CHROMATOGRAPHY MEDIUM WITH BOUND MICROGLOBULES AND METHOD FOR THE PREPARATION THEREOF

Abstract

The present invention relates to a chromatography medium which can be used in affinity chromatography and to a method for the preparation thereof.

Claims

1. A chromatography medium comprising: a porous matrix; and nonporous microglobules, the nonporous microglobules being bound on the inner and outer surfaces of the porous matrix, and the average radius of the microglobules being not more than 30% of the average pore diameter of the porous matrix.

2. The chromatography medium as claimed in claim 1, wherein the nonporous microglobules are bound on the inner and outer surfaces of the porous matrix by physical means or by covalent means.

3. The chromatography medium as claimed in claim 1, wherein the permeability of the chromatography medium is at least 40% of the permeability of the porous matrix without bound microglobules.

4. The chromatography medium as claimed in claim 1, wherein the nonporous microglobules are substantially spherical oligomers and/or polymers which are constructed from at least one monomer selected from the group consisting of glycidyl (meth)acrylate, substituted or unsubstituted alkyl (meth)acrylates and their derivatives, styrene and its derivatives, 2-vinyl-4,4-dimethylazlactone, substituted or unsubstituted N-alkyl(meth)acrylamides and their derivatives and substituted or unsubstituted N-N-dialkyl(meth)acrylamides and their derivatives.

5. The chromatography medium as claimed in claim 1, wherein the degree of grafting P of the chromatography medium is from 25% to 40%, given by: $P=\frac{m_m-m_0}{m_0}.Math.100.Math.\%$ where m.sub.m is the mass of the chromatography medium and m.sub.0 is the mass of the porous matrix without microglobules.

6. The chromatography medium as claimed in claim 1, wherein the relative degree of grafting P.sub.rel of the chromatography medium is at most 0.25 g/cm.sup.3, given by: $P_{\mathrm{rel}}=\frac{P*}{\mathrm{Por}}$ where P is the degree of grafting of the chromatography medium in %, is the density of the porous matrix without microglobules in g/cm.sup.3, and Por is the porosity of the porous matrix without microglobules in %.

7. The chromatography medium as claimed in claim 1, wherein the nonporous microglobules comprise additional chromatographically active centers or ligands which are bound to the microglobules or immobilized thereon.

8. A method for preparing a chromatography medium as claimed in claim 1, comprising: providing a porous starting matrix; providing a polymerization solution comprising at least one monomer, a bi-, tri- or multifunctional crosslinker, a polymerization initiator and a solvent or solvent mixture, the at least one monomer, the bi-, tri- or multifunctional crosslinker and the polymerization initiator being completely soluble in the solvent or solvent mixture; and initiating a polymerization in the polymerization solution in the presence of the porous starting matrix to form nonporous microglobules, the nonporous microglobules being insoluble in the solvent or solvent mixture and being bound to the inner and outer surfaces of the porous starting matrix; the average radius of the microglobules being not more than 30% of the average pore diameter of the porous starting matrix.

9. The method for preparing a chromatography medium as claimed in claim 8, wherein the at least one monomer is selected from the group consisting of glycidyl (meth)acrylate, substituted or unsubstituted alkyl (meth)acrylates and their derivatives, styrene and its derivatives, 2-vinyl-4,4-dimethylazlactone, substituted or unsubstituted N-alkyl(meth)acrylamides and their derivatives and substituted or unsubstituted N-N-dialkyl(meth)acrylamides and their derivatives.

10. The method for preparing a chromatography medium as claimed in claim 8, wherein the bi-, tri- or multifunctional crosslinker is selected from the group consisting of ethylene glycol dimethacrylate, trimethylpropane trimethacrylate, divinylbenzene and N-N-methylenebisacrylamide.

11. The method for preparing a chromatography medium as claimed in claim 8, wherein the polymerization initiator is selected from the group consisting of 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone, azobis(isobutyronitrile), 4,4-azobis(4-cyanovaleric acid), 1,1-azobis(cyclohexanecarbonitrile), benzoyl peroxide, 2,2-bis(tert-butylperoxy)butane, 1,1-bis(tert-butylperoxy)cyclohexane, tert-butyl hydroperoxide, tert-butyl peroxyisopropyl carbonate, cyclohexanone peroxide and 2,4-pentanedione peroxide.

12. The method for preparing a chromatography medium as claimed in claim 8, wherein the solvent or solvent mixture is selected from the group consisting of cyclohexanol/dodecan-1-ol, octan-2-one, n-butyl acetate, p-xylene, toluene, ethyl acetate, benzonitrile, cyclohexanone, dodecan-1-ol, acetonitrile/ethanol/water, decan-1-ol and isopropanol/decan-1-ol.

13. The method for preparing a chromatography medium as claimed in claim 8, wherein the total volume of monomer and crosslinker, based on the total volume of the polymerization solution, is not more than 20% by volume.

14. The method for preparing a chromatography medium as claimed in claim 8, wherein the total volume of the crosslinker, based on the total volume of the polymerization solution, is not more than 6% by volume.

15. The method for preparing a chromatography medium as claimed in claim 8, wherein the concentration of the polymerization initiator in the polymerization solution is preferably from 1 to 3% by weight.

16. The method for preparing a chromatography medium as claimed in claim 8, wherein a macroscopically observable phase separation occurs after not more than 30 seconds.

Description

[0087] The figures show:

[0088] FIG. 1: SEM images of the chromatography medium according to the invention as per inventive modification 2 (top/center) and of the porous starting matrix before the modification (bottom).

[0089] FIG. 2: SEM images of chromatography media as per the modification protocols of PA documents 2 (top left), 5 (top right), 1 (bottom left) and 4 (bottom right).

[0090] FIG. 3: Reduction in permeability as per Example 3.

[0091] FIG. 4: Breakthrough curves of chromatography media according to the invention and of Sartobind A, plotted as percentage breakthrough against dynamic binding capacity DBC.

[0092] FIG. 5: Plot of the specific surface areas after inventive modification and before modification.

[0093] FIG. 6: Plot of the dynamic capacity of chromatography media according to the invention against their specific surface area.

[0094] FIG. 7: Pore-size distribution of chromatography media according to the invention and of unmodified starting matrices (top); plot of porosity of chromatography media according to the invention against the hydrodynamic diameter of tracer molecules (bottom).

[0095] FIG. 8: Plot of the permeability (the flow rate), the degree of grafting and the dynamic binding capacity against the polymerization time of the modification.

[0096] FIG. 9: Plot of the permeability against the relative degree of grafting.

[0097] FIG. 10: Calibration curve for Example 9.

[0098] FIG. 11: Plot of the dynamic ligand utilization against the respective specific surface area of chromatography media according to the invention.

[0099] FIG. 12: Plot of the static ligand utilization against the respective specific surface area of chromatography media according to the invention.

[0100] FIG. 13: Plot of the amount of immobilized ligand against the respective specific surface area of chromatography media according to the invention.