another embodiment, the present invention relates to the use of affinity ligands of the present invention and conjugates affinity ligand - matrix containing the ligands of the present invention, for the separation, isolation, purification, characterization, identification or quantitative evaluation of proteins or proteinaceous materials, such as immunoglobulins or their subclasses, fragments, precursors or derivatives, regardless of whether they occur from natural or recombinant sources, for example, immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA) or subclass, fragments, precursors or derivatives originating from natural and recombinant sources. In another embodiment, the present invention relates to the separation, isolation, purification, characterization, identification or quantification of immunoglobulins in any way in which these immunoglobulins put on conjugates affinity ligand - matrix of this izaberete the invention also relates to a method for separation or purification of protein materials providing for affinity chromatography using as a biospecific ligand of General formula (a), ligand described above.

In another embodiment, the present invention relates to the use of affinity ligands of the present invention and conjugates affinity ligand - matrix containing the ligands of the present invention, for the separation, isolation, purification, characterization, identification or quantitative evaluation of insulin, their analogs, derivatives, fragments, and precursors originating from natural and recombinant sources, as well as factor VII or human growth hormone or analogues, derivatives, fragments, and precursors.

For example, was designed library, containing more than 60 different ligands. This library was skanirovali using partially purified precursor of insulin, and selected ligands were immobilized on agarose by solid-phase synthesis. Using appropriate hardware binding matrix optimized length and nature of the spacer elements of the group, ligand density, and so on , resulting in a received 3 prototype that differ in their dynamic amotivational yeast fermentation broth by holding one stage, simulating affinity purification under very mild conditions.

Thus, a particularly preferred use of affinity ligands of the present invention and conjugates affinity ligand - matrix containing the affinity ligands of the present invention is the purification of insulin, their analogs, derivatives, fragments, and precursors originating from natural and recombinant sources. Particularly preferred conjugates - affinity ligand - matrix for such use contain a ligand selected from the following ligands:

< / BR>
< / BR>
< / BR>
where the ligand is associated with the matrix carrier in position And, as described above, optionally via spacer elements of the leg, located between the matrix and ligand represented by the General formula (b), defined above. The preferred ligand is 11a, and the preferred matrix carrier is not necessarily activated agarose, cellulose, silica or glass.

In another embodiment, the present invention relates to the separation, isolation, purification, characterization, identification or quantification of insulin or insulin analogues, derivatives thereof, and any predecessors sposoby ligand - the matrix of the present invention at a pH in the range from 4.0 to 9.0, and then removed, elute or desorbed by reducing the pH to 3.99 or lower or increase to 9.01 or higher. The elution procedure may be, for example, the alternative implemented by lowering the ionic strength or by adding co-solvents such as organic solvents.

In more detail, the present invention is described in the following examples. These examples are only for illustrative purposes and should not be construed as a limitation of the present invention.

Example 1

This example illustrates the synthesis of a typical affinity ligand, carried out by the reaction of halogenated heterocyclic compounds of General formula (II) with compounds of General formula (III) and (IV).

19,8 parts of aniline was dissolved in 50 g of acetone, and this solution was introduced into the suspension, obtained by pouring the solution to 36.8 hours the acid chloride cyanuric acid in 200 hours of acetone in a mixture containing 50 g of ice and 50 hours of water. The mixture was stirred for 2 hours, during which the pH was maintained between 6 and 7 by adding a solution containing 16,8 including sodium bicarbonate and 300 hours of water. After this time, the precipitated 2-aniline-4,6-dichloro-1P> The solution is 2.74 hours tiramina in a mixture of 50 hours of acetone and 10 am, water was added to the solution 4,82 including 2-aniline-4,6-dichloro-1,3,5-triazine in 100 hours of acetone. The resulting mixture was heated to a temperature of 50oC and kept at this temperature for 2 h, while maintaining the pH from 6 to 7 by adding a solution 1,68 including sodium bicarbonate in 30 hours of water. After this time, the precipitated 2-aniline - 4-[ (4'-hydroxyphenyl)-ethylamino]-6-chloro-1,3,5 - triazine was filtered, washed with water and dried in vacuum.

Example 2

This example illustrates the method of immobilization product of example 1 in the solid-phase carrier.

10 o'clock agarose bearing amino groups, transferred to acetone solvent by solvent exchange using 100 PM 30% aqueous solution of acetone, then 100 hours 70% aqueous acetone solution, and then 100 hours of acetone. 1 h 2-aniline-4- [ (4'-hydroxyphenyl)-ethyl - amino]-6-chloro-1,3,5-triazine was dissolved in 20 hours of acetone and heated to a temperature of 50oC. This warm acetone solution was added to acetone suspensions derived agarose carrying amino groups. To the resulting suspension was added a solution of 0.42 g sodium bicarbonate in 5 o'clock water, and the mixture was stirred for 16 h at 50oC. after this time is on acetone. Then derivationally affine media transferred back into the water by rinsing first with 70% aqueous acetone solution, followed by 30% aqueous solution of acetone and finally with water.

Example 3

This example illustrates the use of the new affine matrix described in example 2, as a specific chromatographic matrices for immunoglobulins.

1,25 including human plasma was diluted to 3.75 hours aqueous phosphate buffer of pH 7 and a concentration of 10 millimoles per liter, and then was applied to a column containing 1 o'clock affine matrix, obtained as described in example 2. Then affinity column was washed with phosphate buffer up until the UV-monitoring of drilling fluids did not specify that all unbound protein was removed. Then the affine matrix were washed in citrate buffer having a pH of 3.8 and a concentration of 0.2 millimoles per liter, up until UV monitoring washing solution did not specify that the removal of the bound protein was finished. It was found that the protein contained in the washing solution, is immunoglobulin G.

Table 1 also presents examples of new affinity ligands of the present invention, which can byto in example 1, by the corresponding number of halogenated heterocyclic compounds presented in column II of that table; and, with the replacement of 19.8 parts of aniline used in example 1, an appropriate amount of amine, are presented in column III of the table; and replacing 2,74 including tiramina used in example 1, an appropriate amount of amine, are presented in column IV of this table. Non of the examples listed in column I of this table.

These ligands are described in the examples 4-38, can be attached to an agarose matrix method, described in detail in example 2, and used for the purification of immunoglobulin G by the method described in example 3.

Example 39

6 hours of Ethylenediamine was added to a solution of 2.5 hours 2-aniline-4-[ (4'-hydroxyphenyl)-ethylamino] -6-chloro-1,3,5-triazine in 20 hours of toluene, and the mixture was heated at 100oC for 16 hours and Then the toluene is evaporated from the mixture under reduced pressure, and the residue is washed 5 times with 100 hours of water, and then dried at 70oC.

Table 2 also presents examples of new affinity ligands of the present invention, which can be obtained by the method described in example 39 but replacing 2,5 hours 2-aniline-4- [ (4'-hydroxyphenyl)-ethylamino]- 6-CHL is the PMC II of table 2, and with the replacement of ethylene diamine used in example 39, the corresponding amount of diamine or aminoketone connection presented in Column III of table 2.

Example 58

This example illustrates the method of immobilization of the compound obtained in example 39, the solid-phase carrier.

60 hours of agarose, bearing an epoxy group, washed with 300 hours of water, and then 300 g solution consisting of 5,95 tsp bicarbonate of potassium, dissolved in 180 hours of methanol and 120 h of water. 1 h compounds obtained as described in example 39 was dissolved in 60 g of methanol and added to a solution of 40 hours of an aqueous solution of potassium bicarbonate. Then 100 hours of this aqueous solution of methanol was added to the resulting suspension of 60 hours agarose carrying epoxy groups, and the resulting suspension was gently stirred at room temperature for 16 hours, the resulting matrix on the basis of agarose was washed first with a mixture of 360 hours of methanol and 240 hours of water, and then 600 hours of water.

Example 59

Repeating the procedure of example 3, except that the column containing the 1 o'clock affine matrix, which was obtained as described in example 2 was replaced with a column containing 1 o'clock affine matrix, obtained as described in example 58. Protein m example illustrates the immobilization product, and obtained in example 39, the solid-phase carrier.

80 hours agarose then washed 1050 including water, 1050 hours 30% aqueous solution of acetone, 1050 hours 70% aqueous acetone and 1425 hours of acetone. 80 hours of agarose and then suspended in 100 hours of acetone, and the resulting suspension was heated to 37oC. To agarose suspension was added 15 p.m para-toluensulfonate dissolved in 15 hours of pyridine and 25 hours of acetone, and the resulting mixture was stirred for 8 h at 37oC. Then the activated agarose was washed 225 hours of acetone, 225 hours 70% aqueous acetone, then 225 hours 30% aqueous solution of acetone, and finally, 225 including water.

45 o'clock this activated matrix was washed 225 hours of a solution containing 2,3 tsp bicarbonate of potassium, dissolved in a mixture of 178 hours of methanol and 47 hours of water. To activated agarose suspension was added a solution of 0.75 h compound obtained as described in example 39, in a mixture of 45 hours of methanol and 30 hours of water containing 1.5 g potassium bicarbonate, and then the mixture was left for 16 h at room temperature. The obtained affine matrix was washed 270 hours of methanol and 180 hours of water containing 9 g potassium bicarbonate, and then 450 hours of water.

Before using, 115 including the obtained affine matrix suspender 3, but with the replacement of the column containing the 1 o'clock affine matrix, receipt of which is described in example 2, the column containing the 1 o'clock affine matrix, receipt of which is described in example 60, the protein contained in the wash solution at a pH of 3.8 was again identified as immunoglobulin G.

Example 62

10 o'clock affine matrix, obtained as described in example 60, was kept in phosphate buffer with pH 8.0, for 16 hours Repeating the procedure of example 3, except that there used matrix is replaced with an equivalent amount of this new affine matrix, and instead of human plasma used in example 3 used the cell-free supernatant from cell culture medium containing murine immunoglobulin. The protein contained in the wash solution with a pH of 3.8, was again identified as the murine immunoglobulin M

Table 3 also presents examples of new binding affinity of the ligands of the present invention, which can be obtained by the method described in example 58, but with substitution of a new affinity ligand used in example 58, by the corresponding number of new affinity ligand specified in column II of table 3, and with the replacement 39 PM agarose, activerow the ke III of table 3, and activated reagent specified in column IV of table 3.

Example 70

Repeated the procedure for obtaining the ligand described in example 1, except that 2,74 including tiramina was subjected to reaction with 4,84 including 2-phenoxy-4,6-dichloro-1,3,5 - triazine instead 4,82 including 2-aniline-4,6-dichloro-1,3,5-triazine with obtaining 2-phenoxy-4- [(4'-hydroxyphenyl)-ethylamino]-6-chloro - 1,3,5-triazine, which can be used instead of 2-aniline-4-[ (4'-hydroxyphenyl)-ethyl - amino]-6-chloro-1,3,5-triazine to obtain affine matrix method described in example 2. Similarly, immunoglobulin G may be a standard way isolated from human plasma by the method described in example 3.

Example 71

Instead 4,84 including 2-phenoxy-4,6-dichloro-1,3,5 - triazine used in example 70, were used 5,16 including 2-phenylthio-4,6-dichloro-1,3,5-triazine, the result of which was obtained 2-phenylthio-4-[ -(4'-hydroxyphenyl)-ethylamino]-6 - chloro - 1,3,5 - triazine, which can be similarly used for the purification of immunoglobulin G.

Example 72

This example illustrates an alternative method of producing conjugates affinity ligand - matrix, as described in example 2 and example 58.

10 am, agarose, carrier aminoethylamino-g is ment to 0.5 mol per liter. To this suspension was added 2.5 g solution containing 1 teaspoon of acid chloride cyanuric acid in 10 g acetone, and then the mixture was stirred for 1 h at a temperature of 0-5oC. the resulting agarose activated by dichlorotriazines, then washed with 50 hours 50% aqueous solution of acetone, 50 hours water, 50 hours 50% aqueous solution of acetone and 100 hours of water. 20 hours washed agarose activated by dichlorotriazines, was added to 20 hours of an aqueous solution containing 1 o'clock tiramina 200 hours of water, and the resulting suspension was stirred for 16 h at room temperature. After this time, the received derivational matrix was washed with 200 hours and water was added to 20 hours of an aqueous solution containing 0,55 parts of aniline dissolved in 200 hours of water. The resulting suspension was stirred for 16 h at 90oC, then, derivational matrix was washed with 200 hours of water.

Example 73

By repeating the procedure of example 3, but replacing the column containing the 1 o'clock affine matrix, receipt of which is described in example 2, the column containing the 1 o'clock affine matrix, receipt of which is described in example 72, the protein contained in the wash solution with a pH of 3.8 was again identified as immunoglobulin G.

Example 74

tion, which can then be skanirovaniya to determine their protein-binding properties.

1 tsp agarose bearing amino groups, was mixed with 1 tsp 1 M phosphate potassium buffer, pH 7.0, and were left to settle under gravity. Buffered Amin the agarose was transferred to a reaction vessel and mixed at 0-5oC with 0.5 hours 0.5 M phosphate potassium buffer, pH 7.0, and 0.5 hours of acetone. Then added 1/4 tsp solution containing 1 teaspoon of acid chloride cyanuric acid in 10 g acetone, and the mixture was stirred for 1 h at 0-5oC, after which the mixture was filtered and then washed 10 am, 50% acetone, 6 o'clock water, 6 hours 50% acetone and 10 o'clock water, resulting in a received agarose activated 2,4-dichloro-sym-triazine-6-yl.

1 tsp agarose activated 2,4-dichloro-sym-triazine-6-yl, were added to 5 molar equivalents of amine compounds, presented in column II of table 4 and dissolved in 2-3 hours of a solution containing 1 teaspoon of acetone and 1 tsp of water, and then brought to neutral pH by adding sodium hydroxide. The suspension was stirred for 24 h at 30oC. the resulting agarose activated monochloro-triazine-6 - yl, then washed 10 hours of dimethylformamide, 10 o'clock solution containing ti.

1 hour monochlor-sym-triazine-6-yl-activated agarose was added to 5 molar equivalents of amine compounds presented in column III of table 4 and dissolved in 2-3 hours of a solution containing 1 teaspoon of dimethylformamide and 1 tsp of water, and then brought to neutral pH by adding sodium hydroxide. The suspension is stirred for 72 h at 85-95oC. the resulting conjugates affinity ligand - matrix is then washed 10 hours of dimethylformamide, 10 o'clock of a solution containing 3 h propan-2-ol and 7 o'clock 0.2 M sodium hydroxide, and 10 o'clock water, and then left for sedimentation under gravity. Synthesized a library of conjugates of affinity ligand - matrix of the present invention. The examples listed in column I of table 4.

Example 128

This example illustrates a method of screening, which can be identified protein-binding properties of conjugates of affinity ligand - matrix described in example 74.

The chromatographic column with 1 ml of the full volume was Packed by conjugates of affinity ligand - matrix examples 75-127. These speakers were balanced by intensive washing with 10 ml of 50 mm phosphate-sodium chloride buffer, pH 8.0. For each chromatographic column neprobivali 10 ml of 50 mm phosphate - sodium buffer, pH 8.0, to remove unbound immunoglobulin G. the Bound IgG was suirable by intensive wash each column with 5 ml of 50 mm citrate-sodium buffer, pH 3.0. The IgG content in the leaching and elution fractions was determined by measuring optical density at 280 nm against a buffer control. Analysis of the obtained results

revealed that all the conjugates of affinity ligand - matrix examples 75-127 been associated with human IgG. It conjugates affinity ligand - matrix examples 92, 99, 101, 102, 103, 111, 113 and 119 has been achieved almost quantitative elution of the bound IgG, as a result, we can assume that these conjugates have exceptional efficiency in separation, selection or purification of human IgG.

Example 129

Selective binding and elution of recombinant coagulation factor VIIa (rFVIIa) is plotted on the affine matrix of example 181, from the cell culture medium.

Procedure:

0,85 ml settled volume affine matrix, obtained as described in example 181, Packed in h mm column (Pharmacia HR 5/5) and balanced 20 ml buffer A: 20 mm Tris, 50 mm NaCl, 5 mm CaCl2, pH 8.0. 5 ml of the supernatant, air-conditioned cell culture KSS enriched the rFVIIa was suirable using 5 ml of buffer A: 20 mm Trinitrate sodium, 50 mm Tris, pH 8.0.

The flow rate during chromatography was 0.3 ml/min

The stream flowing from the column was passed through the device with a UV monitor and collected in 1 ml fractions, and each fraction was analyzed for content of rFVIIa and full of protein by analytical reversed-phase vysokorazryeshayuscyei liquid chromatography (RP-URGH).

The results:

Using a UV monitor revealed that the majority of UV (280 nm) is absorbed material was during application of the supernatant fraction and subsequent washing with buffer A. In subsequent elution buffer In, there was a marked peak, which corresponded to the expected size of the deposited amount of rFVIIa.

OF VRGH analysis of the collected fractions showed that 90% of the applied amount of rFVIIa was in fractions during elution buffer C. the Purity of rFVIIa in these fractions was higher than 95%.

The results showed that in this case is achieved by selective binding of rFVIIa enriched culture media used ligand.

Example 130

Cleaning In-circuit1-29insulin a-chain1-21Well-To - insulin on the conjugate affinity ligand - matrix of example 171

255 mg is abilitati predecessor was added 10 drops of 1 M acetic acid. Then added 0.2 M potassium citrate, pH 5.5 to full volume 510 ml with obtaining a solution of 0.12 mg/ml (PF-URGH analysis). The measured pH was $ 5.53, ionic strength was about 30.0 MS/cm, and the redox potential was 273 mV.

400 ml of the above solution was applied on the column, Pharmacia K (1.6 x 6 cm), Packed 12 ml of the above conjugate matrix and equilibrated in 0.2 M potassium citrate, pH 5.5, at 1.8 ml/min at ambient temperature. This column was washed with 50 ml of 0.2 M potassium citrate, pH 5.5, and then suirable 0.1 M acetic acid at 1.8 ml/min Were collected 12 fractions of 5.0 ml

The column was cleaned with 50 ml of 0.5 M NaOH and regenerates 50 ml of 0.1 M citric acid, 60% (vol./about.) the ethanol.

Samples for OFF-URGH analysis were diluted with 2 M acetic acid immediately prior to analysis (see tab. 5).

Thus, the total yield was 83%.

The purity of the product was determined with the help OF VRGH analysis and amounted to 94%. The remaining impurities were also insulin.

Example 131

Cleaning des-ThrB30-insulin on the conjugate affinity ligand - matrix of example 171

150 mg of sediment des-ThrB30-insulin (INS-J-009) suspended in 50 ml of H2O. For the dissolution of SSP is the acidity 0,076 mg/ml (PF-URGH analysis). The measured pH was 5,52, ionic strength was 30 MS/cm, and the redox potential was 264 mV. 400 ml of the above solution was applied on the column, Pharmacia K (1.6 x 6 cm), Packed 12 ml of the above conjugate matrix and equilibrated in 0.2 M potassium citrate, pH 5.5, at 1.8 ml/min at ambient temperature. This column was washed with 50 ml of 0.2 M potassium citrate, pH 5.5, and then suirable 0.1 M acetic acid at 1.8 ml/min Were collected in 10 fractions of 5.0 ml

The column was cleaned with 50 ml of 0.5 M NaOH and regenerates 50 ml of 0.1 M citric acid, 60% (vol./about.) the ethanol.

Samples for OFF-URGH analysis were diluted with 2 M acetic acid immediately prior to analysis (see tab. 6).

Thus, the total yield was 71%.

The purity of the product determined with the help OF VRGH-analysis was 91%, and the remaining impurities were insulin.

Example 132

Cleaning In-circuit1-29insulin a-chain1-21Well-To - insulin on the conjugate affinity ligand - matrix of example 145

Two liters of broth was centrifuged (party 628) was brought to pH 5.5 by adding 5 M NaOH, filtered through filter Leitz Tiefenfilter (Seitz EK), and then filtered through filter Leitz Tiefenfilter (Seitz cm, and the redox potential was 316 mV.

1000 ml of the above solution was applied on the column, Pharmacia K (1.6 x 6 cm), Packed 12 ml of the above conjugate matrix and equilibrated in 0.2 M potassium citrate, pH 5.5, at 1.8 ml/min at ambient temperature. The column was washed with 50 ml of 0.2 M potassium citrate, pH 5.5, and then suirable 0.1 M acetic acid at 1.8 ml/min Were collected 11 fractions of 5.0 ml

The column was cleaned with 50 ml of 0.5 M NaOH and regenerates 50 ml of 0.1 M citric acid, 60% (vol./about.) the ethanol.

Samples for OFF-URGH analysis were diluted with 2 M acetic acid immediately prior to analysis (see tab. 7).

Thus, the total yield was 63%.

The purity of the product determined with the help OF VRGH analysis, amounted to 88%. The remaining impurities were insulin.

Example 133

Cleaning In-circuit1-29insulin a-chain1-21Well-To - insulin on the conjugate affinity ligand - matrix of example 171

Two liters of broth was centrifuged (party 628) was brought to pH 5.5 by adding 5 M NaOH, filtered through filter Leitz Tiefenfilter (Seitz EK), and then filtered through filter Leitz Tiefenfilter (Seitz EKS). The concentration, measured by means OF URGH, sostavlyal 316 mV.

1000 ml of the above solution was applied on the column, Pharmacia K (1.6 x 6 cm), Packed 12 ml of the above conjugate matrix and equilibrated in 0.2 M potassium citrate, pH 5.5, at 1.8 ml/min at ambient temperature. The column was washed with 50 ml of 0.2 M potassium citrate, pH 5.5, and then suirable 0.1 M acetic acid at 1.8 ml/min Were collected 11 fractions of 5.0 ml

The column was cleaned with 50 ml of 0.5 M NaOH and regenerates 50 ml of 0.1 M citric acid, 60% (vol./about.) the ethanol.

Samples for OFF-URGH analysis were diluted with 2 M acetic acid immediately prior to analysis (see tab. 8).

Thus, the total yield was 74%.

The purity of the product determined with the help OF VRGH analysis, amounted to 86%. The remaining impurities were insulin.

Example 134

Purification of the b-chain (1-29) EEAEC-insulin a-chain (1 - 21) AAC-insulin on the conjugate affinity ligand - matrix of example 171

Centrifuged yeast broth (party Y44) was filtered through a filter Letz Tiefenfilter (Seitz EK) with a concentration of 0.35 mg/ml (PF-URGH analysis). The measured pH was 5,27, ionic strength was 7,38 MS/cm, and the redox potential was 221 mV.

120 ml of the above solution was applied E. potassium, pH 5.5, at 1.8 ml/min at ambient temperature. The column was washed with 50 ml of 0.2 M potassium citrate, pH 5.5, and then suirable 0.1 M acetic acid at 1.8 ml/min Were collected 11 fractions of 5.0 ml

The column was cleaned with 50 ml of 0.5 M NaOH and regenerates 50 ml of 0.1 M citric acid, 60% (vol./about.) the ethanol.

Samples for OFF-URGH analysis were diluted with 2 M acetic acid immediately prior to analysis (see tab. 9).

Thus, the total yield was 79%.

The purity of the product was determined with the help OF VRGH analysis and was 93%. The remaining impurities were also insulin.

Example 135

Cleaning In-circuit1-29[AspB28]-insulin a - chain1-21Well-To-insulin on the conjugate affinity ligand-matrix of example 171

Centrifuged broth (party GSG9414) was brought to pH 5.5 by adding 5 M NaOH, filtered through filter Leitz Tiefenfilter (Seitz EK), and then filtered through filter Leitz Tiefenfilter (Seitz EKS). The concentration, measured by means OF VRGH was 0.02 mg/ml Measured ionic strength was 17.0 MS/cm, and the redox potential was 308 MB.

800 ml of the above solution was applied on the column, Pharmacia K (1.6 x 6 cm), Packed 12 ml videocassete. This column was washed with 50 ml of 0.2 M potassium citrate, pH 5.5, and then suirable 0.1 M acetic acid at 1.8 ml/min Were collected 12 fractions of 5.0 ml

The column was cleaned with 50 ml of 0.5 M NaOH and regenerates 50 ml of 0.1 M citric acid, 60% (vol./about.) the ethanol.

Samples for OFF-URGH analysis were diluted with 2 M acetic acid immediately prior to analysis (see tab. 10).

Thus, the total yield was 75%.

The purity of the product determined with the help OF VRGH analysis was 84%, while the remaining impurities were insulin.

Example 136

In this example, furthermore, illustrated by way of screening, by means of which it can be identified protein-binding properties of conjugates of affinity ligand - matrix of the present invention.

Library of conjugates of affinity ligand - matrix of the present invention was synthesized as described in example 74, except that 5 molar equivalents of amine compounds, presented in column II of table 4, is replaced with the corresponding amount of amine compounds, presented in column II of table 5, and 5 molar equivalents of amine compounds presented in column III of table 4, replacement is avana library conjugates affinity ligand - the matrix of the present invention. Examples of which is specified in column I of table 5.

The chromatographic column with total volume of 1 ml was Packed by conjugates of affinity ligand - matrix examples 137-180. The column was balanced by intensive washing with 10 ml of 0.2 M sodium acetate, 0.1 M nitrilosides buffer, pH 5.0. 12 ml of the clarified culture fluid containing 50 mg/ml of the precursor of human insulin, were applied to each chromatographic column, which was then washed with 12 ml of 0.2 M sodium acetate, 0.1 M nitrilosides buffer, pH 5.0 to remove unbound material. Associated precursor of human insulin was suirable by intensive washing each column of 3 ml of 2 M acetic acid. The content of the precursor of human insulin through the stream, washing and elution fractions was determined by high-performance liquid chromatography (URGH) using a reversed-phase column with silica C18 (4 x 250 m) and solvents containing buffer A (0.2 M sodium sulfate, 40 mm phosphoric acid and 10% (vol./about.) acetonitrile, pH 2) and buffer (50% (vol./about.) acetonitrile) and filed a flow rate of 1 ml per minute, and in the ratio of 55% buffer a to 45% buffer C. BP is LASS="ptx2">

The analysis of the results showed that all the conjugates of affinity ligand - matrix examples 139, 140, 145, 148, 153, 159, 162, 163, 164, 166, 167, 170, 171, 173 associated with the precursor of human insulin, which was polyiron under the conditions described in this example. On this basis it should be noted that the conjugates of affinity ligand - matrix examples 139, 140, 145, 148, 153, 159, 162, 163, 164, 166, 167, 170, 171, 173 possess exceptional efficiency in the separation, the separation and purification of the precursor of human insulin (see table. 11).

Example 181

This example illustrates the method of synthesis of conjugates of affinity ligand - matrix of the present invention, which are effective for the purification of factor VII.

Conjugate affinity ligand - matrix of the present invention was synthesized as described in example 74, except that 5 molar equivalents of amine compounds, presented in column II of table 4, was replaced with a corresponding amount of 2 - aminobenzimidazole and 5 molar equivalents of amine compounds presented in column III of table 4, was replaced with 3-amino-2-naphthoic acid. This conjugate affinity ligand - matrix can be used for the purification of factor VIla example 129 this is and the conjugate affinity ligand - the matrix of example 171

50 ml of ion-exchange purified precursor of insulin (In - circuit1-29-EEAEC-insulin-And-chain1-21Well-To-insulin at 2.2 mg/ml) was applied on the column, Pharmacia K (1.6 x 6 cm), Packed 12 ml conjugate matrix and equilibrated in 0.2 M potassium citrate, pH 5.5, at 1.8 ml/min at room temperature. This column was washed with 50 ml of 0.1 M potassium citrate, 0.2 M potassium sulfate, pH 5.5, and then suirable 0.1 M acetic acid at 1.8 ml/min Were collected 5,0 ml-fractions.

The column was cleaned with 50 ml of 0.5 M NaOH and regenerates 50 ml of 0.1 M citric acid, 60% (vol./about.) the ethanol.

Samples for OFF-URGH analysis were diluted with 2 M acetic acid immediately prior to analysis (see tab. 12).

Was demonstrated dynamic binding capacity of 9.5 mg/ml matrix with the output of the predecessor 88%.

1. Conjugates affinity ligand - matrix containing the ligand of General formula (a)

< / BR>
where R1represents a hydrogen atom, an alkyl group containing 1-6 carbon atoms, hydroxyalkyl group containing 1-6 carbon atoms, tsiklogeksilnogo group, amino group, phenyl group, naftalina group, 1-phenylpyrazole group indazol group, benzothiazolyl group, benzoxazol the ash, benzthiazole, benzoxazole or benzimidazole ring optionally substituted by one or more substituents independently selected from the group comprising alkyl groups containing 1-6 carbon atoms; alkoxygroup containing 1-6 carbon atoms; acyloxy or alluminare containing 1-6 carbon atoms; amino groups, hydroxyl groups; carboxyl groups; sulfonylurea group; carbamoyl group; sulfamoyl group; alkylsulfonyl group containing 1-6 carbon atoms; or halogen atoms;

Y is an oxygen atom, a sulfur atom or a group N-R2;

Z is an oxygen atom, a sulfur atom or a group N-R3;

R2and R3each independently represents a hydrogen atom; an alkyl group containing 1-6 carbon atoms; hydroxyalkyl group containing 1-6 carbon atoms; benzyl group or phenylethylene group;

R4, R5and R6each independently represents a hydrogen atom; hydroxyl group; alkyl group containing 1-6 carbon atoms; alkoxygroup containing 1-6 carbon atoms, amino group; acyloxy or alluminare containing 1-6 carbon atoms; carboxyl group, sulfoxyl group, carbamoyl and Odin from X - the nitrogen atom and the other X is a nitrogen atom or a carbon atom bearing a chlorine atom or a cyano;

Q represents a benzene, naphthalene, 1-phenylpyrazole, indazol, benzthiazole, benzoxazole or benzimidazole ring;

n = 0 - 6, an integer;

p = 0 - 20, integer,

and where the ligand is associated with the matrix carrier in position And not necessarily by means of spacer elements located between the matrix and ligand, provided that formula (a) does not include the reaction products of compounds 4-chloro-2,6-di(phenylamino)-1,3,5-triazine-3'-sulfonic acids, 4-chloro-2,6-di(phenylamino)-1,3,5-triazine-3', 2"-disulfonate, and 4-chloro-2-(4"-aminophenylamino)-1,3,5-triazine-3',2"-disulfonate with Dextran T 500.

2. Conjugates affinity ligand - matrix p. 1, where the optional spacer elements group, located between the ligand and the matrix represented by the General formula (b)

-T-[-L-V-]m-,

where T is an oxygen atom, a sulfur atom or a group N-R7where R7is a hydrogen atom or an alkyl group containing 1-6 carbon atoms;

V represents an oxygen atom, a sulfur atom, a group-COO-group, CONH, or NHCO group, or group-RHO3N-, a group NH-aralen-SO2-CH2-CH2or a group N-R8where R8- at the Yu hydrocarbon bond, containing 2-20 carbon atoms;

m = 0 or 1.

3. Conjugates affinity ligand - matrix, which are represented by the General formula (1)

< / BR>
where R1, Y, Z, R2, R3R4, R5, R6, X, Q, n, and p are defined above;

T is an oxygen atom, a sulfur atom or a group N-R7;

V is an oxygen atom, a sulfur atom, a group-COO-group, CONH, or NHCO group or group-RHO3N-, a group NH-aralen-SO2-CH2-CH2or a group N-R8;

R7and R8each independently a hydrogen atom or an alkyl group containing 1-6 carbon atoms;

L represents an optionally substituted hydrocarbon linkage containing 2 to 20 carbon atoms;

m = 0 or 1;

M - the remainder of the matrix carrier.

4. Conjugates affinity ligand - matrix according to any one of the preceding paragraphs, where M is the residue matrix-carrier, which may be any compound or material contained in or out in the form of particles, soluble or insoluble, porous or non-porous, which can be used in combination with affinity ligands for the formation of new conjugates affinity ligand - matrix according to any one of the preceding paragraphs and which provides a convenient way of separating affine according to any one of the preceding paragraphs, where R1represents phenyl or naftalina group, each of which is optionally substituted on the benzene or naphthalene ring by one or more groups independently selected from hydroxyl and carboxyl groups.

6. Conjugates affinity ligand - matrix according to any one of the preceding paragraphs, where R2- the hydrogen atom.

7. Conjugates affinity ligand - matrix according to any one of the preceding paragraphs, where R3- the hydrogen atom.

8. Conjugates affinity ligand - matrix according to any one of the preceding paragraphs, where R4is a hydrogen atom, hydroxyl group, carboxyl group or amino group.

9. Conjugates affinity ligand - matrix according to any one of the preceding paragraphs, where R5represents a hydrogen atom, hydroxyl group, carboxyl group or amino group.

10. Conjugates affinity ligand - matrix according to any one of the preceding paragraphs, where R6is a hydrogen atom, hydroxyl group, carboxyl group or amino group.

11. Conjugates affinity ligand - matrix according to any one of the preceding paragraphs, where R7- the hydrogen atom.

12. Conjugates affinity ligand - matrix according to any one of the preceding paragraphs, where T is an oxygen atom where R2defined above.

14. Conjugates affinity ligand - matrix according to any one of the preceding paragraphs, where Z Is N-R3where R3defined above.

15. Conjugates affinity ligand - matrix according to any one of the preceding paragraphs, where each X is a nitrogen atom.

16. Conjugates affinity ligand - matrix according to any one of the preceding paragraphs, where Q is a benzene or naphthalene ring.

17. Conjugates affinity ligand - matrix according to any one of the preceding paragraphs, where n = 0 or 2.

18. Conjugates affinity ligand - matrix according to any one of the preceding paragraphs, where p = 0 or 2.

19. Conjugates affinity ligand - matrix according to any one of the preceding paragraphs, where m = 0 or 1.

20. Conjugates affinity ligand - matrix according to any one of the preceding paragraphs, where L is ethyl, through hydroxypropyl, boutelou, pentelow, hexeline, octillo or decile group, and V and m are defined above.

21. Conjugates affinity ligand - matrix according to any one of the preceding paragraphs, where V is an oxygen atom or the NH group, a L and m is defined above.

22. Conjugates affinity ligand - matrix according to any one of the preceding paragraphs, where m = 1, a L and V defined above.

23. Conjugates affinity ligand consistent with the agarose, silicon dioxide, cellulose, glass, small, pearl, hydroxyethylmethacrylate, polyacrylamide, stradivariuses, Hyper D, fluorosurfactants.

24. Conjugates affinity ligand - matrix according to any one of the preceding paragraphs, where M represents the agarose, optional activated trasylol, sulphonylchloride, Totila, vinylsulfonate, or epoxy.

25. Conjugates affinity ligand - matrix according to any one of the preceding paragraphs, selected from

i)

< / BR>
ii)

< / BR>
iii)

< / BR>
iv)

< / BR>
v)

< / BR>
vi)

< / BR>
vii)

< / BR>
viii)

< / BR>
where M is defined above.

26. Way to generate new conjugates affinity ligand - matrix according to any one of the preceding paragraphs, including interaction in any order halogenated geterotsiklicheskikh compounds of General formula (II)

< / BR>
where X have the meanings indicated above;

W is a halogen atom,

(i) with a compound of General formula (III)

R1-(CH2)p-Y-H

where the symbols R1, p, and Y are defined above

(ii) with a compound of General formula (IV)

< / BR>
where R4, R5, R6, Q, Z and n are defined above and

(iii) or with optional derivateservlet matrix-carrier of the General formula (V)

H-T-hand - the matrix according to any one of the preceding paragraphs, including interaction in any order halogenated heterocyclic compounds of General formula (II) p. 26: (i) with a compound of General formula (III) under item 26, (ii) with a compound of General formula (IV) p. 26 and (iii) with the link of General formula (VI)

H-T-L-V-H,

where L, V and T are defined above,

obtaining compounds of General formula (VII)

< / BR>
where R1, R4, R5, R6, T, Q, L, V, X, Y, Z, m, n and p are defined above,

and subsequent reaction of compounds of General formula (VII) matrix carrier.

28. New affinity ligands of General formula (XII)

< / BR>
where R1, R4, R5, R6Q, X, Y, Z, n and p above;

the halogen atom is fluorine, chlorine, bromine or iodine, provided that formula (XII) does not include such compounds as 4-chloro-2,6-di(phenylamino)-1,3,5-triazine-3'-acid, 4-chloro-2,6-di(phenylamino)-1,3,5-triazine-3', 2"-disulfonate and 4-chloro-2-(4"-aminophenylamino)-1,3,5-triazine-3', 2"-disulfonate.

29. The method of linking the new affinity ligands of General formula (XII) by p. 28 matrix new formula (V) p. 26 interaction of new affinity ligands to the matrix at temperatures from -20C to 121C optionally in the presence of the agent, with the , R5, R6, Q, X, Y, Z, m, n and p above,

j = 2 to 20, an integer.

31. The method of obtaining the above-mentioned new affinity ligands of General formula (XIII) by p. 30 by reacting compounds of General formula (XII) by p. 28 with alkylenediamines General formula H2N-(CH2)j-NH2at 0 - 100C in the presence of an agent that binds acid.

32. New affinity ligands of General formula (XIV)

< / BR>
where R1, R4, R5, Q, X, Y, Z, m, n and p above;

q = 0 or 1;

j = 2 to 20, an integer.

33. A method of obtaining a new affinity ligands of General formula (XIV) p. 32 by reacting compounds of General formula (XII) by p. 28 with aminohydroxylation General formula H2N-(CH2)j-(CO)qHE2at 0 - 100C, optionally in the presence of an agent that binds acid.

34. New affinity ligands of General formula (VIII)

< / BR>
where R1, R4, R5, R6, L, Q, T, V, X, Y, Z, m, n and p are defined above;

R9represents a hydrogen atom or an alkyl group containing 1-6 carbon atoms;

R10carbonyl group, a methylene group, a group-NH-CH2- or a group-S-CH2-.

35. New affinity ligands of General formula (XV)
includes compounds 4-chloro-2,6-di(phenyl-amino)-1,3,5-triazine-3'-sulfonic acids, 4-chloro-2,6-di(phenyl-amino)-1,3,5-triazine-3', 2"-disulfonate, and 4-chloro-2-(4"-aminophenylamino)-1,3,5-triazine-3',2"-disulfonate.

36. New affinity ligands of General formula (XVI)

< / BR>
where R1, R4, R5, R6, Q, n and p above;

j = 2 to 20, an integer.

37. Affinity ligands according to any one of paragraphs.28, 30, 32, 34, 35 or 36, where R1represents phenyl or naftalina group, each of which is optionally substituted on the benzene or naphthalene ring by one or more groups independently chosen from hydroxyl groups and carboxyl groups.

38. Affinity ligands according to any one of paragraphs.28, 30, 32, 34, 35 or 36, where R4is a hydrogen atom, hydroxyl group, carboxyl group or amino group.

39. Affinity ligands according to any one of paragraphs.28, 30, 32, 34, 35 or 36, where R5is a hydrogen atom, hydroxyl, carboxyl or amino group.

40. Affinity ligands according to any one of paragraphs.28, 30, 32, 34, 35 or 36, where R6is a hydrogen atom, hydroxyl, carboxyl or amino group.

41. Affinity ligands according to any one of paragraphs.28, 30, 32, 34, 35 or 36 where Q is a benzene or naphthalene ring.

42. Affinity ligands according to any one of paragraphs.28, 30, 32 or 34, where X is the nitrogen atom.

43. A28, 30, 32, 34, or 35, where Z is the group-NH-.

45. Affinity ligands according to any one of paragraphs.28, 30, 32, 34, 35 or 36, where n = 0 or 2.

46. Affinity ligands according to any one of paragraphs.28, 30, 32, 34, 35 or 36, where p = 0 or 2.

47. Affinity ligands according to any one of paragraphs.30, 32 or 36, where j = 2, 4 or 6.

48. Affine ligands on p. 34, where L is ethyl, boutelou, or hydroxyl group.

49. Affine ligands on p. 34, where T is the group-NH-.

50. Affine ligands on p. 34, where V is the group-NH-.

51. Affine ligands on p. 34, where m = 1.

52. New affinity ligands of General formula (XI)

< / BR>
where j = 2 to 20, an integer.

53. Affinity ligands according to any one of paragraphs.28-51 selected from the group including

< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
54. Affinity ligands according to any one of paragraphs.28, 30, 32, 34-53, applicable to obtain conjugates affinity ligand - matrix.

55. The method of linking the new affinity ligands of General formula (VII) p. 27, of General formula (XIII) under item 30, the General formula (XVI) according to p. 36, and the General formula (XI) by p. 52 carbohydrate matrix, or matrix of organic polymer by reacting a specified carbohydrate matrix, or matrix of organic polymer trigger and the Ghent, linking acid.

56. The method of linking the new affinity ligands of General formula (XIV) p. 32 carbohydrate matrix, or matrix of organic polymer by condensation with the specified matrix.

57. The method of linking the new affinity ligands of General formula (VII) under item 21, the General formula (XIII) under item 30, the General formula (XVI) according to p. 36, and the General formula (XI) by p. 52 matrix of the metal oxide, glass or silicon dioxide, optionally with a coating of organic polymer, by reacting the matrix of the metal oxide, glass, or silicon dioxide having an optional coating, with an activating agent and subsequent reaction of this activated matrix with the new affinity ligand, optionally in the presence of an agent that binds acid.

58. The method of linking the new affinity ligands of General formula (XIV) p. 32 matrix of the metal oxide, glass or silicon dioxide, with an optional coating of organic polymer by condensation with the specified matrix.

59. The method of linking the new affinity ligands of General formula (XV) by p. 35, and the General formula (XII) by p. 28 matrix of General formula (V) p. 26 through the interaction of new affinity ligands to the matrix when tempenny ligand - the matrix obtained according to any one of paragraphs.26, 27, 29, 54-58.

61. Conjugates affinity ligand - matrix according to any one of paragraphs.1-25 applicable for affinity chromatography in order to separate, separation, purification, identification or quantification of protein materials.

62. Conjugates affinity ligand - matrix p. 61, wherein the protein materials are IgG, IgM, IgA, insulin, factor VII or human growth hormone, their analogues, derivatives and fragments, and precursors.

63. The method of carrying out affinity chromatography for the separation or purification of the protein material, characterized in that as a biospecific ligand using conjugate under item 1, corresponding to the formula (a).

64. Conjugates affinity ligand - matrix p. 61, wherein the protein materials are immunoglobulins or their subclasses, fragments, precursors or derivatives, regardless of whether they come from natural or recombinant sources.

65. Conjugates affinity ligand - matrix p. 61, wherein the protein materials are immunoglobulin G(IgG), immunoglobulin M (IgM), immunoglobulin A (IgA), or their subclasses, fragments, precursors or derivatives of jugate affinity ligand - the matrix p. 61, wherein the protein materials are insulin or insulin analogs, derivatives, fragments, and precursors regardless of whether they come from natural or recombinant sources.

67. Conjugates affinity ligand - matrix p. 61, wherein the protein materials are FVII or its analogs, derivatives, fragments and precursors, regardless of whether they come from natural or recombinant sources.

68. Conjugates affinity ligand - matrix p. 61, characterized in that the ligand is selected from the following ligands:

< / BR>
< / BR>
< / BR>
where the ligand is attached to the matrix-carrier in position (A) through optional spacer elements of the group represented by General formula (b), defined above.

69. Conjugates affinity ligand - matrix p. 68, wherein the ligand is a ligand 11a.

70. Conjugates affinity ligand - matrix p. 68 or 69, wherein the matrix carrier is not necessarily activated agarose, cellulose, silica or glass.

71. The chromatography method of immunoglobulins, including their application to conjugates affinity ligand - Mat, Loiret or desorbed by reducing the pH to 4.9 or below.

72. The method according to p. 71, characterized in that during chromatography carry out the separation of immunoglobulins.

73. The method according to p. 71, characterized in that during chromatography carry out the selection of immunoglobulins.

74. The method according to p. 71, characterized in that during chromatography carry out the purification of immunoglobulins.

75. The method according to p. 71, characterized in that during chromatography carry out the identification of immunoglobulins.

76. The method according to p. 71, characterized in that during chromatography carry out a quantitative assessment of immunoglobulins.

77. Method chromatography of insulin, their analogues, derivatives and precursors, including their application to conjugates affinity ligand - matrix, characterized in that the printing performed on the conjugate according to any one of paragraphs. 1 and 3 at pH 4,0 - 4,9, and then removed, elute or desorbed by reducing the pH to 3.99 or lower or raise pH to 9.01 or higher.

78. The method according to p. 77, characterized in that during chromatography carry out the separation of insulin, their analogues, derivatives and precursors.

79. The method according to p. 77, characterized in that during chromatography suceuses fact, when chromatography carry out the purification of insulin, their analogues, derivatives and precursors.

81. The method according to p. 77, characterized in that during chromatography carry out the identification of insulin, their analogues, derivatives and precursors.

82. The method according to p. 77, characterized in that during chromatography carry out a quantitative assessment of insulin, their analogues, derivatives and precursors.

83. The method of isolation of recombinant FVIIa from within the cell by cultural selective binding of FVIIa with conjugate affinity ligand - matrix

< / BR>
where M is the rest of the agarose,

when FVIIa is applied to the specified conjugate affinity ligand - matrix in the presence of 5 mm CA2+and then elute.

 

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