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WO1996025992A9 - Supports polymeres poreux passives et procedes de preparation et d'utilisation de ces derniers - Google Patents

Supports polymeres poreux passives et procedes de preparation et d'utilisation de ces derniers

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Publication number
WO1996025992A9
WO1996025992A9 PCT/US1996/001891 US9601891W WO9625992A9 WO 1996025992 A9 WO1996025992 A9 WO 1996025992A9 US 9601891 W US9601891 W US 9601891W WO 9625992 A9 WO9625992 A9 WO 9625992A9
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WO
WIPO (PCT)
Prior art keywords
matrix
chromatographic media
passivated
porous
εaid
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PCT/US1996/001891
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English (en)
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WO1996025992A1 (fr
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Priority to AU49215/96A priority Critical patent/AU4921596A/en
Publication of WO1996025992A1 publication Critical patent/WO1996025992A1/fr
Publication of WO1996025992A9 publication Critical patent/WO1996025992A9/fr

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  • This invention relates generally to modified porous solid supports and processes for the preparation and use of same.
  • passivated porous supports are disclosed which are characterized by a reversible high sorptive capacity substantially unaccompanied by non-specific adsorption of or interaction with biomolecules such as proteins, polysaccharides or oligo- or polynucleotides.
  • the passivated porous supports of the present invention exhibit other characteristics highly desirable in chromatographic applications, such as high porosity, physical rigidity, high charge density, and chemical stability under a variety of extreme conditions.
  • the passivated porous supports of the present invention may also be used advantageously in a high flow, high efficiency mass transfer chromatographic technique which may be carried out in a fluidized-bed, packed-bed, or other mode of operation.
  • Polyfunctional macromolecules such as proteins
  • ion-exchange chromatography proteins are separated on the basis of their net charge. For instance, if a protein has a net positive charge at pH 7 it will bind to a negatively charged ion- exchange resin packed in a chromatography column. The protein can be released, for example, by decreasing the pH or adding cations that compete for binding to the column with the positively charged groups on the protein. Thus, proteins that have a low density of net positive charge, and thus a lower affinity for the negatively charged groups of the column, will tend to emerge first, followed by those having a higher charge density.
  • the ion-exchange resins which are used in these procedures are solids possessing ionizable chemical groups.
  • cation-exchangers which contain acidic functional groups such as sulfate, sulfonate, phosphate or carboxylate
  • anion- exchangers which contain functional groups such as tertiary and quaternary amines.
  • ionizable functional groups may be inherently present in the resin or they may be the result of the chemical modification of the organic or mineral solid support.
  • Organic ionic-exchangers which are made from polysaccharide derivatives, e.q., derivatives of agarose, dextran and cellulose, etc. , have been used for both laboratory and industrial scale ion-exchange chromatography.
  • these ion-exchangers have many disadvantages.
  • polysaccharide-derived ion-exchangers are not very mechanically stable and are not resistant to strong acids. This instability limits the length of the column and, also, limits the flow rate through the column.
  • such ion-exchangers have limited sorption capacity due to the limited number of ionic or ionizable groups that can be attached to the polysaccharide.
  • these polysaccharidic derivatives are poor adsorbents for use in rapid fluidized-bed separations because of the low density of the material.
  • a fluidized bed it is desirable to pass the fluid without simultaneously washing out the particles. Therefore, it is generally desirable to have as great a density difference as possible between the solid support particles (e.q. , silica) and the fluidizing medium.
  • the intrinsic high density of inorganic sorbents based on passivated mineral substrates facilitates packing and rapid decantation into chromatographic columns. Dense packing prevents formation of empty spaces and channeling when using packed beds.
  • fluidization of dense particles in aqueous suspension is possible at high flow rates that, in turn, are very desirable when dealing with large scale applications. Operation of fluidized beds at high superficial flow velocities is generally not possible with low-density organic or polymeric sorbents, which can be eluded from fluidized beds at relatively low liquid flow rates.
  • synthetic polymers are mechanically more stable than inorganic supports, and the former are more resistant to strong acidic conditions.
  • they suffer disadvantages as well, such as limited capacity, limited solute diffusivity and thus, limited productivity.
  • These synthetic polymers also suffer to some extent from the problem of non-specific adsorption of biomolecules, such as proteins. Untreated mineral supports such as silica are also inadequate in many chromatographic protein separation applications because of such non-specific adsorption.
  • Non-specific adsorption is caused by the interaction of a protein with the surface of the support — be it organic or inorganic in nature.
  • silica is an acidic compound, and the negatively charged silanol groups present at the solid/liquid interface tend to create a separate ion- exchange interaction between the surface of silica and the protein.
  • Non-specific adsorption is also caused by hydrogen bonding that takes place between, e.g., amino groups present in the amino acid residues of proteins and these same silanols present at the silica surface.
  • Such non-specific interactions create separation problems during chromatography — e.g . , poor protein recovery and/or inadequate resolution.
  • both polysaccharides and most hydroxyl containing synthetic sorbents are sensitive to the cleaning solutions used in industrial settings, which often include strong oxidizing agents such as hypochlorite or peracetic acid and which may be characterized by extremes of pH.
  • the porous configurations of the coated, composite membrane structures claimed by Steuck are essentially identical to those of the corresponding uncoated porous membrane substrates, implying that the polymer of Steuck is applied as a thin surface layer or coating that does not interfere with the porosity or flow properties of his composite membranes. Moreover, Steuck does not disclose the concept of a "passivating layer” or the use of monomers capable of functioning as "passivating” monomers within the meaning of the present invention as discussed in more detail below.
  • Varady et al. in U.S. Patent No. 5,030,352, disclose pellicular support materials useful as chromatography media which are obtained by applying various thin hydrophilic coatings to the surfaces of hydrophobic polymer substrates (e.g., polystyrene). Varady's surface coatings are applied by first exposing the surfaces of the hydrophobic substrate to a solution of a solute characterized by interspersed hydrophilic and hydrophobic domains; contact between surface and solute takes place under conditions that promote hydrophobic-hydrophobic interaction between solute and substrate, with the result that solute molecules are adsorbed onto the surface of the substrate as a thin coating that is ultimately crosslinked in place.
  • hydrophobic polymer substrates e.g., polystyrene
  • Varady's coating materials may further comprise reactive groups capable of being derivatized to produce various materials useful in ion- exchange, affinity, and other types of chromatographic and adsorptive separations.
  • the hydrophilic, functional coating of Varady's invention is limited to a thin adherent film on the surface of the hydrophobic support.
  • the morphology of this coating layer is a direct and unavoidable consequence of the stated method of its deposition — i.e., by the crosslinking of adjacent solute molecules adsorbed onto the surface of the hydrophobic substrate.
  • Varady's coating method is at least partially effective in reducing the non-specific binding of proteins to the substrate
  • the sorption capacity of the chromatographic materials so produced is necessarily limited and inferior to those of the media produced by the process of the present invention.
  • the method of the present invention causes the formation of a crosslinked and functional gel that extends out into and substantially fills the pores of the support.
  • the static and dynamic sorption capacities of the chromatographic media are not limited by the porous surface area of the substrate, as is the case with the pellicular material of Varady's invention.
  • U.S. Patent No. 4,415,631 to Schutijser discloses a resin consisting of inorganic silanized particles onto which is bonded a cross-linked polymer comprised of copolymerized vinyl monomers and which contains amide groups.
  • the invention specifies that the inorganic porous support, including silica, must be silanized prior to coating.
  • the silanization treatment provides the inorganic porous support with reactive groups so that the copolymer can be covalently bonded to the silica surface.
  • Nakishima et al. in U.S. Patent 4,352,884, also discloses the use of silica as a porous substrate.
  • the silica is coated with a polymer made up of acrylate or methacrylate monomer and a copolymerizable unsaturated carboxylic acid or a copolymerizable unsaturated amine.
  • Nakashima et al. use an already preformed polymer to coat the support.
  • Nakashima et al. in a separate and distinct step, utilize a crosslinking agent in a subsequent curing process.
  • the present invention provides a passivate porous support comprising a porous solid matrix having interior and exterior surfaces and innate (i.e., inherently present) groups that render the matrix susceptible to undesirable non-specific interaction with biological molecules, and a polymer network derived from a passivation mixture comprising effective amounts of a main monomer, a passivating monomer different from the main monomer, and a crosslinking agent, the mixture having been allowed to come into intimate contact with the surfaces of the matrix for a sufficient period of time such that on polymerization of the mixture the innate groups of the matrix become deactivated, resulting in the minimization or substantial elimination of the above-mentioned undesirable non-specific interactions.
  • innate i.e., inherently present
  • the passivated porous supports of the present invention are further characterized by reversible high sorptive capacity for biological molecules including proteins. Furthermore, the passivated porous supports of the present invention enjoy exceptional chemical stability on exposure to strongly acidic or alkaline media and/or strong oxidizing solutions such as those that are frequently utilized during cleaning of industrial manufacturing equipment.
  • the primary objective of the present invention concerns the passivation of porous solid matrices that possess innate undesirable groups that render the matrix susceptible to non ⁇ specific interactions (e.g. , adsorption) with biological molecules" in particular, proteinaceous substances.
  • porous solid matrices are amenable to passivation by the general method of the present invention.
  • porous matrices include, but are not limited to, (i) mineral oxide supports, (ii) "stabilized” mineral oxide supports rendered chemically resistant to leaching by the application of thin protective coatings of hydrophobic polymers to their surfaces, and (iii) porous matrices comprised solely of organic/polymeric materials, in particular hydrophobic polymers.
  • mineral oxide supports such as silica, alumina, and the like
  • passivated supports that exhibit desirable characteristics, such as high sorptive capacity, high density and good resolving (chromatographic) properties, unaccompanied by undesirable non-specific interactions that would otherwise be due largely to innate hydroxyl groups present on the surfaces of mineral oxides (e.g. , silanols in the case of silica supports) .
  • transition metal oxides such as zirconium, titanium, chromium and iron oxides are considered in the present invention to be within the scope of the term "mineral oxide" supports.
  • the non ⁇ specific interactions include either electrostatic interactions, hydrogen bonding, or both.
  • the passivating monomer (alternatively described herein as the "neutralizing” monomer) is chosen to dampen, “neutralize”, or “deactivate” such non-specific binding interactions; that is, one selects a passivating monomer that is capable of interacting with the innate groups of mineral oxide substrates either electrostatically or via hydrogen-bonding or both.
  • the passivating monomer can also act as the main monomer (i.e., said passivating or neutralizing monomer is chemically identical to the main monomer) , but such situations are limited to those in which the neutralizing monomer is an acrylamide-based monomer that possesses at least one polar substituent, preferably an ionizable (e.g. , tertiary amino, carboxylic acid, sulfonic acid, etc.) or ionic (e.g., ammonium, phosphate, etc.) substituent.
  • an ionizable e.g. , tertiary amino, carboxylic acid, sulfonic acid, etc.
  • ionic e.g., ammonium, phosphate, etc.
  • acrylate-based monomers cannot serve both as the passivating (neutralizing) monomer and as the main monomer — in part, because the acrylate-based monomers are less stable than the acrylamide-based monomers, particularly under strongly acidic or alkaline conditions.
  • a passivating or neutralizing monomer in combination with the main monomer and crosslinking agent, allows for the formation of a three-dimensional polymer network comprising a thin passivation region or layer that is substantially adjacent to the matrix surface, which polymer network extends into and throughout the porous volume of the substrate matrix and which passivation layer is made up primarily of units of the passivating or neutralizing monomer engaged in interactions with the innate groups of the substrate matrix.
  • This thin passivation region or layer is additionally held in close proximity to the matrix surface by a lattice of main monomer units which extends from the passivation layer to the exposed exterior surfaces of the resulting "passivated" porous support.
  • the crosslinking agent acts to tether the respective polymeric (or copolymeric) chains to one another, thereby creating a stable three-dimensional polymer (i.e., "gel") network that is surprisingly effective in minimizing or eliminating undesirable non-specific binding interactions between biological molecules and the non-passivated porous solid matrix.
  • a passivated porous support comprising a porous solid matrix having interior and exterior surfaces and innate groups that render the matrix susceptible to undesirable, nonspecific interaction with biological molecules, and a polymer network derived from a passivation mixture comprising effective amounts of an acrylamide or methacrylamide monomer further substituted with at least one polar ionic or ionizable substituent, which monomer is capable of functioning both as a main monomer and as a passivating or neutralizing monomer, and a crosslinking agent, the mixture having been allowed to come into intimate contact with the surfaces of the matrix for a sufficient period of time such that on polymerization of the mixture, the innate groups of the matrix become deactivated, resulting in the substantial elimination of the above-mentioned undesirable non-specific interaction.
  • porous matrices comprised of hydrophobic polymer substrates (as opposed to mineral oxide matrices) are concerned, it is a further object of the present invention to reduce the nonspecific binding associated with exposure of such hydrophobic polymer surfaces to proteinaceous solutions.
  • porous synthetic polymeric solid matrices comprised of such materials as polystyrene, polysulfone, polyethersulfone, polyolefins (e.g., polyethylene and polypropylene) , polyacrylate, polyvinyl acetate (and partially hydrolyzed versions thereof) , and the like, exhibit non-specific binding associated with hydrophobic-hydrophobic (among other types, e.g., hydrogen-bonding) interactions.
  • hydrophobic synthetic polymer matrices are passivated by the incorporation of passivating ("neutralizing") monomers that are capable of associating with and consequently deactivating innate non-polar hydrophobic groups exposed on the matrix surface.
  • the passivating monomers of the present invention adsorb upon (and consequently cover) the hydrophobic groups on the surface by virtue of their containing long-chain saturated hydrocarbons, olefinic hydrocarbon groups, aromatic groups, or like hydrophobic domains that interact with and become appreciably bound to their hydrophobic counterparts on the matrix surface as a consequence of the hydrophobic- hydrophobic interaction between them.
  • passivated porous supports exhibiting exceptional stability in alkaline media are provided.
  • These passivated resins comprise porous solid matrices precoated with a thin film of a synthetic organic polymer, such as polystyrene or polystyrene substituted with nonionic, ionic, or ionizable functional groups.
  • a synthetic organic polymer such as polystyrene or polystyrene substituted with nonionic, ionic, or ionizable functional groups.
  • the methods of the present invention can be advantageously applied to the passivation of chromatographic support media comprised of porous mineral oxide particles (e.g., silica and alumina), the interior and exterior surfaces of which have previously been coated with a thin, protective layer of a coating polymer.
  • This protective polymer coating is applied for the purpose of improving the chemical stability of the underlying mineral oxide material (e.g., against leaching or other chemical decomposition at alkaline, acidic, or strongly oxidizing conditions) .
  • strongly alkaline aqueous media e.g., 0.5 M sodium hydroxide solutions
  • conventional silica supports can suffer significant weight loss (of order 50%) associated with leaching of the material over repeated cleaning cycles (e.g., 100 cycles).
  • the approach to substrate stabilization taken in one embodiment of the present invention involves coating the alkaline-sensitive porous mineral oxide substrate matrix with a soluble polymer that substantially encapsulates the mineral oxide matrix and thereby minimizes or prevents contact between the mineral oxide substrate and potentially destructive chemical cleaning solutions (e.g., caustic).
  • the protective polymer coating is applied in the form of a thin surface layer upon the pore wall surfaces in order to avoid significantly decreasing the porous volume or blocking the mouths of pores.
  • the protective polymer coating layer is readily applied, for example, by (i) first dissolving the protective polymer (e.g.
  • the passivating monomers useful in this embodiment of the present invention adsorb upon (and consequently cover) the hydrophobic groups on the surface by virtue of their containing long-chain saturated hydrocarbons, olefinic hydrocarbon groups, aromatic groups, or like hydrophobic domains that interact with and become appreciably bound to their hydrophobic counterparts on the matrix surface as a consequence of the hydrophobic-hydrophobic interaction existing between them.
  • the present invention utilizes base matrices having the following characteristics: an initial average particle size ranging from about 5 to about 1000 microns; an initial porous volume ranging from about 0.2 to about 2 cm 3 /gram; an initial surface area ranging from about 1 to about 800 m 2 /gram; and an initial pore size ranging from about 50 to about 6000 angstroms.
  • the base matrix is characterized by: an initial average particle size ranging from about 10 to about 300 microns, although passivated supports having narrow particle size ranges, such as about 15-20, about 15-25, about 30-45, about 50-60, about 80-100, and about 100-300 microns, are most preferred.
  • Preferred ranges for other characteristics include an initial porous volume ranging from about 0.8 to about 1.2 cm 3 /gram; an initial surface area ranging from about 10 to about 400 m 2 /gram; and an initial pore size ranging from about 1000 to about 3000 angstroms.
  • the density of the porous solid matrix obviously varies with its chemical nature, being higher for mineral oxide (e.g., silica) substrates and lower for polymeric ones (e.g., polystyrene).
  • the size exclusion limit varies somewhat from one type of passivated porous ⁇ upport to another, but generally falls in the range of about 500 to about 2,000,000 daltons, preferably, 50,000 to about 500,000.
  • the sorptive capacity can also be manipulated, depending on the amount of main monomer incorporated in the polymer network, and ranges between about 1 milligram to about 300 milligrams of solute or biological molecule per unit volume (ml) of passivated support bed — preferably at least about 50 mg/ml, and mo ⁇ t preferably about 100 mg/ml.
  • Yet another object of the present invention relates to the passivation of non-passivated porous solid matrices while maximizing the openness (e.g., gel porosity and pore size) of the resulting passivated porous support.
  • open gel morphologies have the advantage of permitting high sorption capacities to be achieved without affording excessive resistance to the transport of solutes such as proteins through the gel.
  • the polymerization of the passivation mixture is effected in the presence of an effective amount of a pore inducer.
  • number of additives are suitable as pore inducers, including, but not limited to, polyethylene glycol, polyoxyethylene, polysaccharide, and the like.
  • the polymerization of the passivation mixture can be effected in the presence of an effective pore-inducing amount of a polar solvent.
  • a polar solvent for example, the polymerization can be carried out in alcohol, a cyclic ether, a ketone, a tertiary amide, a dialkyl sulfoxide, or mixtures thereof.
  • polar solvents include, but are not limited to, methanol, ethanol, propanol, tetrahydrofuran, dimethylsulfoxide, dimethylformamide, acetone, dioxane, or mixtures thereof.
  • polymerization is effected in the presence of an effective amount of a polymerization initiator, for example, thermal initiators such as ammonium persulfate/tertiary amine, nitriles or transition metal ⁇ .
  • thermal initiators such as ammonium persulfate/tertiary amine, nitriles or transition metal ⁇ .
  • thermal initiators such as ammonium persulfate/tertiary amine, nitriles or transition metal ⁇ .
  • thermal initiators such as ammonium persulfate/tertiary amine, nitriles or transition metal ⁇ .
  • thermal initiators such as ammonium persulfate/tertiary amine, nitriles or transition metal ⁇ .
  • other examples include 2,2'-azobi ⁇ (2- amidinopropane) hydrochloride, potassium persulfate/dimethylaminopropionitrile, 2,2'- azobis(isobutyronitrile) , 4,4
  • Photochemical initiators may also be used, such as isopropylthioxantone, 2(2'-hydroxy-5'- methylphenyl) benzoltriazole, 2,2'-dihydroxy-4- methoxybenzophenone, riboflavin, and the like.
  • Polymerization begins, as is known in the art, e.g., with agitation, exposure to heat, or expo ⁇ ure to a ⁇ ufficient amount of radiant energy.
  • the object of the pre ⁇ ent invention to provide further passivated porous supports in which the main monomer of the polymer network comprises a vinyl monomer having at least one polar substituent.
  • substituent may further be ionic, non-ionic, ionizable, or in the case of a vinyl monomer having more than one polar sub ⁇ tituent, such substituents may be a combination of such substituents.
  • affinity chromatography it is preferred in affinity chromatography that the main monomer on polymerization, as part of the polymer network, have an affinity for a pre ⁇ elected biological molecule.
  • the further modification of the polymer network to incorporate specific ligands capable of binding to biological molecules of interest is not precluded.
  • neutralizing monomers are provided which can interact with the innate groups of the matrix surfaces in the same manner as the non-specific interaction (e.g., electrostatically, via hydrogen bonding or both in the case of mineral oxide matrices — or via hydrophobic-hydrophobic interaction in the case of synthetic polymeric matrices) .
  • substituent ⁇ can be polar, cationic, anionic or hydrophobic depending on the particular application at hand.
  • suitable neutralizing monomers for porous mineral oxide matrices comprise a vinyl monomer having at least one polar ionic or ionizable sub ⁇ tituent.
  • the substituent has the capacity to bear a po ⁇ itive charge.
  • neutralizing monomers are selected to provide near-surface pas ⁇ ivating regions and polymer networks that are effective in deactivating-polar groups on the surfaces of non- passivated matrices (e.g., in deactivating hydroxyl groups on the surface ⁇ of porou ⁇ mineral oxide matrice ⁇ ) .
  • neutralizing monomer ⁇ u ⁇ eful in the pa ⁇ sivation of porous mineral oxide matrices may be selected from diethylaminoethyl methacrylamide, diethylaminoethyl acrylamide, methacrylamido propyltrimethyl ammonium halide, triethylaminoethyl acrylamide, trimethylaminoethyl methacrylate, polyethyleneglycol dimethacrylate, dimethylaminoethyl methacrylate, polyethyleneglycol divinyl ether, or polyethyleneglycol diacrylate.
  • suitable passivating monomers for use in the pas ⁇ ivation of hydrophobic polymer ⁇ urface ⁇ whether said polymer is present as a protective ⁇ urface coating on a mineral oxide matrix or a ⁇ the bulk, ⁇ tructural-material in the case of a porous polymeric chromatographic support matrix — will typically comprise vinyl monomers having at lea ⁇ t one substantially non-polar or hydrophobic sub ⁇ tituent.
  • this ⁇ ubstituent comprises a hydrocarbon-rich functional group or moiety that imparts hydrophobicity to a portion of the passivating monomer.
  • the hydrophobic character will result from the presence in the pas ⁇ ivating monomer of a saturated (e.g., aliphatic) or unsaturated (e.g. , aromatic) hydrocarbon substituent, and may further be described as straight-chain, branched, cyclic, or heterocyclic.
  • Long-chain alkyl functional groups are particularly u ⁇ eful a ⁇ substituents in this class of passivating monomers, which further contain one or more vinylic, acrylic, acrylamide, or allylic monomers.
  • These passivating monomers are typically employed at concentrations in the reaction mixture of from about 0.1 to 1.0%.
  • Cros ⁇ linking agent ⁇ u ⁇ eful in the present invention comprise vinyl monomer ⁇ having at lea ⁇ t one other polymerizable group, such as double bond, a triple bond, an allylic group, an epoxide, an azetidine, or a strained carbocyclic ring.
  • Preferred crosslinking agents having two double bonds include, but are not limited to, N,N'- ethylenebi ⁇ -(acrylamide) , N,N'-methylenebis(methacrylamide) , diallyl tartardiamide allyl methacrylate, diallyl amine, diallyl ether, diallyl carbonate, divinyl ether, 1,4- butanedioldivinylether, polyethyleneglycol divinyl ether, and 1,3-diallyloxy-2-propanol.
  • the amount of neutralizing monomer is chosen to be sufficient to counteract the innate group ⁇ pre ⁇ ent on the ⁇ urface of the non-passivated matrix.
  • the surfaces of the matrix are contacted (e.g., by dropwise addition) with a solution of the pas ⁇ ivation mixture.
  • the passivation mixture is prepared as an aqueous solution and, as mentioned above, may in addition contain effective amounts of a pore inducer.
  • the volume (in ml) of the passivation mixture solution is adjusted to correspond approximately to the weight (in grams) of the non-pas ⁇ ivated porou ⁇ solid matrix.
  • Yet another object of the present invention is related to a method of separating a desired biological molecule from a sample containing same comprising: (a) loading a column packed with the passivated porous support having an affinity for a preselected biological molecule with a sample containing the preselected biological molecule; and (b) passing an eluent solution through the loaded column to effect the separation of the preselected biological molecule.
  • the sample may be introduced to the column in any number of ways, including as a solution. Chromatographic separations employing these passivated supports in fluidized-bed modes of operation are also within the scope of the invention.
  • the methods of the present invention are effective to isolate or separate a broad range of biological molecules, including peptides, polypeptides, and proteins (such as in ⁇ ulin and human or bovine serum albumin) , growth factors, immunoglobulins (including IgG, IgM, and therapeutic antibodie ⁇ ) , carbohydrates (such as heparin) and polynucleotides (such as DNA, RNA, or oligonucleotide fragments) .
  • proteins such as in ⁇ ulin and human or bovine serum albumin
  • growth factors such as immunoglobulins (including IgG, IgM, and therapeutic antibodie ⁇ )
  • carbohydrates such as heparin
  • polynucleotides such as DNA, RNA, or oligonucleotide fragments
  • Eluent solutions suitable for use in the present invention are well known to those of ordinary skill in the art.
  • a change in ionic strength, pH or solvent composition may be effective in "stepwise" elution processes.
  • eluent solutions may comprise a salt gradient, a pH gradient or any particular ⁇ olvent or solvent mixture that is specifically useful in displacing the pre ⁇ elected biological molecule.
  • Such method ⁇ are generally known to those engaged in the practice of protein chromatography.
  • Still another object of the present invention relates to a chromatographic method for the separation of biological molecules comprising passing a sample containing a mixture of biological molecules through a column packed with the passivated porous support disclosed herein.
  • a method of preparing a passivated porous solid support comprising: (a) contacting a porous ⁇ olid matrix, having interior and exterior surfaces and innate groups that render the matrix ⁇ u ⁇ ceptible to undesirable non-specific interaction with biological molecules, with a passivation mixture comprising effective amounts of a main monomer, a passivating or neutralizing monomer different from the main monomer, and a crosslinking agent; and (b) effecting the polymerization of the mixture to form a polymer network within the pores of said porous solid matrix, such that the innate groups of the matrix become deactivated, to provide a passivated porous solid support that is substantially free of undesirable non-specific interactions.
  • FIG. IA is a graph which schematically repre ⁇ ents the chromatographic separation of a protein mixture consi ⁇ ting of (1) cytochrome, (2) bovine hemoglobin, (3) ovalbumin, and (4) beta-lactoglobin on a cationic pa ⁇ ivated porous ⁇ upport.
  • the condition ⁇ of the experiment were as follows:
  • Fig. IB is a graph which schematically represents the chromatographic separation of a protein mixture con ⁇ i ⁇ ting of (1) ovalbumin, (2) beta-lactoglobulin, (3) cytochrome c and (4) ly ⁇ ozyme on an anionic pa ⁇ ivated porou ⁇ ⁇ upport.
  • the condition ⁇ of the experiment were a ⁇ follows:
  • Fig. 2 represents a comparison between the chromatographic separations of a protein mixture consisting of (1) ovalbumin, (2) beta-lactoglobulin, (3) cytochrome c, and (4) lysozyme using an anionic passivated porous support and an anionic nonpa ⁇ ivated matrix.
  • the conditions of the experiment were as follows:
  • Second Buffer acetate 50 ml, pH 6.5
  • Fig. 3A shows ⁇ a graph of u ⁇ eful relative ⁇ orption capacity ver ⁇ u ⁇ flow rate for variou ⁇ porou ⁇ ⁇ upport ⁇ including the porou ⁇ ⁇ upport ⁇ of the present invention passivated with a cationically charged polymer network (i.e., a passivated porous support useful as an anion-exchange resin) .
  • Fig. 3B shows a graph of productivity versus flow rate for the various porou ⁇ ⁇ upport ⁇ ⁇ hown in Fig. 3A.
  • Fig. 4 shows a graph of the absolute sorptive capacity (in mg/ml) as a function of flow rate of a variety of ⁇ olid 5 ⁇ upports, including a pas ⁇ ivated porou ⁇ ⁇ upport of the pre ⁇ ent invention.
  • Fig. 5 is a schematic illustration of the putative architecture of the three-dimensional polymer network formed within and extending from the internal surface ⁇ of an 10 individual pore in a porous solid matrix upon polymerization of the passivation mixture of the present invention.
  • a primary component of the pas ⁇ ivation mixture of the present invention is the main monomer.
  • the appropriate amount of main monomer (or other solute) for use in the main monomer is the main monomer.
  • the volume of the monomer solution is effectively the volume of the solution of a pa ⁇ ivation
  • main monomer 25 mixture containing main monomer, neutralizing monomer, and crosslinking agent.
  • concentrations of the main monomer range from about 5% to about 50% (i.e., 5-50 gram ⁇ of main monomer per 100 mL of monomer solution) .
  • concentrations of the main monomer are from about 7% to about
  • the main monomer i ⁇ defined a ⁇ including any monomer known to tho ⁇ e skilled in the art which can be utilized for the preparation of an adsorbent useful in a chromatographic separation (e.g.,
  • Such monomers include, but are not limited to, non-ionic monomers, ionic monomers, hydrophilic monomer ⁇ , hydrophobic monomer ⁇ , and reactive monomer ⁇ .
  • Reactive monomers are monomers having special functional groups that enable them to react chemically with other molecules that are subsequently immobilized irreversibly on the polymer network. This procedure is the basis of affinity chromatography, the chemically attached molecule being referred to as the "ligand".
  • the main monomers of the present invention can be aliphatic, aromatic or heterocyclic; however, they must possess a polymerizable double bond; for example, the main monomers can be acrylic, allylic, vinylic or the like.
  • anionic polymer ⁇ are used to create anionic ⁇ orbents (i. e. , cation-exchange supports).
  • the functional groups i.e., the substituents on the vinyl monomer
  • the functional groups are preferably: carboxylic groups (e.g., acrylic acid, N-acryloyl-aminohexanoic acid, N- carboxymethylacrylamide) , sulfonate groups (e.q. acrylamidomethyl-propane sulfonic acid) , or phosphate groups (e.g. N-phosphoethyl-acrylamide) .
  • Cationic polymers used to create cationic sorbents may contain the following functional groups: sub ⁇ tituted amino group ⁇ (e.g., diethylaminoethyl methacrylamide, diethylaminoethyl acrylamide, methacrylamidopropyltrimethylammonium halide, triethylaminoethyl acrylamide, trimethylaminoethyl methacrylate, polyethyleneglycol dimethacrylate, dimethylaminoethyl methacrylate, polyethyleneglycol divinyl ether, or polyethyleneglycol methacrylate) , or heterocyclic amines (e.g., 2-vinylpyridine, vinylimidazole, 4-vinyl- pyridine) .
  • sub ⁇ tituted amino group ⁇ e.g., diethylaminoethyl methacrylamide, diethylaminoethyl acrylamide, methacrylamidopropyl
  • Nonionic polymers may be comprised of: acrylamide, hydroxy-containing acrylamide derivatives (e.q. N-tris-hydroxymethyl-methy1-acrylamide, methylolacrylamide, dimethylacrylamide, 2-hydroxyethylacrylamide, N-acryloyl- morpholine) , methacrylamide, hydroxy-containing methacrylamide derivatives, heterocyclic neutral monomer ⁇ (e.q. vinylpyrrolidone, N-acryloyl-morpholine) , or hydroxy- containing acrylate ⁇ and methacrylate ⁇ (e.g. hydroxyethylacrylate or hydroxyethyl methacrylate, hydroxyphenyl methacrylate, 4-vinylphenol, and 2- hydroxypropylacrylate) .
  • acrylamide hydroxy-containing acrylamide derivatives
  • methacrylamide, hydroxy-containing methacrylamide derivatives e.q. N-tris-hydroxymethyl-methy1-acrylamide, methylolacryl
  • Hydrophobic monomers useful in creating sorbent ⁇ for hydrophobic chromatography include octyl-acrylamide or - methacrylamide, phenyl-aerylamide, butyl-acrylamide, benzyl- acrylamide, and triphenylmethy1-acrylamide.
  • Activated monomer ⁇ useful in creating preactivated sorbents include glycidylacrylate or -methacrylate, acrolein, acrylamidobutyraldehyde dimethylacetal, acrylic-anhydride, acryloyl chloride, N-acryloxy ⁇ uccinimide, and allyl- chloroformate.
  • the pa ⁇ ivation mixture further compri ⁇ e ⁇ an appropriate amount of a passivating or neutralizing monomer capable of neutralizing the non-specific adsorption properties of innate sites on the surface of the porous solid support.
  • a passivating or neutralizing monomer capable of neutralizing the non-specific adsorption properties of innate sites on the surface of the porous solid support.
  • the amount of neutralizing monomer to be used is preferably an amount sufficient to counteract approximately up to an equivalent number of Si-OH groups pre ⁇ ent at the exterior and interior surfaces of said support.
  • the amount of neutralizing monomer should be about 0.5% to about 6% (w/v), preferably about 1.5 to about 3% (i.e., about 1.5-3 grams of neutralizing monomer per 100 ml of monomer solution) .
  • Suitable neutralizing monomers for use in the present invention may be monomer ⁇ bearing a po ⁇ itive charge at a neutral pH; example ⁇ include monomers containing a cationic amine group, such as substituted amines or pyridine and the like.
  • the cationic neutralizing monomers must have at least one double bond, such as vinyl, acrylic, or allylic monomers.
  • cationic monomers or monomers which are able to engage in hydrogen bonding are al ⁇ o useful as neutralizing monomers in a particular embodiment of the present invention.
  • Preferred neutralizing cationic monomers of the present invention include, but are not limited to, diethylaminoethyl acrylamide, diethylaminoethyl methacrylamide, diethylaminoethyl methacrylate, methacrylamide propyltrimethyl ammonium halide, triethylaminoethyl acrylamide, triethylaminoethyl methacrylate and copolymers thereof.
  • Polyoxyethylene-containing monomers can also be used. Thi ⁇ latter group can interact with polar group ⁇ (via hydrogen bonding) .
  • Preferred neutralizing monomers able to induce hydrogen bonding are polyoxyethylene monomers like poly(ethylene glycol) n -dimethylacrylate, where "n" is between about 50 and about 1000.
  • Preferred neutralizing hydrophobic monomers include, but are not limited to, N-alkylacrylamide in which the alkyl groups are branched, N-alkylacrylamide methylene chains having up to about 20 carbon atom ⁇ in the alkyl moiety, and N-arylacrylamide derivatives, like N-benzylacrylamide, N,N- (1,l-dimethy1-2-phenyl)ethyl-acrylamide, N-triphenyl methylacrylamide, or N,N-dibenzyl acrylamide.
  • Specific representative passivating monomers useful in treating polymeric or polymer-coated matrices include, but are not limited to, N-tert-octylacrylamide (TOA) , N-(l- ethylundecyl)-acrylamide (MUA) , N-(1 ,1,3,5-tetramethyl)- octylacrylamide (TMOA) , Triton-X-100-methacrylate (TWMA) , and polyethyleneglycol-dimethacrylate (PEG-DMA) .
  • TOA N-tert-octylacrylamide
  • UOA N-(l- ethylundecyl)-acrylamide
  • TMOA N-(1 ,1,3,5-tetramethyl)- octylacrylamide
  • TWMA Triton-X-100-methacrylate
  • PEG-DMA polyethyleneglycol-dimethacrylate
  • Hydrophobic adsorption sites present on the internal surface ⁇ of ⁇ ome organic (i.e., polymeric) porous matrices like poly ⁇ tyrene — or on protective polymer coating ⁇ deposited on porou ⁇ mineral oxide matrices — are neutralized using hydrophobic passivating monomers incorporating these aromatic and aliphatic hydrophobic moieties or substituents.
  • a bifunctional crosslinking agent is added to the mixture comprising the neutralizing and main monomers.
  • the crosslinking agent allows the three-dimensional insoluble polymeric network to form within the pore volume of the porou ⁇ matrix.
  • the polymer formed would be linear and thus soluble.
  • the amount of crosslinking agent should be about 0.1% to about 10% (w/v).
  • the amount of crosslinking agent can be calculated based on the total weight of main monomer and neutralizing monomer in use.
  • the amount of crosslinking agent is from about 3 to about 10 percent by weight of the total weight of main and neutralizing monomers.
  • the cros ⁇ linking agent ⁇ u ⁇ ed in the present invention are acrylic, vinylic or allylic monomers that possess at least two polymerizable functional group ⁇ .
  • Preferred cro ⁇ slinking agent ⁇ have at lea ⁇ t two double bonds and include, but are not limited to, N,N'-methylene-bis- acrylamide, N,N'-methylene-bismethacrylamide, diallyl tartradiamide, allyl methacrylate, diallyl amine, diallyl ether, diallyl carbonate, divinyl carbonate, divinyl ether, 1,4-butanedioldivinylether, and l,3-diallyloxy-2-propanol.
  • porous mineral oxide matrices u ⁇ ed in the present invention include but are not limited to silica, alumina, transition metal oxides (including but not limited to titanium oxide, zirconium oxide, chromium oxide, and iron oxide) and any other ⁇ imilar ceramic material including silicon nitride and aluminum nitride.
  • the preferred mineral moietie ⁇ of the pre ⁇ ent invention include silica, zirconium oxide, and titanium oxide.
  • the most preferred mineral moiety is porous silica of a particle size of about 5 ⁇ m to about 1000 ⁇ m, having a porous volume of about 0.2 to about 2 cm 3 /gr, a pore size of about 50 to about 6000 A, and a surface area of about 1 to about 800 m 2 /gr. At this time, most all of the aqueous solution will have been absorbed by the mineral support, leaving a substantially dry, solid porous matrix.
  • non-aqueous dispersing medium include non-polar organic solvents known to those skilled in the art.
  • non-aqueous medium for suspending the treated matrix may include but are not limited to mineral and vegetable oils, aromatic solvents, aliphatic low molecular weight solvents, or chlorinated solvents.
  • the most preferred non-aqueous media include toluene, methylene chloride, and hexane.
  • a polymerization starter is added to the mixture, now in a non-aqueou ⁇ medium, in order to initiate polymerization of the monomers within the ⁇ ilica pore ⁇ .
  • concentration of initiator is from about 0.1% to about 2%, preferably about 0.8% to about 1.2%.
  • the polymerization initiator can be added to the initial solution of passivation mixture prior to addition of that mixture to the porous solid matrix.
  • an initiator combination of ammonium persulfate and tetramethylethylenediamine (TMEDA) can be introduced ⁇ eparately.
  • One component (the water-soluble persulfate salt) is combined with the aqueous mixture of main monomer, neutralizing monomer, and cros ⁇ linking agent, while the other component (TMEDA) i ⁇ combined with the non-aqueou ⁇ dispersing medium.
  • TEDA water-soluble persulfate salt
  • the persulfate/TMEDA combination is particularly useful because TMEDA displays appreciable solubility in water.
  • the TMEDA is able to penetrate the pore ⁇ of the treated ⁇ upport and thereby initiate polymerization, particularly upon heating.
  • Typical polymerization initiator ⁇ known to tho ⁇ e ⁇ killed in the art can be u ⁇ ed in the pre ⁇ ent invention.
  • these initiators may be capable of generating free radicals.
  • Suitable polymerization starters include both thermal and photoinitiators.
  • Suitable thermal initiator ⁇ include but are not limited to ammonium persulf- ate/tetramethylethylene diamine (TMEDA), 2,2'-azobis-(2- amidino propane) hydrochloride, potassium persulfate/dimethylaminopropionitrile, 2,2*-azobis(isobutyro- nitrile) , 4,4'-azobis- (4-cyanovaleric acid), and benzoyl- peroxide.
  • TEDA ammonium persulf- ate/tetramethylethylene diamine
  • 2,2'-azobis-(2- amidino propane) hydrochloride potassium persulfate/dimethylaminopropionitrile
  • Preferred thermal initiators are ammonium persulfate/ tetramethyethylenediamine and 2,2'- azobis(isobutyronitrile) .
  • Photo-initiators include but are not limited to: isopropylthioxantone, 2-(2'-hydroxy-S'- methylphenyl)benzotriazole, 2,2'-dihydroxy-4- methoxybenzophenone, and riboflavin. It is further contemplated that riboflavin be used in the pre ⁇ ence of TMEDA.
  • the persulfate i ⁇ preferably added prior to the addition of the non-aqueou ⁇ medium, since persulfate is much more soluble in water than in non-aqueous disper ⁇ ing media.
  • the polymerization step can take place in the presence of a pore inducer.
  • the pore inducer ⁇ of the pre ⁇ ent invention allow polymerization to take place without ⁇ ubstantially reducing the porosity of the solid support.
  • Suitable pore inducers also referred to as porogens, used in the present invention include but are not limited to polyethylene glycols, polyoxyethylenes, polysaccharides such as dextran, and polar ⁇ olvent ⁇ .
  • Polar solvent ⁇ include those commonly used in chemical synthesis or polymer chemistry and known to those skilled in the art.
  • Suitable polar solvents include alcohols, ketones, tetrahydrofuran, dimethylformamide, and dimethylsulfoxide.
  • Preferred polar solvents are ethanol, methanol, dioxane, and dimethylsulfoxide.
  • Porous polymeric matrices amenable to passivation by the methods of the present invention include, but are not limited to polystyrene, polysulfone, polyethersulfone, various cellulose esters (e.g., cellulose acetate, cellulose nitrate), polyolefins (e.g., polyethylene and polypropylene), polyvinylacetate (and partially hydrolyzed versions thereof) , polyacrylates, polyvinylidene fluoride, polyacrylonitrile, polyamides, polyimides, and various blends, mixtures, and copolymers thereof.
  • Procedure ⁇ for the manufacture of porous particles and other structures (e.g., microporous membranes) from such polymers are generally known in the art.
  • the polymer surface to be pa ⁇ ivated is in the form of a thin, protective coating residing upon the pore walls of mineral oxide ⁇ ub ⁇ trate that is thu ⁇ ⁇ tabilized again ⁇ t leaching
  • the polymer will generally consi ⁇ t of a linear, high-molecular-weight polymer capable of being dissolved in a suitable organic solvent.
  • a coating ⁇ olution of linear poly ⁇ tyrene e.g., with an average molecular weight 400 kil ⁇ daltons
  • a chlorinated hydrocarbon ⁇ uch as methylene chloride is conveniently prepared by dis ⁇ olving the polymer in a chlorinated hydrocarbon ⁇ uch as methylene chloride.
  • Typical concentration ⁇ of polymer in the coating solution range from about 2% (w/v) to about 20% (w/v)
  • the ideal concentration is determined by achieving a balance between effectivenes ⁇ in preventing or minimizing leaching of the mineral oxide substrate (which argues for higher polymer concentrations) and the constriction of pore ⁇ and partial lo ⁇ of porous volume (and sorption capacity) that can occur at higher polymer concentrations.
  • protective coatings of polystyrene are deposited on porous ⁇ ilica, a poly ⁇ tyrene concentration of about 10% (w/v) is preferred.
  • the coating is applied by first impregnating the porous support with the solution of protective coating and then removing the solvent vehicle by evaporation.
  • a modified polymerization procedure is advantageously employed where polymeric surfaces are to be passivated, which procedure entails a so-called "dry polymerization" procedure as opposed to that described above involving an oil-phase dispersing medium.
  • the porous matrix impregnated with aqueous pas ⁇ ivating mixture i.e., monomer ⁇ olution
  • undergoe ⁇ the polymerization reaction while in the form of an apparently "dry” and free- flowing powder typically agitated (e.g., by stirring or fluidization) in a closed, inert (e.g., nitrogen) atmosphere.
  • the dry polymerization reaction is typically conducted a-t a temperature from about 60 to 90°C, at a pressure of 1 to 2 bar ⁇ , and for a period ranging from about 2 hours to overnight.
  • aqueou ⁇ pa ⁇ ivating mixture in a careful, metered fashion (e.g., dropwise) to the porous matrix, so that little or no exces ⁇ liquid-pha ⁇ e pa ⁇ ivating mixture i ⁇ pre ⁇ ent.
  • organic co- ⁇ olvents e.g., ethanol, dimethyl ⁇ ulfoxide, and the like
  • the crosslinking agent is conveniently added to the final monomer mixture in the form of an aqueou ⁇ 10% ethanol solution.
  • the initiators i.e., polymerization catalysts
  • the initiators employed in this dry polymerization process are necessarily water-soluble and are generally thermally activated.
  • a representative thermally activated polymerization initiator is azo-bi ⁇ -amidinopropane.
  • polymeric and polymer-coated mineral oxide matrices may be treated with hydrophilic polymers such as polyoxyethylene (POE) and polyvinylpyrrolidone (PVP) prior to effecting the polymerization and crosslinking of the monomer solution within the pore ⁇ of the ⁇ upport.
  • hydrophilic polymers such as polyoxyethylene (POE) and polyvinylpyrrolidone (PVP)
  • PEO polyoxyethylene
  • PVP polyvinylpyrrolidone
  • the polymerization process of the present invention creates a three-dimensional lattice or cro ⁇ -linked polymer network that extend ⁇ away from the pore-wall ⁇ urface ⁇ of the porou ⁇ solid matrix.
  • the three-dimensional shape of the polymer lattice is believed to be ⁇ ub ⁇ tantially identical to the shape of the pore which it fill ⁇ ( ⁇ ee Figure 5) , with the passivating layer oriented adjacent to and continuous (i.e.
  • Thi ⁇ configuration prevents "neutralizing” or “deactivating" pieces of the polymer network from eluting from the ⁇ upport during regular u ⁇ e — for example, when the pa ⁇ ivated porou ⁇ support is exposed to vigorous washing or cleaning conditions, such as high acidic pH, high alkaline pH, high ionic ⁇ trength, and ⁇ trong oxidizing condition ⁇ .
  • Thi ⁇ crosslinked polymer network creates a novel chromatographic ⁇ orbent which can then be u ⁇ ed, for example, in a proce ⁇ for ⁇ eparating and purifying variou ⁇ biomolecule ⁇ , including macromolecule ⁇ .
  • the passivated porous supports of the present invention manifest chromatographic characteristic ⁇ that are unparalleled under ⁇ everal criteria, particularly in terms of dynamic sorptive capacity as a function of flow rate.
  • the passivated porou ⁇ supports of the present invention show little decrease in useful sorptive capacity from a static condition up to flow rate ⁇ approaching 200 cm/h. Compare, for example, the behavior of prior art "gel"-type material ⁇ with the supports of the present invention, as illu ⁇ trated in the graphs of Fig. 3A, 3B, and 4 (described further in Example 16) .
  • the absolute capacities of the passivated porous ⁇ upport ⁇ of the pre ⁇ ent invention are considerably greater than even those attained with other types of solid I support ⁇ (e.g., SpherodexTM) that exhibit a similar insensitivity to high flowrates.
  • solid I support ⁇ e.g., SpherodexTM
  • a plot of the absolute capacity vs. flowrate of various solid supports unambiguously show ⁇ that the passivated solid support ⁇ of the present invention combine a very high absolute sorption capacity (expres ⁇ ed as mg/ml) with a relative insensitivity to solution flowrate ⁇ .
  • the polymeric network of the present invention extends outwardly into the pore volume itself in the manner of a three-dimen ⁇ ional lattice, as opposed to a two- dimensional coating limited strictly to the pore wall ⁇ urface area.
  • a schematic diagram of ⁇ uch a ⁇ tructure, as it is thought to exist, is illustrated in Fig. 5, where a biological molecule of interest (depicted as a spherical object) is also shown interacting with the lattice.
  • porogens pore-inducers
  • a method of performing chromatographic ⁇ eparation ⁇ characterized by high sustained sorptive capacity independent of flowrate and rapid, efficient mass transfer is achieved with the pas ⁇ ivated porous supports of the present invention, which supports include an open, flexible three-dimensional network or lattice of crosslinked polymer chains extending within and throughout the pores of the ⁇ upport matrix.
  • the ⁇ eparation and purification process usually involves at least two steps. The first step is to charge a packed or fluidized bed column containing the pas ⁇ ivated porous ⁇ olid support with a solution containing a mixture of biomolecules, at lea ⁇ t one of which it is desired to ⁇ eparate and recover in at least partially purified form. The second step is to pass an eluent solution through said column to effect the release of said biomolecules from the column, thereby causing their separation.
  • Stepwise elution can be effected, for example, with a change in solvent content, salt content or pH of the eluent solution.
  • gradient elution techniques well known in the art can be employed.
  • proteins reversibly bound to cation exchange media can generally be eluted by increasing the pH to alkaline values ( ⁇ ubject to limits as ⁇ ociated with the chemical stability of the protein)
  • immunoglobulins bound to protein A or like adsorbent ⁇ may be eluted by decrea ⁇ ing the pH to acidic values.
  • Porosity factor i the ratio between elution volume (Ve) of a protein (e.g., BSA in our case) and the total volume (Vt) of the packing bed determined under physiochemical condition ⁇ (e.g. , high ionic strength) in which no interaction exists between the protein and the porous support.
  • Sorption capacity is the amount of adsorbed protein in "mg" per unit volume (ml) of pas ⁇ ivated porous support bed determined under particular conditions: for cationic sorbents: 50 m M Tris-HCl, pH 8.6. for anionic sorbents: 50 MM Acetate, pH 6.5.
  • Jon exchange capacity is the number of ionizable groups in ⁇ eq per unit volume (ml) of passivated porous support bed determined by titration.
  • EXAMPLE 1 Preparation of a porous cation-exchange resin.
  • acrylamidomethyl propane sulfonic acid (AMPS) sodium salt and 1 g of N,N'-methylene-bisacrylamide (MBA) are dis ⁇ olved in 50 ml of di ⁇ tilled water.
  • 500 mg of ammonium persulfate are added at room temperature.
  • porous silica 40 to 100 ⁇ m diameter ⁇ , 1000 to 1500 A pore diameter, 20 to 35 M 2 /g ⁇ urface area and 1 CM 3 /g porou ⁇ volume
  • MATAC methacrylamidopropyl trimethyl ammonium chloride
  • MSA N,N'-methylene-bis-acrylamide
  • N,N,N',N'-tetramethylenediamine is added to polymerize the monomer solution inside the silica pores.
  • Ion-exchange capacity 114 ⁇ eq of quaternary ammonium groups per ml of resin. - No visible pre ⁇ ence of acidic ( ⁇ ilanol) group ⁇ on titration curve.
  • Sorption capacity for bovine serum albumin (BSA) gi mg/ml resin.
  • Example 2 Three 80 ml solutions each containing two monomers (MAPTAC and MBA) are prepared according to Example 2, using varying amounts of MBA: 0.5 g, 1 g and 2 g.
  • the anion-exchange resin ⁇ differ by the following properties:
  • Amount of MBA 0.5 g lg 2g Ionic charges per ml of resin: 36 ⁇ eq 114 ⁇ eq 218 ⁇ eq
  • Sorption capacity per ml (BSA) 35 mg 91 mg 72 mg
  • MBMA N,N'-methylene-bis-methacrylamide
  • DMSO dimethylsulfoxide
  • Ion-exchange capacity 201 ⁇ eq of quaternary amino group ⁇ per ml of resin.
  • Sorption capacity for BSA 112 mg/ml.
  • Three different resins are prepared according to Example 4 with differing amounts of MBMA as a crosslinking agent.
  • the amount of MBMA is varied as follows: 0.5 g, 1 g and 2 g. Paraffin oil is used as the non-aqueous (organic) solvent at
  • Amount MAPTAC 20g 20g 20g
  • Amount MBMA 0.5g ig 2g
  • Porosity factors 0.52 0.52 0.51 for BSA (V e /V t ) .
  • All of the above resins are stable to oxidizing agents, such as hypochlorites and peraceticacid.
  • silica having a surface area of 5 m 2 /g i ⁇ 50% lower than when u ⁇ ing a ⁇ ample having a surface area of 25 m 2 /g.
  • aqueous solutions of monomers are composed of: MAPTAC: 3 g (monomer to neutralize the silanol groups of silica)
  • the final properties of the final cation-exchangers obtained are a ⁇ follows: - Quantity of AMPS: 7 g 10 g
  • EXAMPLE 8 Preparation of a strong cation-exchange resin with MBRA as crosslinker.
  • MBMA 0.5 g of MBMA are dissolved in 50 ml of DMSO while stirring. To this ⁇ olution 30 ml of aqueous ⁇ olution containing 10 g of AMPS i ⁇ added as well as 6 ml of a 50% aqueous solution of MAPTAC.
  • the final volume is adjusted to 100 ml prior the addition of 1 g of ammonium per ⁇ ulfate at room temperature.
  • the final cation-exchange re ⁇ in shows the following characteristics: ion-exchange groups per 123 ⁇ eq ml of re ⁇ in: - sorption capacity for 128 mg cytochrome c: porosity factor 0.82 for lysozyme re ⁇ i ⁇ tance to oxidizing Excellent even at a agent ⁇ (NaoCl) : concentrated form (1/10 dilution of commercial) concentrated product.
  • the volume of the solution is then adju ⁇ ted to 100 ml, the pH adjusted to about 4.5, and 1 g of ammonium per ⁇ ulfate i ⁇ added at room temperature.
  • a non-aqueous water-immi ⁇ cible ⁇ olvent e.g., paraffin oil, toluene, or methylene chloride
  • Sorption capacity for cytochrome c 118 mg
  • the monomers comprising the initial ⁇ olution are the following:
  • composition of the solutions are:
  • V e /V, for bovine albumin is respectively 0.71, 0.74 and 0.61.
  • the resin is utilized to immobilize either a dye (Cibacron Blue F3GA) or heparin.
  • the obtained resins show the following characteristics:
  • the exclusion limit is actually larger when PEG is added.
  • EXAMPLE 12 Further separations of protein mixtures by ionic resins. Two resins are used to show their ability to separate protein mixtures rapidly and efficiently: a cationic resin (quaternary ammonium resin from Example 5) . an anionic sulfonated resin (see Example 8) .
  • the cationic resin (201 ⁇ eq quaternary amino groups/ml) is packed in a column of 1 cm in diameter and 8 cm in length and then equilibrated with a 0.05 M Tris-HCl buffer, pH 8.5. A ⁇ ample containing 1 mg of cytochrome c, hemoglobin, betalactoglobulm and ovalbumin is injected and ⁇ eparated under a salt gradient.
  • the artionic resin (138 ⁇ eq S0 3 groups/ml) i ⁇ packed in a column of 1 cm in diameter and 7 cm in length and then equilibrated with a 0.05 M acetate buffer, pH 4.5. A sample containing ovalbumin, betalactoglobulm, cytochrome c and lysozyme is injected and separated under a salt gradient.
  • Two aqueous solutions of monomer (100 ml each) are prepared according to Example 1 differing essentially by the presence of the cationic monomer MAPTAC.
  • Silicas chosen are the following:
  • the degree of passivation is estimated by the measurement of non-specific adsorption of lysozyme.
  • Non-specific ads 55 mg 13 mg 13 mg 0 mg 0 mg 15 mg 0 mg 0 mg (Lyzozyme)
  • the level of nonspecific adsorption for lysozyme (a strong cationic protein) is high when the MAPTAC is absent.
  • the non-specific adsorption for silica with large surface ares (X 075, 100m 2 /g) is higher (55 mg/ml of 5 resin) than the non-specific adsorption for silica X 015 (25 m 2 /g; 15 mg/ml of resin) .
  • a certain proportionality exists between the surface area and the original level of non-specific absorptions.
  • the amount of MAPTAC to decrease the level of non ⁇ specific adsorption down to zero is also proportional to the 0 surface area available: 1.5% of MAPTAC is necessary with silica X 015 (25 m 2 /g) whereas at least 6% is necessary to passivate silica X 075 (100 M 2 /g) •
  • EXAMPLE 15 Preparation of an Anion Exchange Resin Based on Polystyrene. 5 10 g of methacrylamidopropyltrimethylammonium chloride, 2 g of N-(l,1-dimethy1-2-phenyl)ethylacrylamide and 2 g of N,N,- methylene-bis-methacrylamide are dissolved in 30 ml of dimethyl sulfoxide. The volume of the solution is then increased to 50 ml by adding 20 ml of water. Under stirring, 0.3 g of 2,2,- o azobis-(2-amidinopropane)- hydrochloride is added at room temperature.
  • porous polystyrene 50 g of porous polystyrene (50-150 ⁇ m beads diameter, 300-400
  • the excess of monomer solution is thus s eliminated by filtration under vacuum.
  • the impregnated polystyrene beads are introduced into a closed container and heated at 80-90°C for five hours to polymerize the monomer solution within the pores of the polystyrene matrix.
  • the performance characteristics of the passivated porous support are compared with those of other support materials under high solution flow rates (e.q., approaching 100 cm/h).
  • the relative sorption capacity and productivity characteristics of DEAE-SpherodexTM, DEAE-Trisacryl PlusTM, DEAE- TrisacrylTM, DEAE-Agarose-based sorbent, and passivated porous supports of the present invention are illustrated in Figs. 3 A and 3 B.
  • the absolute sorption capacities at flow rates approaching 200 cm/h are compared for these supports in Fig. 4.
  • the data of Fig. 4 are generated for a 50 mM Tris buffer (pH 8.6) solution of BSA (5 mg/ml).
  • the useful sorption capacity decreases by half or more at flow rates between about 50 cm/h to about 100 cm/h for Trisacryl, Trisacryl Plus and the Agarose-based sorbent.
  • the degree to which the useful sorption capacity of the passivated porous supports of the present invention e.g. , the passivated support of Example 2 or 4
  • the degree to which the useful sorption capacity of the passivated porous supports of the present invention compares favorably with DEAE-SpherodexTM even at flow rates approaching 100 cm/h (i.e., the useful sorption capacity remains substantially unchanged as a function of flow rate) .
  • Fig. 3 B the productivity, a measure of the amount of material processed in the separation procedure per unit time, of the respective supports are compared in Fig. 3 B.
  • the performance of the passivated porous supports of the present invention compares favorably with the DEAE -SpherodexTM sorbent.
  • the passivated porous supports of the present invention are clearly superior to DEAE-SpherodexTM, however, when their sorption capacities are compared on an absolute basis, as shown in Fig. 4.
  • EXAMPLE 17 Preparation of an Anion-Exchange Resin using a Surface-Protected (i.e., Precoated) Silica Passivated Porous Support
  • Polystyrene pellets (10 g, average molecular weight about 400,000 daltons) are dissolved in 100 ml of methylene chloride and then added dropwise to 100 g of porous silica (40-100 gm diameter, 2000-3000 A pore diameter, 10 m 2 /g surface area and about 1 cm 3 /g porous volume) . After about 30 minutes shaking the mixture is dried under an air stream at room temperature until total evaporation of the chlorinated solvent (i.e., until a constant weight is observed) . The obtained dry powder is then heated at 190° C overnight to permit the polystyrene to form a homogeneous thin layer on the surfaces (internal and external) of the silica.
  • MATAC methacrylamidopropyl trimethyl ammonium chloride
  • N,N'-methylene-bismethacrylamide 20 g
  • ammonium persulfate 20 ml of distilled water.
  • the two solutions are then mixed together at room temperature and added dropwise to 100 g of polystyrene-coated silica, obtained as described above.
  • paraffin oil 250 ml is added to the mixture, along with 2 ml of N,N,N' ,N'-tetramethylethylenediamine to polymerize the monomer solution inside the silica pores.
  • the resulting suspension is then heated at 60-70°C to induce polymerization.
  • the passivated resin is then recovered by filtration.
  • the oil is eliminated with an extensive washing with water containing 0.1-0.5% of a non-ionic detergent and then stored in a saline buffer at neutral pH.
  • the product resin shows very similar ion-exchange characteristics as those described in Example 2. Additionally, its sensitivity in strong alkaline media is much improved as measured by its weight loss after one night of contact with 0.5 M sodium hydroxide.
  • the passivated resin of this example lost only about half as much weight as an artionic resin prepared from silica having an unprotected surface area.
  • the polystyrene can be coated on the surfaces of the matrix by polymerizing the vinyl monomer in situ, thus assuring that the internal surfaces of even the smallest pores of the matrix are coated with protective polymer.
  • the conditions for the polymerization of the vinyl monomer are well known to those of ordinary skill (e.g. , see. Kirk-Othmer Concise Encyclopedia of Chemical Technology, Wiley-Interscience Publication, New York, pp. 1115-1117) . After such an in situ polymerization, it is preferred that the coated support be heated overnight at 190 ⁇ C, as described above, to provide a homogeneous thin-film layer over the matrix.
  • polystyrene may also contain substituents, particularly at the 4-position of the phenyl ring, which can be non-ionic or ionizable.
  • substituents particularly at the 4-position of the phenyl ring, which can be non-ionic or ionizable.
  • carboxylic acids, carboxylic acid esters or amides, sulfates, phosphates, N,N- dialkylcarboxamides, lower alkylamines, N,N dialkylamines, quaternary ammonium groups, and the like can be present on the polymer.
  • a 4-iodo substituent on all or a portion of the phenyl groups of polystyrene would allow a large host of other functional group to be introduced by known methods (e.g., formation of aryllithium, Grignard, or copper reagents followed by quenching with carbon dioxide or alkylation) .
  • passivation of the porous solid matrix having a thin-film coating of a synthetic organic polymer can also be achieved by other variations in the procedure disclosed in the present invention, such as the method of Example 15.
  • Particle size 40-100 40-100 40-100 Porous volume (cm 3 /g) 1 1 1 Pore size (Angstroms) 3000 1250 300 Surface area (m2/g) 10 25 100 surface area is seen to increase as the pore size decreases, while porous volume remains essentially constant.
  • the characteristics of the passivated ("Q-CPI") anion- exchange support prepared from these silica base materials are summarized in the following table: Silica Matrix X-005 X-015 X-075 Particle size (microns) 40-100 40-100 40-100 Ionic groups (microeq/ml) 111 133 183 BSA capacity (mg/ml) 130 125 82 Sorption efficiency 1.17 0.94 0.45
  • the ion-exchange capacity (i.e., number of ionic groups) and BSA sorption capacity are seen to be relatively constant; in fact, these values decrease somewhat as the surface area of the silica support is increased) .
  • ion- exchange and BSA sorption capacities do not increase as the surface area of the silica increases (i.e., from left to right in the table) .
  • EXAMPLE 19 Preparation of an Anion-Exchange Resin Based on a Surface-Protected (i.e., Polystyrene- Precoated) Passivated Porous Silica Support
  • Polystyrene pellets (10 g, average molecular weight approximately 400 kD) were dissolved in 10 ml of methylene chloride and then added dropwise to 100 g of porous silica.
  • the silica was characterized by a particle diameter of 40 to
  • N-1-methylundecyl-acrylamide (MUA) were dissolved in 100 ml of pure ethanol, and the solution was added dropwise to 100 g of the polystyrene-coated silica obtained as described above. After shaking for about 30 minutes, the material was placed in a nitrogen stream under conditions that resulted in complete evaporation of the ethanol (again, as observed by attainment of constant solids weight) .
  • N,N'-methylene-bis-methacrylamide was dissolved in 20 ml of dimethylsulfoxide.
  • 20 g of methacrylamidopropyltrimethylammonium chloride (MAPTAC) were added, and the total volume of the solution was adjusted to 80 ml by the addition of distilled water.
  • 0.5 g of azo-bis-amidino-propane (as initiator) was dissolved in 10 ml of distilled water and then added to the solution of monomers. The volume of the latter was then adjusted to 100 ml with water; 90 ml of this solution were then added dropwise to the polystyrene-precoated silica.
  • This material i.e., monomer-solution-impregnated polystyrene-precoated silica
  • the product so obtained was then washed extensively with water and water-compatible solvents to remove any unpolymerized material and other reaction byproducts.
  • the cationic (i.e., anion-exchange) resin so prepared exhibited a fixed-charge density (i.e., ion-exchange capacity) of 150 microequivalents/ i of quaternary amino groups. Its capacity for reversibly absorbing BSA was 125
  • EXAMPLE 20 Preparation of a Cationic Resin Based on a 0 Porous Polystyrene Matrix
  • Porous polystyrene beads characterized by a particle diameter of 50 to 70 microns, a pore diameter of 1000
  • the mixture was stirred in a closed vessel under a nitrogen pressure at 85°C for at least 2 hours. After this period, the product beads were removed and 0 washed extensively with acidic, alkaline, and aqueous alcohol solutions to remove reaction byproducts and uncopolymerized materials.
  • the anion-exchange resin product obtained in this manner was very hydrophilic and contained cationic groups at a density of 124 microequivalents/ml of settled resin volume. Protein sorption capacity as measured by uptake of bovine serum albumin (BSA) was between 30 and 50 mg/ml of settled resin, depending on operating conditions.
  • BSA bovine serum albumin
  • Example 21 differs from the preceding Example 20 in its incorporation of the passivating monomer MUA into the mixture polymerized within the pores of the polystyrene support.
  • porous polystyrene beads characterized by a particle diameter of 50 to 70 microns, a pore diameter of 1000
  • the anion-exchange resin product obtained in this manner contains cationic groups at a density of about 115 microequivalents/ml of settled resin volume. Protein sorption capacity as measured by uptake of bovine serum albumin (BSA) is about 80 mg/ml of settled resin.
  • BSA bovine serum albumin
  • the resin is stable over a wide range of pH values (from 1 to 14) and can be used advantageously in the chromatographic separation of various protein mixtures.
  • EXAMPLE 22 Preparation of a Passivated Anionic Resin Based on a Porous Polystyrene Matrix
  • Example 22 differs from the preceding Example 21 in two respects: (i) its replacement (on a 1-for-I basis by weight) of an anionic monomer (acrylamido-methyl-propane sulfonic acid sodium salt) for the cationic monomer (MAPTAC) , used in the passivating mixture polymerized within the pores of the porous polystyrene support, and (ii) its use of N-(1,1,3,5- tetramethyloctyl)-acrylamide as opposed to N-lmethyl-undecyl- acrylamide (MUA) as the passivating or neutralizing monomer.
  • an anionic monomer acrylamido-methyl-propane sulfonic acid sodium salt
  • MUA N-(1,1,3,5- tetramethyloctyl)-acrylamide
  • MUA N-lmethyl-undecyl- acrylamide
  • porous polystyrene beads with a particle diameter of 50 to 70 microns, a pore diameter of 1000 Angstroms, and a porous volume of 1.6 cm 3 /g, are obtained from Polymer Laboratories, Inc. Five grams of these porous crosslinked polystyrene beads are washed extensively with ethanol and dried under vacuum.
  • 61 mg of methylene-bis-methacrylamide are o dissolved in 3.76 ml of dimethyl sulfoxide. To this are added 2.44 ml of an aqueous solution containing 1.3 g of acrylamido-methyl-propane sulfonic acid sodium salt and 25 mg of azo-bis-a idino-propane. To this solution, which is stirred gently under a nitrogen atmosphere at 4°C, are added _ 1.5 ml of pure ethanol containing 50 mg of N-(1,1,3,5- tetramethylocty1)-aerylamide as a passivating ("neutralizing") monomer.
  • This solution is then added dropwise to the dry polystyrene beads until it is totally absorbed within the porous volume of the beads. After 30 0 minutes of shaking, the mixture is stirred in a closed vessel under a nitrogen pressure at 85°C for 2 hours or more. After this period, the product beads are removed and washed extensively with acidic, alkaline, and aqueous alcohol solutions to remove reaction byproducts and uncopolymerized materials.
  • the cation-exchange resin product obtained in this manner is very hydrophilic and contains anionic (sulfonate) groups at a density of about 100 microequivalents/ml of settled resin volume. Protein sorption capacity as measured by uptake of lysozyme is about 95 mg/ml of settled resin.
  • the anionic resin is stable over a wide range of pH values (from 1 to 14) and can be used advantageously in the chromatographic separation of various protein mixtures.
  • EXAMPLE 23 Preparation of an Anion-Exchange Resin Using a surface-Protected (i.e., Pre-coated) and POE- Passivated Porous Silica support
  • Polystyrene pellets (10 g, average molecular weight approximately 400 kD) were dissolved in 10 ml of methylene chloride and then added dropwise to 100 g of porous silica.
  • the silica was characterized by a particle diameter of 40 to 100 microns, a pore diameter of 2000 to 3000 Angstroms, a surface area of 10 m 2 /g surface area, and a porous volume of about 1 cm 3 /g. After about 30 minutes of shaking, the mixture was dried under an air stream at room temperature until total evaporation of the chlorinated solvent had occurred, as evidenced by attainment of a constant particle weight. The dry powder was then heated overnight at 190-200°C.
  • This polystyrene-coated silica was then suspended in 200 ml of an aqueous solution of 5% polyoxyethylene (POE) with an average molecular weight of about 600 kD. The mixture was stirred gently for about 5 hours at 85°C and then the excess solution was removed by filtration. The silica beads were then washed extensively with water to remove the excess POE; the beads were finally rinsed twice with pure ethanol and dried.
  • POE polyoxyethylene
  • N,N'-methylene-bis-methacrylamide was dissolved in 20 ml of dimethylsulfoxide under stirring.
  • 20 g of methacrylamidopropyltrimethylammonium chloride was added, and the total volume of the solution was adjusted to 80 ml by the addition of distilled water.
  • 0.5 g of azo-bis-amidino-propane was dissolved in 10 ml of water and then added to the solution of monomers. The latter was then adjusted to a total volume of 100 ml with water.
  • Ninety milliliters of this solution were then added dropwise to the precoated POE-treated dry silica.
  • the cationic (i.e., anion-exchange) resin so obtained exhibited an ion-exchange capacity of 170 microequivalents/ml of quaternary ammonium groups and displayed a reversible BSA sorption capacity of-115 mg/ml. No non-specific binding was evident during a chromatographic separation conducted with the material.
  • EXAMPLE 24 Preparation of an Anion-Exchange Resin using a surface-Protected (i.e., Pre-coated) and PVP- Passivated Porous Silica Support
  • Polystyrene pellets (10 g, average molecular weight approximately 400 kD) are dissolved in 10 ml of methylene chloride and then added dropwise to 100 g of porous silica with characteristics described in the previous example. After about 30 minutes of shaking, the mixture is dried under an air stream at room temperature until total evaporation of the chlorinated solvent has occurred, as evidenced by attainment of a constant particle weight. The dry powder is then heated overnight at 190-200°C.
  • This polystyrene-coated silica is then suspended in 200 ml of an aqueous solution of 5% polyvinylpyrrolidone (PVP) with an average molecular weight of about 400 kD. The mixture is stirred gently for about 5 hour ⁇ at 85°C and then the excess solution is removed by filtration. The silica beads are then washed extensively with water to remove the excess POE; the beads are finally rinsed twice with pure ethanol and dried.
  • PVP polyvinylpyrrolidone
  • the silica, impregnated with monomer solution, is then placed in a closed vessel at 80°C and the polymerization is effected under nitrogen for two hours.
  • the product so obtained is washed extensively with water and water-compatible solvents at acidic and alkaline pH values to eliminate any unpolymerized materials and reaction by products.
  • the cationic (i.e., anion-exchange) resin so obtained exhibits an ion-exchange capacity of about 160 microequivalents/ml of quaternary ammonium groups and displays a reversible BSA sorption capacity of about 120 mg/ml. Little or no non-specific binding is evident during a chromatographic separation conducted with the material.
  • Polystyrene pellets (10 g, average molecular weight approximately 400 kD) are dissolved in 10 ml of methylene chloride and then added dropwise to 100 g of porous silica with the following characteristics: a particle diameter of 25 to 60 microns, a pore diameter of 3000 Angstroms, a surface area of 15 m 2 /g surface area, and a porous volume of about 1 cm 3 /g. After about 30 minutes of shaking, the mixture is dried under an air stream at room temperature until total evaporation of the chlorinated solvent has occurred, as evidenced by attainment of a constant particle weight. The dry powder is then heated overnight at 190-200°C.
  • This polystyrene-coated silica is then suspended in 200 ml of an aqueous solution of 5% polyoxyethylene and stirred gently for about 5 hours at 85°C. The excess solution is removed by filtration. The silica beads are then washed extensively with water to remove the excess POE; the beads are finally rinsed twice with pure ethanol and dried.
  • This silica, impregnated with monomer solution, is then placed in a closed vessel at 80°C and the polymerization is effected under nitrogen for a period of at least 3 hours.
  • the polyanionic product so obtained i ⁇ then washed extensively as described in the immediately preceding examples.
  • the resin so obtained exhibits an ion-exchange capacity of about 100 microequivalents/ml of sulfonate groups and displays a reversible lysozyme sorption capacity of about 130 mg/ml.
  • EXAMPLE 26 Preparation of a Cation-Exchange Resin using a surface - Protected (i.e.,. Precoated) Porous Silica Support
  • porous silica particles having a particle diameter of 25 to 60 microns, an average pore diameter of 3000 Angstro ⁇ , a surface area between 5-20 m 2 , and a porous volume of about 1 cm 3 /g were coated with polystyrene in the manner described in Example 24.
  • PEI polyethylenimine
  • regular ethanol 95%)
  • BDGE butanedioldiglycidylether
  • the total volume of the solution was adju ⁇ ted to 120 ml by the addition of regular ethanol.
  • Thi ⁇ PEI-ethanol solution was then mixed with a 100 g of the polystyrene coated porous silica described above. After approximately 30 minutes of mixing, the ethanol wa ⁇ evaporated by circulating nitrogen or air at 40°-45°C. Once the ethanol was eliminated, the mixture is heated to 80°-85°C to permit cros ⁇ linking of the PEI by the BDGE.
  • the pH of the solution was then adjusted to between 6.5 to 7.5 and demineralized water was added up to 100 ml/or to a volume corresponding to the porous volume of the coated ⁇ ilica.
  • Thi ⁇ solution was then added dropwise to the polystyrene-polyethylenimine coated silica.
  • the monomer solution was added while the coated silica was under agitation. After the monomer ⁇ olution wa ⁇ added, the mixture wa ⁇ agitated for an additional 30 minute ⁇ and then checked for the pre ⁇ ence of a dry aspect with no aggregates. Slowly an exces ⁇ of nitrogen wa ⁇ injected up to a pre ⁇ ure of about one bar. The ve ⁇ el wa ⁇ then clo ⁇ ed.
  • the mixture wa ⁇ heated under agitation up to 80°-85°C and maintained at thi ⁇ temperature for two hour ⁇ . The heating wa ⁇ then ⁇ topped and the mixture wa ⁇ ⁇ tirred gently overnight.

Abstract

L'invention a pour objet des supports solides poreux modifiés et des procédés de préparation et d'utilisation de ces derniers. En particulier, l'invention concerne des supports poreux passivés qui sont caractérisés par une forte capacité de sorption réversible ne s'accompagnant sensiblement d'aucune adsorption non spécifique de biomolécules ni d'aucune interaction avec ces dernières. La passivation est effectuée au moyen d'un mélange de passivation comprenant un monomère principal, un agent de passivation comprenant de la polyéthylénimine et un agent de réticulation . Lors de la polymérisation, ce mélange se traduit par l'élimination sensible de l'interaction non spécifique avec les biomolécules indésirable.
PCT/US1996/001891 1995-02-22 1996-02-13 Supports polymeres poreux passives et procedes de preparation et d'utilisation de ces derniers WO1996025992A1 (fr)

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