GRAFT COPOLYMER FOR CATION-EXCHANGE CHROMATOGRAPHY

Abstract

The invention relates to chromatographic separating materials having improved binding capacity for biological constituents in cell culture supernatants, or animal or human body fluids, in particular for monoclonal antibodies. The present invention likewise relates to the preparation of separating materials of this type, and to the use thereof, in particular for the removal of charged biopolymers from corresponding liquids.

Claims

1-16. (canceled)

17. A separating material for ion exchange chromatography based on an organic, hydroxyl-containing polymer base support to the surface of which copolymers are covalently bonded, wherein a) the base support contains aliphatic hydroxyl groups, b) the covalently bonded copolymers are bonded to the support via a terminal monomer unit, c) the copolymers contain at least two different monomer units, d) the monomer units are linked in a linear manner, e) the copolymer contains at least one monomer unit which has a negative charge in the form of a sulfonic acid or carboxylic acid and additionally contains ester or amide groups and alkyl and/or alkylene groups and in total a maximum of 8 C atoms, but no aryl groups, or which carries a negative charge in the form of a sulfonic acid or carboxylic acid and in addition contains alkyl and/or alkylene groups, but no aryl groups, where the copolymer has at least one monomer unit having a negative charge, either of formula (1), ##STR00020## in which R.sup.1, R.sup.2 and Y each, independently of one another, denotes H or CH.sub.3, R.sup.3 denotes R.sup.4SO.sub.3M or R.sup.4COOM, R.sup.4 denotes a straight-chain or branched alkylene having 2 to 4 C atoms, and M denotes H, Na, K or NH.sub.4, or of formula (2), ##STR00021## in which R.sup.7 and R.sup.8 each, independently of one another, denotes H or CH.sub.3, or R.sup.7 denotes COOM if Z=M and R.sup.8H, denotes M, R.sup.4COOM or R.sup.4SO.sub.3M, R.sup.4 denotes straight-chain or branched alkylene having 2 to 4 C atoms, and M denotes H, Na, K or NH.sub.4, or at least n each case one monomer unit of formula (1) and of formula (2), and f) the copolymer contains at least one monomer unit which has, as hydrophobic group, a straight-chain or branched alkyl having 4 to 18 C atoms or corresponding aryl groups and contains amide groups, where the copolymer contains at least one monomer unit of formula (1) which has a hydrophobic group which imparts a hydrophobic character on the copolymer, ##STR00022## in which R.sup.1 denotes H or COOM, R.sup.2 denotes H or CH.sub.3, Y and R.sup.3 each, independently of one another, denotes straight-chain or branched alkyl having up to 18 C atoms, in which Y and R.sup.3 together carry at least 6 C atoms, or Y denotes H, and R.sup.3 denotes straight-chain or branched alkyl having 6 to 18 C atoms, or Y denotes H, and R.sup.3 denotes aryl or R.sup.6-aryl, or denotes H or CH.sub.3, and R.sup.3 denotes R.sup.4CONHX, X denotes straight-chain or branched alkyl having 6 to 18 C atoms, aryl or R.sup.6-aryl R.sup.4 denotes straight-chain or branched alkylene having 2 to 4 C atoms, R.sup.6 denotes a straight-chain or branched alkylene having 1 to 4 C atoms, in which a methylene group is optionally replaced by 0 and is optionally substituted by COOM, and M denotes H, Na, K or NH.sub.4, and g) the ratio of the monomer units having a negative charge to the monomer units containing a hydrophobic group is in a range of 99:1 to 10:90.

18. A separating material according to claim 17, wherein e) the copolymer comprises at least one monomer unit having a negative charge selected from the group consisting of 2-acrylamido-2-methylpropanesulfonic acid, 2-acrylamidoethanesulfonic acid, carboxymethylacrylamide, carboxyethylacrylamide, carboxypropylacrylamide, carboxymethylmethacrylamide, carboxyethlymethacrylamide, carboxypropylmethacrylamide, maleic acid, acrylic acid and methacrylic acid, and f) the copolymer comprises at least one monomer unit containing a hydrophobic group of formula (1) ##STR00023## in which R.sup.1 denotes H, R.sup.2 denotes H or CH.sub.3, Y denotes H, and R.sup.3 denotes aryl or R.sup.6-aryl, or Y denotes H or CH.sub.3, R.sup.3 denotes R.sup.4CONHX, X denotes aryl or R.sup.6-aryl, R.sup.4 denotes methylene, ethylene, propylene, R.sup.6 denotes a straight-chain or branched alkylene having 1 to 4 C atoms, in which a methylene group is optionally replaced by 0 and is optionally substituted by COOM, and M denotes H, Na, K or NH.sub.4.

19. A separating material according to claim 17, wherein e) the copolymer comprises at least one monomer unit having a negative charge selected from the group consisting of 2-acrylamido-2-methylpropanesulfonic acid, 2-acrylamidoethanesulfonic acid, carboxymethylacrylamide carboxyethylacrylamide, carboxypropylacrylamide, carboxymethylmethacrylamide, carboxyethlymethacrylamide, carboxypropylmethacrylamide, maleic acid, acrylic acid and methacrylic acid, and f) the copolymer comprises at least one monomer unit containing a hydrophobic group of formula (1) ##STR00024## in which R.sup.1 denotes H, R.sup.2 denotes H or CH.sub.3, Y denotes H, and R.sup.3 denotes phenyl, benzyl, phenylethyl or phenoxyethyl, or Y denotes H or CH.sub.3, R.sup.3 denotes R.sup.4CONHX, X denotes phenyl, benzyl, or phenylethyl, and R.sup.4 denotes methylene, ethylene, propylene, acryloylphenylglycine or acryloylphenylalanine.

20. A separating material according to claim 17, wherein a) the copolymer comprises 2-acrylamido-2-methylpropanesulfonic acid and/or 2-acrylamidoethanesulfonic acid as the monomer unit having a negative charge, and b) the ratio of the monomer units having a negative charge to the monomer units containing a hydrophobic group is in a range OF 70:30 to 30:70.

21. A separating material according to claim 17, wherein a) the copolymer comprises acrylic acid and/or methacrylic acid as the monomer unit having a negative charge, and b) the molar ratio of the monomer units having a negative charge to the monomer units containing a hydrophobic group is in a range between 95:5 to 70:30.

22. A separating material according to claim 17, wherein a) the copolymer comprises a monomer selected from the group consisting of 2-acrylamido-2-methylpropanesulfonic acid and 2-acrylamidoethanesulfonic acid as the monomer unit having a negative charge, and b) a monomer selected from the group consisting of acrylic acid and methacrylic acid, and c) the molar ratio of the monomer units having a negative charge to the monomer units containing a hydrophobic group is in a range between 95:5 to 30:70.

23. A separating material according to claim 17, wherein the monomer unit which carries a negative charge contains carboxylic acid groups and some of the carboxylic acid groups are converted to amide groups by coupling with an amine, such that the total amount of amine is in a molar ratio of 0.01 to 100:1 to the carboxyl groups in the monomer units and the amine is an alkyl-, aryl- or arylalkylamine having 6 to 18 C atoms selected from the group consisting of aniline, benzylamine, 4-fluorobenzylamine, 4-methoxybenzylamine, napthylmethylamine, phenacylamine, phenylethylamine, phenoxyethylamine, tryptamine and tyramine.

24. A separating material according to claim 17, wherein the copolymers are capable of interacting and bonding to proteins and antibodies.

25. A separating material according to claim 17, wherein the copolymers are capable of adsorbing biopolymers in dissolved form from a liquid having an electrolytic conductivity which is higher than 6 mS/cm and a pH greater than 4.

26. A separating material according to claim 17, wherein the copolymers are capable of adsorbing antibodies from an aqueous liquid having a pH of 5.5 and having an electrolytic conductivity which is higher than 6 mS/cm.

27. A separating material according to claim 17, wherein the copolymers are provided as flexible tentacles independently extending from the support surface.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0453] FIG. 1 shows diagrammatically two preparation variants,

[0454] FIG. 2 shows viscosity radius data, and

[0455] FIG. 3 shows a chromatogram.

TABLE-US-00002 TABLE 2 Coupling of amines to a graft copolymer comprising 2-acrylamido-2-methylpropanesulfonic acid and acrylic acid, static binding capacity (SB) of polyclonal human IgG (Gammanorm) at pH 6.5 and 75 or 150 mM sodium chloride per ml of gel sediment. Amine SB 75 mM NaCl SB 150 mM NaCl Batch Precursor Solvent mmol Amine(s) (ratio) mg of IgG/ml mg of IgG/ml SO3.sup.[1] None 6.4 0.3 COO.sup.[2] None 1.2 0.2 05PP131.sup.[3] None 1.5 0.3 05PP157 05PP131 Water 10 4-Methoxybenzylamine 16.6 Not determined 05PP143 05PP131 Water 60 Phenoxyethylamine 17.4 Not determined 05PP158 05PP131 Water 10 4-Fluorobenzylamine 17.4 Not determined 06PP069 06PP066 DMF/water 60 Octylamine 20.2 5.2 05PP152 05PP131 Water 5 Phenacylamine hydrochloride 23.4 Not determined 05PP151 05PP131 Water 30 Aniline 26.5 Not determined 06PP080 06PP066 DMF/water 60 Napthylmethylamine 27.9 11.4 06PP054 06PP066 Water 60 Benzylamine/tyramine (90:10) 28.1 12.5 05PP132 05PP131 Water 60 Benzylamine 30.2 Not determined 06PP051 06PP066 Water 60 Benzylamine/ethanolamine (90:10) 32.7 12.9 06PP119 06PP066 Water 60 Benzylamine 37.9 20.4 06PP118 06PP066 DMF/water 60 Tryptamine 42.3 25.4 06PP086 06PP066 Water 60 Phenylethylamine 45.5 18.1 .sup.[1]Commercially available Fractogel EMD SO.sub.3.sup. (M), .sup.[2]Commercially available Fractogel EMD COO.sup. (M), .sup.[3]Graft copolymer before coupling reaction with hydrophobic amine.

TABLE-US-00003 TABLE 3 Dynamic binding capacity (DB) of polyclonal human IgG (Gammanorm) at 150 mM sodium chloride and pH 5.5 or pH 6.5 per ml of packed gel and chemical composition of the graft polymers. DB.sup.[1] DB.sup.[2] DB pH 5.5 pH 5.5 Charged pH 6.5 (recovery) (recovery) SO3 Benzyl groups mg of mg of mg of groups groups (titration) Batch IgG/ml IgG/ml IgG/ml mol/g mol/g mol/g Fractogel EMD SO.sub.3.sup. (M) 1.1 0.4 (n.d.) n.d. Fractogel EMD COO.sup. (M) 0.5 0.6 (n.d.) n.d. 05SW136 1.4 28.0 (96%) n.d. 240 422 393 06SW297 0.9 42.9 (83%) 50.5 (96%) 122 2016 06SW085 n.d. 34.7 (93%) 36.9 (98%) 107 187 497 06PP262 1.2 68.1 (82%) 76.1 (93%) 521 467 1366.sup.[3] 06PP345 0.5 15.7 (85%) 22.7 (100%) 657 353 1480.sup.[3] 06PP346 1.3 61.2 (77%) 67.4 (89%) 582 530 1303.sup.[3] [.sup.1]Elution with buffer B at pH 5.5 .sup.[2]Elution with buffer B at pH 9.0 .sup.[3]Calculated from the titration result of the precursor (06PP189 mol/g or 06PP292 with 1963 mol/g) and the benzyl group density n.d. not determined