1. Field of the Invention
The present invention relates to a porous, non-particulate, convectively permeable polysaccharide matrix, on the surface of which there is fixed a grafted-on polymer derived from at least one ethylenic monomer compound having functional groups, wherein the polysaccharide matrix is prepared by grafting a porous, non-particulate, convectively permeable polysaccharide starting matrix with the at least one ethylenic monomer compound having functional groups in the presence of an organic acid having at least one carboxylic acid group and/or having at least one acidic XH group, where X=—O, —S, or —N, and of a transition metal or lanthanide compound, and also to processes for preparing the polysaccharide matrix and to the use of a polysaccharide matrix of this kind.
2. Description of the Related Art
The filtration, purification or removal of biomolecules such as proteins, amino acids, nucleic acids, viruses or endotoxins from liquid media is of great interest to the biopharmaceutical industry. Porous, non-particulate polysaccharide matrices, particularly in the form of adsorption membranes, are used especially in such processes in which the adsorbands are present in the liquid phase at very low concentrations in relation to the capacity of the matrix, and so, based on the unit area of the matrix, a large volume of the liquid phase can be processed until the capacity is exhausted. A typical application is the adsorption of deoxyribonucleic and ribonucleic acids (RNA and DNA), viruses, host cell proteins and endotoxins, in order to remove these contaminants from antibody-containing solutions using positively charged membranes.
The open pore structure of said membranes permits the adsorption of large adsorbands in the pore interior. For conventional gels, adsorption in these cases is limited to the outer particle surface. Therefore, membranes are used with success especially in the purification of large adsorbands such as DNA, RNA, blood coagulation factor VIII (FVIII) and viruses. However, for the adsorption of smaller adsorbands, the smaller inner surface of the membranes and the resulting lower capacity is disadvantageous compared to chromatography gels.
DE 39 29 648 C1 and EP 0 490 940 B1 disclose processes for grafting onto nitrogen-containing polymers, more particularly polyamides. They are based on the hydrogen of NH groups of the polymers being replaced by a halogen, preferably by a chlorine atom from organic hypohalites, organic N-halogen compounds or tetrachloromethane, and nitrogen free-radical formation is subsequently effected by reaction with a reducing agent. Any desired ethylenic monomers can then be grafted onto the free-radical nitrogen sites. The disadvantage of these membranes prepared according to these processes, which membranes have been proven to be useful per se, is that they have markedly lower hydraulic permeabilities than the starting membrane.
EP 0 527 992 B1 therefore proposes solving the problem of the reduced hydraulic permeability by coating membranes comprising a first polymer (e.g., cellulose hydrate, polyvinylidene difluoride (PVDF) or cellulose hydrate) with a solution of a second polymer, preferably an N-chlorinated nylon derivative, and by subsequently grafting the membrane thus coated with ethylenic monomers. A disadvantage of this process is that the ethylenically unsaturated monomers cannot be grafted onto the first polymer directly and that the structural prerequisite for the grafting reaction is thus the coating of the first polymer with the solution of the second polymer before grafting can take place. This process is complicated and cost-intensive owing to the multiplicity of its individual steps.
Ion exchangers based on hydroxyl-containing supports (e.g., Fractogel® TSK from Merck), on the surface of which epoxy-containing methacrylic acid derivatives are grafted, are known from EP 0 722 360 B1. The epoxy groups of the grafted-on polymer can be subsequently converted to vicinal diol or 1,2-aminoalcohol functions by NH— or OH-containing reagents.
EP 0 337 144 B1 discloses hydroxyl-containing supports, the surface of which has covalently bonded to it polymers producible by graft polymerization which have the unit [—CR′R″—CR1Y]n, where Y=—CO2R4, —CN, —CHO, —OH, —CH2NH2 or —CH2NR2R3, as repeat unit. The supports are used for fractionating immunoglobulins in human serum and in murine ascitic fluid containing monoclonal antibodies.
EP 1 163 045 B1 discloses a process for preparing cationically modified membranes, wherein a microporous starting membrane, preferably comprising polyethersulphone, is provided with a coating prepared by crosslinking a composition comprising a diallylamine copolymer having epoxy groups and cationic groups, a polyalkyleneamine, and an amine-reactive compound having a cationic group. The amine-reactive compound is preferably a glycidyl compound having ammonium groups.
EP 1 614 459 B1 discloses a process for preparing cationically modified membranes, wherein a microporous starting membrane, preferably comprising (optionally hydrophilic) polyethersulphone, is treated with a mixture of a diallylamine copolymer, a diallyldialkylammonium halide and an acrylic acid monomer having quaternary ammonium groups, and is converted into the cationically modified membrane by heat treatment.
EP 0 538 315 B1 discloses a porous matrix consisting of a porous support having a sponge structure, which support has on its inner and outer surface a grafted-on polymer layer having functional groups, wherein the polymer layer is solvatable by a liquid phase in contact with the matrix such that it can occupy adjustable proportions of the pore volume of the porous support. Precursors used for the porous support are nitrogen-containing polymers, for example nylon derivatives or cellulose hydrate coated with nylon derivatives, onto which monomers, for example in the form of a mixture of hydroxyethyl methacrylate and glycidyl methacrylate, are grafted via N—Cl groups as reactive sites.
Y. Chen et al. disclose in “Polymer Composites”, vol. 28, 2007, pages 47-56, the preparation of a graft copolymer from corn starch and acrylamide as a monomer to be grafted on in the presence of a mixture of cerium(IV) ammonium sulphate as grafting initiator and citric acid. According to IR spectroscopic studies by the authors, for efficient grafting onto the corn starch, oxidative ring cleavage of the hexose building blocks of the corn starch is essential, and subsequently, as a result of reaction with Ce4+ cations, free-radical carbon atoms are generated in the polymer backbone of the starch for the reaction with acrylamide.
EP 1 386 660 B1 discloses processes for isolating immunoglobulins from immunoglobulin mixtures in the pH range of from 2 to 10, wherein use is made of a matrix M-SP1-L, which has in each case a plurality of variable, functional groups SP1-L. Here, M is a chromatography starting matrix to which ligands L having a molecular weight not more than 500 daltons are bonded via spacers SP1. The ligands L are preferably selected from the group consisting of monocyclic or bicyclic (hetero)aromatic compounds, which may optionally carry acidic groups.
EP 0 921 855 B1 likewise discloses processes for isolating immunoglobulins from immunoglobulin mixtures in the pH range of from 2 to 10, wherein use is made of a matrix M to which the functional group SP1-L is bonded. The ligand L, which is bonded to the matrix M by means of a spacer SP1, is selected from the group consisting of benzimidazoles, benzothiazoles and benzoxazoles. The ligand L may optionally carry acidic groups, such as sulphonic or carboxylic acid substituents, on its bicyclic, heteroaromatic moiety.
EP 1 718 668 B1 discloses a process for separating antibodies from at least one contaminant in a solution, wherein use is made of a chromatography resin on which only multimodal ligands are immobilized. Here, multimodal ligands are ligands which can enter into at least two different binding interactions (i.e., an ionogenic and a hydrophobic interaction) with the components to be separated. The ligands have cation-exchanging groups and at least one (hetero)aromatic ring system for these two binding interactions.
An object of the present invention is to provide a porous, non-particulate, convectively permeable polysaccharide matrix, on the surface of which there is fixed a grafted-on polymer and which has a high protein binding capacity, and to provide cost-effective and efficient processes for preparing said polysaccharide matrix. A further object of the invention is to provide a novel use of the polysaccharide matrix for material separation.
USPTO Backgrounds
USPTO Backgrounds