Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
  • G01N33/548—Carbohydrates, e.g. dextran
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39516—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum from serum, plasma
  • A61K39/39525—Purification
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F251/00—Macromolecular compounds obtained by polymerising monomers on to polysaccharides or derivatives thereof
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/26—Conditioning of the fluid carrier; Flow patterns
  • G01N30/38—Flow patterns
  • G01N2030/386—Radial chromatography, i.e. with mobile phase traversing radially the stationary phase
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/60—Construction of the column
  • G01N30/6004—Construction of the column end pieces
  • G01N30/6017—Fluid distributors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/60—Construction of the column
  • G01N30/6091—Cartridges

Definitions

• the present invention relates to carrier supports such as chromatographic supports, and methods for their preparation and use. More particularly, the invention relates to carrier supports compris ing a polymeric carrier grafted to a substrate for affinity chromatography media and for the affinity chromatography media made from these supports. The invention also relates to chromatographic devices which permit radial or tangential flow of a sample relative to the carrier support.
• agarose gels and cross-linked agarose gels have been the most widely used support materials. Their hydrophilicity makes them relatively free of nonspecific binding, but their compressibility makes them less attractive as carriers in large scale processing, such as in manufacturing.
• Controlled-pore glass (CPG) beads have also been used in affinity chromatography. Although high throughputs can be obtained with columns packed with CPG, this carrier is even more expensive than agarose beads.
• Cellulose particles have also been used by immunochemists for synthetic affinity sorbents.
• cellulose particles are formed with more difficulty and therefore, have received less attention in the preparation of affinity sorbents for enzymes.
• Cellulose is perhaps the least expensive of all support matrices.
• Two lesser-used support matrices are polyacrylamide gel beads and Sephadex R gel beads made from dextran and epichlorohydrin. Although convenient methods have been developed for using them, the softness of these beads yields poor column packings, and their low molecular porosity yields a sorbent with poor ligand availability to the ligate.
• a carrier for bio-active materials is also disclosed in Nakashima et al., U.S. Patent No. 4,352,884.
• the Nakashima carrier comprises a substrate coated with a copolymer.
• the substrate may be one of various materials, including inorganic materials such as glass; silica; alumina; synthetic high polymers such as polystyrene, polyethylene, and the like; and naturally occurring high polymers such as cellulose.
• the copolymer is made of a hydrophilic acrylate or methacrylate monomer which is a hydroxy or alkoxyalkylacrylate or methacrylate, and a copolymerizable unsaturated carboxylic acid or amine.
• the base material or substrate is coated with the copolymer by conventional coating or deposition procedures, such as spraying, dipping, phase separation or the like.
• the copolymer may also contain small amounts of a cross-linking agent such as glycidyl acrylate or methacrylate.
• the cross-linking agent allows for cross-linking treatment after the coating process, and provides for the prevention of elution (presumably of the bioactive materials) from the coating layer.
• the amounts of cross-linking agent are quite small, and range between 0.5 and 1% by weight of the total copolymer weight. Such amounts of cross-linking agent are insufficient to cause substantial covalent bonding or grafting of the copolymer onto the underlying substrate.
• the copolymer in Nakashima is thus essentially only physically coating the underlying substrate. Physical coating, however, is accompanied by a series of problems.
• the carrier would not be expected to have an even distribution of the copolymer, would show a multilayered structure, and may have a possible uneven distribution of functional groups.
• Kraemer U.S. Patent No. 4,070,308 which shows copolymers of glycidyl- and amino-containing acrylates, useful as carriers for biologically active substances, such as polysaccharides, enzymes, peptides, hormones, etc.
• the structure of the final product in Kraemer is that of an acrylic copolymer chain covalently modified at a multiplicity of sites thereon with substances such as enzymes, proteins, and the like.
• a fibrous/particulate matrix with addition of cationic polymers to the slurry and cross-linking the polymers to the matrices by oven-drying has yielded a filtration matrix with a positive charge coated on the surface thereof.
• Such charged matrices can be used for filtering minute amounts of particulate impurities from large volumes of liquid by adsorption. (See, for example, Ostreicher, U.S. Patent Nos. 4,007,113 and 4,007,114, as well as U.S. Patent Nos. 4,305,782 and 4,309,247, which are all herein incorporated by reference.)
• silanes which are chemically modified.
• Such silanes may carry functional groups such as DEAE, CM, or affinity chromatography sites. They are mechanically stable, strong and do not swell. However, they are expensive and show very high non-specific adsorption of protein by the silica hydroxy groups.
• Yet another object of the invention is to provide a chromatographic support useful for industrial-scale chromatographic operations.
• Still another object of the invention is to provide industrial processes of affinity chromatography and reverse-phase chromatography.
• Another object of the invention is to provide processes for the preparation of affinity and reverse-phase chromatographic supports and for affinity chromatographic media as well.
• Yet another object of the invention is to provide devices which permit radial or tangential flow of a sample relative to the carrier support.
• a modified polysaccharide material which comprises:
• said synthetic polymer made from a) a polymerizable compound which has a chemical group capable of being covalently coupled directly or indirectly to said polysaccharide; and b) one or more polymerizable compounds contai ning a chemi cal group capable of causing the covalent coupling of said polymerizable compound (b) to an affinity ligand or to a biologically active molecule.
• pre-ligand intermediates affinity molecular separation precursor compounds
• affinity molecular separation materials derived from the aforemen tioned polysaccharide materials, said affinity molecular separation materials comprising polysaccharide substrate, synthetic polymer covalently coupled to the polysaccharide substrate, and an affinity ligand covalently coupled to said synthetic polymer and capable of acting as chromatographic supports for affinity chromatography, reverse-phase chromatography, or as reagents for biochemical reactors.
• Still another object of the invention has been attained by providing molecular separation processes and/or biochemical reaction processes using the aforementioned materials.
• a further object of the invention has been attained by providing devices for the chromatographic separation of at least two components of a sample whereby the device is configured to allow the sample to flow radially or tangentially with respect to the modified polysaccharide material.
• Figure 1 is a partial sectional view of a side elevation of one embodiment of the chromatography column of this invention
• Figure 2 is an enlarged cross-sectional view taken along line 2-2 of Figure 1;
• Figure 3 is a perspective view of the radial flow cartridge with a portion of the solid stationary phase broken away therefrom showing the spirally wound chromatographic media and spacer means therebetween;
• Figure 4 is a cross-sectional view of another embodiment of the invention wherein the chromatography column is in disc configuration
• Figure 5 is a top plan view of the inlet housing member of the invention embodiment in disc configuration
• Figure 6 is a top plan view of the outlet housing member of the invention embodiment in disc configuration
• Figure 7 is a top plan view of one embodiment of the stationary phase of the invention column in disc configuration
• Figure 8 is a side elevation of one embodiment of the stationary phase of the invention column in disc configuration
• Figure 9 is a cross-sectional view of one embodiment of the stationary phase of the invention column in disc configuration depicting a plurality of layers of separation media and spacer means interposed between adjacent layers of said separation media, prior to the sonic welding of the peripheral edges;
• Figure 10 is a cross-sectional view of a preferred configuration for the ion exchange column of the present invention in disc configuration.
• the housing in disc configuration forms a radially outwardly expanding chamber.
• a portion of the spacer means is removed for clarity;
• Figure 11 is a perspective view of one embodiment of a tangential flow cartridge with a portion of the solid stationary phase broken away therefrom showing the spirally wound chromatographic media, spacer means, and impermeable membrane;
• Figure 12 is a cross-sectional view of one embodiment of a tangential flow cartridge within a cylindrical housing
• Figure 13 depicts one method used for constructing the tangential flow cartridge
• Figure 14 is a schematic diagram depicting a comparison of radial and tangential flow of blood in a device
• Figure 15 is a cross-sectional view of another embodiment of the invention which provides for tangential flow across two layers of chromatographic medi a;
• Figure 16 depicts a top plan view of the tangential flow cartridge having dual layers of chromatographic media
• Figure 17 depicts a cross-sectional view of one winding of the dual-layer tangential flow cartridge
• Figure 18 depicts the sample flow relative to the supporting and spacing and channeling means
• Figure 19 depicts a graph of the percent heparin removal versus the surface area for three media (A, protamin B, and C);
• Figure 20 depicts a graph of the percent heparin removal versus the contact time for two media (A and protamin);
• Figure 21 depicts a graph of the amount of heparin bound versus the ligand concentration (protamin) for two surface area quantities
• Figure 22 depicts a graph showing the pressure differential ( ⁇ p) between the inlet and outlet of a tangential flow cartridge versus the flow rate for blood and saline;
• Figure 23 depicts a graph of the percent heparin removal versus volume of blood filtered
• Figure 24 depicts a graph of the pressure differential ( ⁇ p) the inlet and outlet of a tangential flow cartridge versus flow rate for blood and saline;
• Figure 25 depicts a graph of the percent heparin removal versus volume of blood filtered through the tangential flow cartridge.
• Figure 26 depicts a graph of the pressure differential ( ⁇ p) between the inlet and outlet of a tangential flow cartridge, which contains immobilized protein A, for various flow rates.
• Figure 27 depicts a gel electrophoresis of IgG obtained from plasma and blood by purification through a tangential flow cartridge containing immobilized protein A.
• the present invention is related to the discovery and development of materials useful as insoluble supports for a variety of applications including a wide range of chromatographic separations, including affinity separations, or as insoluble supports for bioreactors.
• the support materials are based on a composite of an organic synthetic polymer and a polysaccharide.
• the composite per se is biologically inert.
• the organic synthetic polymer carries chemical groups which are capable of coupling to said polysaccharide, and also carries chemical groups which can provide anchoring capacity for affinity ligands or for biologically active molecules in general.
• the polymer coupled to the polysaccharide may be either a copolymer or a homopolymer.
• the chemical groups capable of coupling to the polysaccharide are the same as the chemical groups useful as anchoring units for affinity ligands or biologically active molecules, the polymer, in this particular form, would be a homopolymer.
• the polymer is a copolymer containing groups capable of coupling to the polysaccharide and also different groups capable of serving as anchoring groups for molecules.
• the polymer is a homopolymer.
• the invention also relates to materials derived from the modified polysaccharide by attaching thereto affinity ligands or biomolecules, to thereby obtain affinity chromatography or bioreactive materials, or attaching thereto hydrophobic substituents for reverse-phase chromatography.
• the invention also relates to mixtures of the aforementioned materials with unmodified polysaccharides, with modified or unmodified particulate material, or mixtures thereof to give a variety of separation media.
• the invention also relates to devices comprising the modified polysaccharide material of the invention which is configured for radial or tangential flow of the sample relative to the support material.
• polysaccharide as used in the specification and claims is meant to include compounds made up of many- -hundreds or even thousands - -monosaccharide units- per molecule. These units are held together by glycoside linkages. Their molecular weights are normally higher than about 5,000 and can range up to millions of daltons. They are normally naturally occurring polymers, such as, for example, starch, glycogen, cellulose, gum arabic, agar, and chitin. The polysaccharide should have one or more reactive hydroxy groups. It may be straight or branched chain. The most useful of the polysaccharides for the purposes of this invention is cellulose.
• the polysaccharide is preferably fully unprotected and carries all of its hydroxy groups in the free state. Some blocking of the hydroxy groups is possible, as for example, by acylation or aminoacylation. Extensive blocking of the hydroxy groups of the polysaccharide, however, is undesirable since the polysaccharide thereby loses its hydrophilic character, which is necessary to provide appropriate chemically compatible interaction with biomolecules. If the polysaccharide becomes too hydrophobic, negative interactions with such molecules as proteins lead to possible nonspecific bonding and denaturation phenomena. Also, if the masking of the polysaccharide hydroxy groups is too extensive, the reactivity of the resulting material with the polymer is greatly diminished. For all of these reasons, it is preferred to retain substantially all hydroxy groups in the free state.
• the polysaccharide may, however, be chemically activated, as seen infra.
• Cellulose is the preferred polysaccharide.
• cellulose it is intended to mean any of the convenient and commercially available forms of cellulose such as wood pulp, cotton, hemp, ramie, or regenerated forms such as rayon. There exists no criticality as to the selection of a suitable form of cellulose.
• Cellulose is a naturally occurring polysaccharide consisting of B(1>4) linked glucose units. In the native state, adjacent cellulose chains are extensively hydrogennbonded, forming microcrystalline regions. These regions are interspersed by amorphous regions with less hydrogen bonding. Limited acid hydrolysis results in preferential loss of the amorphous regions and gives so-called microcrystalline cellulose.
• the cellulose useful in the present invention is either cellulose in the native state or in the microcrystalline state. Also, cellulose derived from cotton 1 inter is better than that derived from wood pulp, as the latter contains lignin.
• Each anhydrous saccharide unit in a polysaccharide molecule may have three or more reactive hydroxy groups. Theoretically, all three or more can be substituted with the polymer. The product from such reaction, however, would have a degree of substitution of three or more, which in case of ion-exchange materials, would render it soluble. Even at levels of substitution below those at which total water solubility occurs, such polysaccharide derivatives become unsuitable as chromatographic supports. Therefore, substitution of the polysaccharide is restricted to the more reactive centers of the amorphous regions and is seldom carried out beyond the level of about 1 mEQ/gm of dry weight in fiber form. At this level of substitution, the native configuration of the polysaccharide structure is only slightly modified, and the low-density, non-uniform exchange sites are readily accessible to large biomolecules.
• the final structure of a molecular support of the invention thus comprises a polysaccharide chain covalently modified at a multiplicity of sites along such chain with the synthetic polymers.
• the polymer which modifies the polysaccharide is either a homopolymer or a copolymer.
• the definition of the polymer as a homo- or copolymer depends on whether the polymerizable compounds (a) and (b) are different.
• the copolymer could be a random, a block, or an alternating copolymer.
• the polymerizable compound (a) may have a group capable of reacting with a hydroxy group of polysaccharide with the formation of a covalent bond.
• Such polymerizable compounds are defined, for example, in U.S. Patent No. 4,070,348 to Kraemer et al., which is herein incorporated by reference.
• the chemical groups are capable of reacting with hydroxy groups at temperatures up to those at which the polysaccharide begins to decompose or depolymerize, e.g., 0o to 120oC, in aqueous solution and thereby form covalent bonds with the oxygen atoms of the hydroxy groups.
• Aqueous solutions comprise pure water or mixtures of water with one or more water-miscible co-solvents such as alcohols, ketones, and the like.
• Hydroxy reactive groups of comonomer (a) are preferably activated carboxy groups such as are known from peptide chemistry or O-alkylating agents, such as alkyl halide or epoxide groups.
• O-alkylating comonomers are acrylic and methacrylic anhydrides, acrylolylmethacrylol N-hydroxy succinimides, ⁇ -iodo-alkyl esters of acrylic or methacrylic acid in which the alkyl group in general contains two to six carbon atoms, allyl chloride, chloromethylstyrene, chloroacetoxy ethyl methacrylate, and compounds having a glycidyl group.
• the latter are ethers or esters formed between a glycidyl alcohol and an unsaturated alcohol or unsaturated carboxylic acid, respectively.
• the glycidyl alcohols are aliphatic and cycloaliphatic alcohols and ether alcohols having from 3 to 18 carbon atoms which are esterified with an ⁇ , ⁇ -unsaturated carboxylic acid, preferably acrylic or methacrylic acid, or are etherified with an olefinically or acetylenically unsaturated alcohol.
• Typical compounds are glycidyl acrylate and methacrylate; 4,5-epoxy-pentylacrylate; 4-(2,3-epoxypropyl)-N-butyl-methacrylate; 9,10-epoxy-stearylacrylate; 4-(2,3-epoxypropyl)-cyclohexyl methyacrylate; ethylene glycol-monoglycidyl etheracrylate; and allyl glycidyl ether.
• Preferred organic synthetic polymers include the homopolymers of glycidyl acrylate and glycidyl methacrylate.
• the active monomer units (a) are sensitive to hydroxy groups, and if they do not react with the polysaccharide offered, they may be transformed, in the presence of water, into hydrophilic carboxy, aldehyde, or hydroxy groups.
• the activated groups are therefore present in the polymeric material in generally greater number than is necessary for the bonding with the polysaccharide.
• the polymerizable compound (a) may be one which does not react directly with hydroxy groups of the polysaccharide, but rather is covalently coupled to the polysaccharide indirectly, via a bridge compound. This is the case when the polysaccharide is first chemically activated as by oxidation and reacted with a compound having, e.g., an epoxy group or a vinyl group capable of reaction with an appropriate functionality of polymerizable comonomer (a).
• the polymerizable comonomer (b), where (b) is different than (a), will vary depending on the ultimate use of the carrier material.
• comonomer (b) carries a chemical group capable of causing the covalent coupling of said comonomer (b) to an affinity ligand, i.e., an "anchoring" group. Since most affinity ligands carry nucleophiles such as hydroxy, amino, thiol, carboxy!ate, and the like, any electrophilic group capable of reacting with such nucleophile can be present in comonomer (b).
• electrophilic groups include, but are not limited to, those described previously as active groups capable of reacting with the hydroxy group of cellulose. They also include activated carboxy groups used in peptide chemistry for the formation of peptide bonds, such as carbonyl chlorides, carboxylic anhydrides, and carboxylic acid azide groups, as well as phenyl esters and aldehydes used for the formation of Schiff (imine) bases.
• R is an ⁇ , ⁇ -unsaturated, polymerizable radical and R' is a C 1 -C 6 alkyl or alkanoyl group, phenyl, or hydroxyphenyl.
• R" may be a direct bond (-) or a C 2 -C 3 alkyl or alkanoyl group.
• Other compounds having activated carboxy! groups include acryloyl and methacryloyl chloride, acrylic and methacrylic anhydride, maleic anhydride, phenyl acrylate and methacrylate, glycidyl acrylate and methacrylate, 4-iodobutylacrylate and methacrylate, and 2-isopropenyl4, 4-dimethyloxazolone-5.
• the last mentioned compound is capable of reacting with the terminal amino group of proteins, either directly or through conversion.
• a very useful potentially electrophilic reactive group in comonomer (b) useful for coupling to an affinity ligand is a group capable of being activated to an electrophilic group with a reagent such as a cyanogen halide. It is known in the art that cyanogen halides react with 1,2-diols to yield activated structures having the following formula (3):
• This structure is then capable of reacting with the nucleophile of an affinity ligand.
• 1,2-diols present in comonomer (b) are various saccharides, including monosaccharides such as glucose, mannose, and galactose; disaccharides such as lactose and maltose; trisaccharides such as raffinose; or, generally, glycosides.
• the 1,2-diol-containing functional group can be attached to the polymerizable comonomer (b) by such reactions as esterification, amide formation, etherification, and the like.
• the reaction of glycidyl acrylate or methacrylate with a saccharide to yield an ether-containing comonomer (b).
• any of the anchoring groups mentioned for comonomers (a) or (b) can also be used.
• Other types of activated groups such as those containing aldehydes or amines can also be used.
• the polymerizable comonomer (b) can be substantially of one type or can be a mixture of one or more types.
• the polymerizable monounsaturated compounds (b) are polymerizable compounds of the formula (4):
• R 1 is hydrogen or methyl
• A is CO, or SO 2 ;
• X is OH, OM (where M is a metal ion), or OR 2 (where R 2 is a straight or branched chain C 1 -C 18 alkyl group), OR 3 OH (where R 3 is a straight or branched chain C 2 -C 6 , alkyl, or aromatic group), ONR 4 R 5 or ON + R 4 R 5 R 6 , where R 4 is the same or different as R 5 which is the same or different as R 6 , and are hydrogen, R 2 , or R 3 OH);
• AX may also have formula (5):
• Y is -CO 2 -, -CH 2 CO 2 -, -SO 3 -, -CH 2 SO 3 -, -OPO 3 H-, -CH 2 PO 4 H-, -CH 2 N(CH 2 COO-) 2 , -CH 2 -NR 4 R 5 , or -CH 2 -N + R 4 R 5 R 6 , or the corresponding free acid, ester or partial ester groups, as described previously.
• the groups R 4 , R 5 ; R 4 , R 6 ; or R 5 , R 6 may form a fiveto seven-membered heterocyclic ring with the nitrogen atom.
• R 4 , R 5 , and R 6 are as previously defined.
• A is CO or SO 2
• X is most preferably O-CH 2 -CH(OH)-CH 2 -Saccharide, where "-Saccharide” is a mono-, di-, or polysaccharide having a group which can be activated for reaction with nucleophilic reactive groups on the affinity ligand or the biomolecule by a cyanogen halide.
• a more preferred comonomer (b) for anchoring materials is the comonomer obtained from reaction of glycidyl acrylate or methacrylate with glucose.
• the average molecular weight of the polysaccharide-modifying polymer is a function of the monomeric mix present therein. It is required to have at least a sufficient number of comonomers (a) units so as to be able to form covalent attachment throughout amorphous regions of the polysaccharide surface.
• the number of comonomers (b) units cannot be too small, since otherwise the exchange capacity, or the anchoring/interacting capacity, is negligible.
• the number of comonomers (b) units can neither be too high, since this would cause great difficulty in the reaction between the reactive groups of comonomer (a) and the polysaccharide.
• the polysaccharidemodifying copolymer carries anywhere between 1 and 500 units of comonomer (a) and most preferably between 20 and 100 units of comonomer (b). This corresponds to molecular weights of between about 100 and 100,000, preferably between 1,000 and 10,000.
• neutral comonomers (c) can also be added to the polymer, if desired.
• These comonomers may be polymerizable unsaturated compounds carrying neutral chemical groups such as hydroxy groups, amide groups, alkyl groups, aryl groups, and the like.
• Preferred among comonomers (c) are C 1 -C 6 alkyl acrylates or methacrylates, or the corresponding hydroxy alkyl acrylates or alkacrylates.
• the function of comonomers (c) may be to increase the presence of hydrophobic or hydrophilic residues in the polymers, so as to provide a desired balance of hydrophilic and hydrophobic groups, if necessary.
• the minimum ratio of comonomer (a) to total comonomer content is important.
• the synthetic polymer should have a sufficient amount of comonomer (a) to permit substantial covalent coupling of the polymer to the polysaccharide. If too little comonomer (a) is present in the polymer, then grafting becomes difficult, if not impossible.
• about 4-12, preferably 5-10% by weight of comonomer (a) relative to the total of (a) plus (b) and (c) (if any is present) is needed. Amounts of about 0.5 to 1 or 2% by weight appear to merely cross-link the polymer, without substantial grafting onto the polysaccharide.
• the upper limit of comonomer (a) in the polymer can be varied up to 99.9% by weight, depending on the desired amount of rigidity, functionality and hydrophilicity. It is preferred to have a predominance of comonomers (b) over comonomers (a). Comonomers (c) may be present in an amount of up to 20% by weight of the total (a) plus (b) plus (c).
• the weight ratio of polysaccharide to the modifying polymer is freely adjustable and varies from 0.1 to 5 weight parts of copolymer to parts by weight of the polysaccharide.
• Crosslinking can be provided either by incorporating into the polymerization recipe a small amount of polyunsaturated comonomer having at least two polymerizable ⁇ , ⁇ - carbon double bonds, such as for example mono- and polyethylene glycol dimethacrylates and diacrylates (with the polyethylene glycol residue containing up to six ethylene groups), ethylene dimethacrylate, ethylene diacrylate, tetramethy!ene dimethacrylate, tetraethylene diacrylate, divinylbenzene, triallyl cyanurate, methylene-bis-acrylamide or -bis-methacrylamide, and the like.
• polyunsaturated comonomer having at least two polymerizable ⁇ , ⁇ - carbon double bonds
• polyunsaturated comonomer having at least two polymerizable ⁇ , ⁇ - carbon double bonds such as for example mono- and polyethylene glycol dimethacrylates and diacrylates (with the polyethylene glycol residue containing up to six
• crosslinking agent is particularly applicable to copolymers made from an aminoalkyl comonomer (b). Because of the presence of a free pair of electrons on the aminoalkyl nitrogen atoms, crosslinking can be carried out with such bifunctional reagents as would react with nitrogen free electron pairs.
• bifunctional reagents such as the diacyl ha! ides, such as Hal-CO-(CH 2 ) n -CO-Hal, or the alkyl dihalides, such as Hal-(CH 2 ) n -Hal, wherein Hal is a halide such as chloride, bromide, or iodide, and n may be anywhere between 2 and 12.
• Other bifunctional reagents capable of reaction with nitrogen atoms can also be used. The advantage of these bifunctional reagents is that they simultaneously cross-link the copolymer, while also providing a cationic charge at the nitrogen centers, thereby ionizing the material.
• the amount of cross-linking agent is best determined empirically. It is to be considered sufficient when the polymer preserves the exchange capacity at a constant value over time, yet would be too high if swelling is prevented, and too much rigidity is obtained in the final materials. Ideally, an amount of cross-linking agent between 5 to 20 mole percent of the synthetic polymer units is sufficient.
• affinity ligand as used throughout the present application and in the claims is meant to include any small or high molecular weight molecule which can be immobilized in a stationary phase and used to purify a complementary binding molecule from a solute phase by affinity chromatography.
• a ligand can be an inhibitor, a cofactor, a prosthetic group, or a polymeric substrate, all of these useful to purify enzymes or holoenzymes.
• ligand/ligate pairs are enzyme/polymeric inhibitors; nucleic acid, single-strand/nucleic acid, complementary strand; hapten or antigen/ antibody; antibody/proteins or polysaccharides; monosaccharides or polysaccharides/lectins or receptors; lectins/glycoproteins or receptors; small target compounds/binding proteins; and binding protein/small target compounds.
• antigen/antibody pairs are used as the ligand/ligate pair, the technique takes the particular name of "immunoaffinity" chromatography.
• biologically active molecule which can be bound to the carriers of the invention can include enzymes, enzyme substrates, inhibitors, hormones, antibiotics, antibodies, antigens, peptides, saccharides, nucleic acids, and the like.
• the only requirement for these molecules is that they have reactive groups thereon which can be covalently coupled to the anchoring chemical groups on the synthetic polymer.
• enzymes such as hydrolases, isomerases, proteases, amylases, and the like. These immobilized enzymes can then be used in biochemical reactors, as is otherwise well known in the art.
• comonomer (b)(2) is usually an acrylate or methacrylate ester of C 6 -C 18 straight or branched chain alcohols, or of aromatic alcohols such as phenol or naphthol.
• the carrier materials of the present invention can be used per se in the same manner as other polysaccharide-based carrier materials of the prior art.
• the polysaccharide material which is preferably in fibrous form after the modification, can be formed into a self-supporting fibrous matrix, such as a fibrous sheet, with affinity chromatography properties, bioreactive or reverse-phase properties.
• the modified fibrous polysaccharide fibrous media can also incorporate unmodified fibers of various different sizes, and, in addition, can also incorporate modified or unmodified particulate material.
• the fibrous media comprises a porous matrix of fiber wherein, because of the nature of the present invention, the fiber is effective for molecule reactions.
• the matrix is substantially homogeneous with respect to each component.
• a particulate may, optionally, be present. When such a particulate is present, is preferred to modify it so that it is also effective for molecular separations or reactions. Such a particulate should be contained in the fibrous phase in an effective amount to achieve the desired separations or reactions.
• the overall media is substantially inert and dimensionally stable.
• the preferred particulates which can be used include all of those substances which can be provided in finely divided form and exhibit chromatographic functionality, i.e., capable of effective molecular separations and/or reactions. Mixtures of such compositions may also be utilized. Exemplary of such particulates are silica, alumina, zirconium oxide, diatomaceous earth, perlite, clays such as vermiculite, carbon such as activated carbon, modified polymer particulates such as other ion-exchange resins, crystalline cellulose, molecular sieves, and the like, the surfaces of which may be modified in a conventional manner.
• Such materials are commercially available under a variety of trademarks such as Biosila, Hi-Flosil, Li Chroprep Si, Micropak Si, Nucleosil, Partisil, Porasil, Spherosil, Zorbax cil, Corasil, Pallosil, Zipax, Bondapak, LiChrosorb, Hypersil, Zorbax, Perisorb, Fractosil, Corning Porous Glass, Dowex, Amberlite resins, and the like.
• trademarks such as Biosila, Hi-Flosil, Li Chroprep Si, Micropak Si, Nucleosil, Partisil, Porasil, Spherosil, Zorbax cil, Corasil, Pallosil, Zipax, Bondapak, LiChrosorb, Hypersil, Zorbax, Perisorb, Fractosil, Corning Porous Glass, Dowex, Amberlite resins, and the like.
• the particle size of the particulate is not critical but influences somewhat the flow rate at which the sample to be treated passes through the material. For radial flow applications, uniform particle sizes greater than about 5 microns are preferred, with about 10-100 microns constituting a practical operational range. Somewhat smaller particle sizes are possible for tangential flow applications where the sample flows across, and not through, the matrix material.
• the amount of the particulate can vary widely up to about 80 wt. % or more of the solid phase. The optimum particulate concentration will vary depending on the molecular separation desired.
• the fibrous media should be capable of immobilizing the particulate contained therein, i.e., capable of preventing significant particulate loss from the stationary phase.
• the media In the case of media configured for radial flow, the media should have a porosity which enables the fluid to pass through the media.
• the modified cellulose materials of the present invention are self-bonding and the addition of extra fibers or binders may not be necessary, it is possible to utilize such extra fibers or binders.
• Other fibers usable for the media include polyacrylonitrile fibers, nylon fibers, wool fibers, rayon fibers and polyvinyl chloride fibers, other cellulose fibers such as wood pulp and cotton, and cellulose acetate.
• One embodiment of the invention is the provision of a fibrous media comprising two different types of celluloses: one a modified cellulose according to the invention and another an unmodified cellulose.
• Another embodiment of the invention which may also be coupled with the aforementioned celluloses, is an unrefined structural fiber which assists in providing sheets of sufficient structural integrity in both the wet "as formed" condition, and in the final dry condition, and also allows handling during processing as well as suitability for the intended end use.
• Such fibers are typically relatively large, with commercially available diameters in the range of 6 to 60 micrometers.
• Wood pulp can also be used and has fiber diameters ranging from 15 to 25 ⁇ m, and fiber lengths of about 0.85 to about 6.5 mm.
• the unrefined self-bonding structural fibers typically have a Canadian Standard Freeness of +400 to +800 ml.
• These long self-bonding fibers may constitute greater than 50% of the fibrous media, by weight, preferably 60-100% of the fibrous media, and most preferably 100%.
• a minor portion of cellulose pulp which has been refined to a Canadian Standard Freeness of between +100 and -600 ml may be incorporated with a major portion of the normally dimensioned cellulose pulp (+400 to +800 ml).
• from about 1 to about 20% of the refined pump and about 50% to about 90% of the unrefined cellulose may be contained in the matrix. Particulate may also be added.
• the particulate materials are millimicron-sized
• modified cellulosic fibrous media carrying modified inorganic support materials such as for example are described in Regnier, U.S. Patent No. 3,983,299, Kirkland et al., U.S. Patent No. 3,795,313, Kirkland et al., U.S. Patent No. 3,722,181, Mazarquil et al., U.S. Patent No. 4,034,139, Talley et al., U.S. Patent No. 4,118,316, Ho Chang et al., U.S. Patent No. 4,029,583 or Regnier, U.S. Patent No. 4,108,603. These are all incorporated herein by reference.
• both the cellulosic fiber and the siliceous particulate are modified, and their interaction provides increased anchoring and/or ion-exchange capacity.
• the addition of particulate material tends to increase the rigidity and strength of the fibrous media and renders it readily useful for industrial applications, especially those involving high pressure.
• the polymer-modified polysaccharide material of the invention can be prepared in various modes. Generally speaking, in one mode, one can first prepare the polymer and then condense the same through its hydroxy reacting groups (if available) to the polysaccharide. Alternatively, in another mode, one can first react the polysaccharide with a hydroxy group-reactive comonomer (a) followed by copolymerization with comonomsr (b) and any other comonomers (e.g., crosslinking comonomers, hydrophobic comonomers, etc.), as desired.
• a hydroxy group-reactive comonomer
• comonomsr comonomsr
• any other comonomers e.g., crosslinking comonomers, hydrophobic comonomers, etc.
• reaction are therefore of two types: 1) coupling of saccharides to hydroxy reactive groups on comonomer (a), and 2) polymerization of polymerizable unsaturated compounds.
• the reaction is conducted under conditions wherein polymerization proceeds prior to covalent coupling.
• a third method of (indirectly) attaching the synthetic polymer to the polysaccharide involves previous chemical activation of the polysaccharide.
• the polysaccharide can be treated with oxidizing agents such as periodate, hydrogen peroxide, eerie or other metallic oxidizing ions or the like.
• oxidizing agents such as periodate, hydrogen peroxide, eerie or other metallic oxidizing ions or the like.
• Reaction of the activated polysaccharide with an amino-containing polymerizable monomeric compound followed by reduction, or an epoxy-containing vinyl monomer will normally yield derivatized polysaccharide carrying unsaturated functionalities along the chain thereof. These unsaturated functionalities can then serve as further attachment positions for conjugating the polymer thereto.
• Another type of chemical activation of the polysaccharide involves reaction with a compound such as a diepoxide or epichlorohydrin, which yields a derivatized polysaccharide-carrying epoxy or o ther groups along the chain thereof. These epoxy or other groups then serve as conjugating positions on the polysaccharide chains.
• Polymerization of comonomers can be carried out by radical chain (free radical), step-reaction, ionic and coordination polymerization. Particularly useful is free radical polymerization.
• the free radical addition polymerization of radical polymerizable comonomers is carried out with free radical initiators using well known steps of initiation, addition and termination.
• a usual procedure is to utilize a substance or substances which produce radicals capable of reacting with the monomers.
• Probably the simplest of all polymerization initiators are the organic peroxides and azo compounds. These substances decompose spontaneously into free radicals in common organic solvents at a finite rate, at temperatures between 50 and 140oC.
• benzoyl peroxide decomposes into two benzoyloxy radicals at 60oC.
• Another example is afforded by the azo compound azo-bis-isobutyronitrile which similarly decomposes into radicals at easily accessible temperatures.
• the necessary energy may also be provided by irradiating the initiator system with ultraviolet light.
• initiation can be provided by irradiating the initiator system in the presence of photo initiators such as benzophenone and its derivatives, benzoin alkyl ethers or derivatives, or acetophenone, with ultraviolet light. It is then necessary that the initiator molecules absorb in the spectral region supplied. In this way radicals can be generated at a finite rate at considerably lower temperatures than are necessary if purely thermal excitation is used.
• bimolecular reactions may produce radicals capable of initiating polymerization. Particularly important are the redox reactions, which occur in aqueous media, and involve electron transfer processes.
• the redox initiators or photochemically induced initiators are particularly preferred in the present invention.
• the amount of initiator is that sufficient t initiate the polymerization reaction.
• Polymerization is carried ou until substantially all of the monomers or comonomers have been incorporated into the polymeric chains. This can be readily ascertained by simple analytical tests on the reaction mixture.
• this polymerization is accomplished almost simultaneously with or immediately prior to the covalent coupling of the polymer to the polysaccharide.
• the coupling and polymerization are performed in the same aqueous phase.
• the condensation of the monomer (or comonomer (a)) with the hydroxy group or groups of polysaccharide is normally carried out by adjusting the temperature of the reaction mixture, or by adding an appropriate acid/base catalyst.
• the most preferred method of carrying out the process is in a "one-pot" system, using a hydroxy reactive monomer (or comonomer (a)).
• All desired monomers and polysaccharide are added to an inert solvent system, such as, e.g., water, alcohols, organics, and the like.
• the polysaccharide and monomers are treated under conditions which will initiate polymerization of the monomers. This can be accomplished, for example, by adding to a well stirred mixture a water solution of an initiator such as ammonium persulfate and sodium thiosulfate, and initiating polymerization from about 15oC to 40oC.
• an initiator such as ammonium persulfate and sodium thiosulfate
• initiator such as ammonium persulfate and sodium thiosulfate
• initiator such as ammonium persulfate and sodium thiosulfate
• initiator such as ammonium persulfate and
• the linking of the formed polymer to the hydroxy groups of polysaccharide is caused by increasing the temperature of the reaction mixture to a temperature sufficient to cause this condensation.
• the temperature is normally around 80-100oC.
• Reaction is then allowed to proceed at the second temperature for a time sufficient to either go to completion, or to achieve modficiation of the polysaccharide to the desired capacity.
• the product is filtered, washed and dried for further treatment, if necessary. Unreacted monomer is preferably washed away with alcohol, unreacted catalyst with aqueous media and polymer with methanol or ethanol. Further reaction of the modified polysaccharide may be by crosslinking.
• Another further reaction of the modified polysaccharide materials would be to anchor the affinity ligands or biologically active molecules to the anchoring groups of the monomer (or comonomer (b)).
• This reaction can be readily accomplished by mixing in an appropriate solvent, normally aqueous, the affinity ligand or biomolecule to be anchored and the modified polysaccharide, and carrying out anchoring for a time and under conditions sufficient to cause covalent coupling therebetween. It may be necessary to activate polysaccharide groups on the monomer (or comonomer (b)) with such materials as cyanogen halides, and to then further treat the activated polysaccharides with the affinity ligands or biomolecules. In this embodiment, it is preferred to first couple the affinity ligand or biologically active molecule to the monomer (or comonomer units (b)), and then bind the resulting polymer or copolymer to the polysaccharide.
• the reactions between the affinity ligand or biologically active molecule and the anchoring groups of the polymer are normally carried out at temperatures of from 0oC to 50oC, may involve the addition of catalysts such as acid or base, or metals, or such other materials as carbo di-imide, or a bi -functional cross-linker such as glutaraldehyde.
• catalysts such as acid or base, or metals, or such other materials as carbo di-imide, or a bi -functional cross-linker such as glutaraldehyde.
• the affinity matrix is produced by grafting a polymeric carrier onto a substrate and then coupling an affinity ligand to the grafted, covalently bound synthetic polymer.
• This pre-coupled structure is referred to below as the pre-ligand structure.
• Suitable substrates include the substrates mentioned above, i.e. polysaccharides such as cellulose. Again, cellulose is the preferred substrate material for use.
• the polymer which is grafted to the substrate may contain functional groups which act as precursor groups for various types of functional groups such as amine and thio. This type of modification provides a means for subsequent derivatization to meet specific needs.
• Typical monomers include ethylenically unsaturated oxirane-containing monomers such as glycidyl acrylate and methacrylate, ethylenically unsaturated hydroxy-containing monomers such as hydroxyethylmethacrylate, and ethylenically unsaturated amide group-containing monomers such as acrylamide.
• Glycidyl methacrylate (GMA) and glycidyl acrylate (GA) are the preferred grafting monomers.
• affinity matrix In order to prepare a satisfactory affinity matrix, especially when used in a radial-flow device, it is important to control the pore size of the grafted polymer so that, even after ligand coupling there will be sufficient space left for the molecules to freely penetrate for ligand binding.
• requirements for such large pore size frequently cause mechanical stability problems.
• Currently available commercial products sold as affinity matrices rely on a carefully controlled degree of crosslinking to provide structural rigidity. However, these lightly cross! inked materials are extremely fragile, and frequently degrade under even conventional mixing techniques. While increased physical stability may be achieved by increasing the crosslinking, the increased linking decreases the porosity of the material.
• this problem of balancing the requirements of structural rigidity and porosity is accomplished with a two step interpenetrating network wherein cellulose or some other natural polymer provides a three-dimensional skeleton within and around which a second network of acrylic polymer is formed.
• the cellulosic or other natural polymer substrate provides the necessary rigidity, permitting the acrylic polymer to be only lightly crosslinkid, this lightly cross! inked polymer possessing the required chemical and porosity properties. Under polymerization conditions similar to those of Examples 3 and 4, crosslinking occurs.
• the synthetic polymer may be formed utilizing any of the polymerization techniques conventional in the art.
• Suspension polymerization is a preferred polymerization technique, the monomer suspended and maintained by continuous stirring of the reaction mixture, optionally with the use of surfactants.
• a free radical initiator is employed which dissolves in the monomer phase and polymerization is achieved by a thermal fragmentation of the catalyst.
• reaction conditions Prior to completion of the polymerization, reaction conditions are altered to facilitate the coupling reaction whereby the polymerization product hydrocarbon chains, with attendant functional groups attached, are then grafted to the natural polymer substrate. During this latter phase of the polymerization, both polymerization and coupling proceeds simultaneously.
• GMA grafted to cellulose represents one preferred affinity matrix.
• GMA provides three functions, the oxirane groups of the GMA monomer providing covalent coupling with the surface hydroxy groups of the cellulose, these same oxirane groups providing crosslinking capability for the synthetic polymer network, and the remaining oxirane groups serving for subsequent ligand coupling.
• Mechanical Rigidity - Cellulose fibers provide good structural strength as a solid support member. These fibers are further strengthened by the strong hydrogen bonding force between the polysaccharide units of the cellulosic fibers, additional strength provided by the highly crystalline structure. Additional mechanical rigidity is provided by crosslinking which occurs between the cellulose and the synthetic polymer.
• Macroporosity Careful selection of fiber diameter, length, degree of fiberization and degree of crosslinking provides for a high degree of control of macroporosity.
• Hydrophilicity The hydroxy! groups in the cellulosic structure provide for a high degree of hydrophilicity. Further, oxidation of the glycidyl groups of the GMA polymer to diols or copolymerization of the latter with a hydroxylcontaining monomer further add to the hydrophilic character of the matrix.
• Chemical Resistivity - Cellulose has a low solubility in conventional solvents; further, solvent resistance is provided by crosslinking the glycidyl groups of the GMA polymer with each other and with the hydroxy groups of the cellulose.
• Structural Integrity - Swelling and shrinking of the matrix is negligibly small due to the grafted crosslinkable monomer, further stability being provided by additional crosslinking with a bifunctional monomer.
• the grafting process further purifies the cellulosic raw materials, decreasing the number of available sites for non-specific binding.
• the cellulose-GMA affinity matrix may be chemically modified as set out in Table I below. The purpose of this chemical modification is to prepare the matrix or preligand structure for coupling with the ligand.
• Suitable oxidizing agents for (1) above of Table I include perchlorate, sulfur trioxide, and periodate, with periodate preferred.
• Suitable aminating agents under (2) above of Table I include compounds having the structural formula NH 2 -R-NH 2 wherein R is a direct bond or (CH 2 ) n .
• Thiolation may be effected by using compounds such as NaSH or KSH. Table II below briefly summarizes oxidation, amination, and thiolation reactions.
• aldehyde reaction product resulting from periodate oxidation as demonstrated at Table II above may be further treated to form a boronate affinity adsorbent according to the following equation:
• Amination with a diamine such as hydrazine proceeds according to the following equation, resulting in the formation of a substituted hydrazine.
• the substituted hydrazine may be converted to the azide form according to the following equation:
• the azide may be converted to an appropriate affinity matrix by reaction with an appropriate ligand, for example, lysine, in accordance with the following equations:
• R 1 and R 2 are C 1 -C 4 alkyl.
• the GMA-cellulose matrix may be thiolated with NaSH.
• the thiol group may then be subsequently activated by reaction with 2,2'-dipyrridine disulfide according to the following equation:
• This substrate will react with sterically accessible-SH groups of protein according to the following equation:
• substrates may be modified to provide an amide functional group, the amide functional group further reacted with a diamine according to the formula:
• the resulting hydrazide may be subsequently treated as with the above amination of the oxirane ring.
• Synthetic polymer-modified substrate with reactive hydroxyl groups may be produced utilizing compounds such as hydroxyl ethylmethacrylate (HEMA) or hydroxyl propylmethacrylate (HPMA) onto an appropriate substrate such as cellulose. Adjacent hydroxyl groups may be activated utilizing CNBr according to the following equation:
• the starting point for designing an affinity matrix for removal of specific enzymes is to examine the structure of the enzyme and, particularly, the structure of the inhibitors of the enzymes.
• the complex formed between an enzyme and its inhibitor provides the best picture of how the enzyme may interact with the specific protein structure. Enzyme inhibition is always competitive and reversible as expressed by the following equation:
• the reactive site residue of the inhibitor fits into the pocket of the enzyme.
• kallikrein and trypsin it is the lysine or arginine residue which interacts with the nucleophilic hydroxyl groups of kallikrein's serine active sites.
• an animal enzyme inhibitor may be extracted from plants. An effective affinity matrix may thus be made by binding the plant inhibitors for enzyme adsorption. It is known that plasma kallikrein inhibitors have been isolated from potatoes and peanuts.
• the resulting substrate product may be further reacted with the amine functionality of a protein to provide an appropriate affinity adsorbent according to the following equation:
• the glycidyl group as a precursor, it is possible to couple another moiety through the glycidyl group.
• the newly linked moiety may be activated with a different activation mechanism for protein coupling as shown below in Table III.
• Proteins are highly reactive toward polyphenols.
• the basic reaction is the addition of any nucleophilic residue on the proteins to the quinone form of the polyphenol through the following route:
• the synthetic polymer-modified substrates of the present invention may be carefully tailored to particular needs by crosslinking the synthetic polymer and/or substrate.
• Crosslinking may be provided either by incorporating into the polymerization recipe a small amount of polyunsaturated comonomer having at least two polymerizable alpha, beta-carbon double bonds, such as for example mono- and polyethylene glycol dimethacrylates and diacrylates (with the polyethylene glycol residue containing up to six ethylene groups), ethylene diemethacrylate, tetraethylene diacrylate, divinylbenzene, triallyl cyanurate, methylene-bis-acrylamide or -bis-methacrylamide, and the like.
• crosslinking agent utilizes the presence of a free pair of electrons on the aminoalkyl nitrogen atoms, where present.
• crosslinking may be carried out with such bifunctional reagents as would react with nitrogen free electron pairs, including the diacyl halides such as Hal-CO-(CH 2 ) n -CO-Hal, or the alkyl dihalides, such as Hal-(CH 2 ) n -Hal, wherein Hal is a halide such as chloride, bromide or iodide, and n may be anywhere between 2 and 12.
• Analogues of these compounds wherein -(CH 2 ) n - is replaced with phenyl are contemplated as well.
• the amount of cross-linking agent is best determined empirically. It is to be considered sufficient when the polymer has achieved the desired structural integrity and porosity. Ideally, an amount of crosslinking agent between 5-20 mole percent of the synthetic polymer units is sufficient.
• Typical ligands include, but are not limited to, DNA blood type antigen, anti-alpha feto protein, C 1 Q, protein A, protein G, polylysine, mucopolysaccharides such as heparin, methylated albumin, tryptophan, phenylalantne, concavaline A, and the like.
• DNA blood type antigen anti-alpha feto protein
• C 1 Q protein A
• protein G protein G
• polylysine mucopolysaccharides
• mucopolysaccharides such as heparin, methylated albumin, tryptophan, phenylalantne, concavaline A, and the like.
• protamines For removal of mucopoly-saccharides such as heparin, low molecular weight basic proteins (protamines) such as protamine sulfate are useful. For removal of kallikrein and PKA, benzamidine is effective. For removal of endotoxins, polymyxin-B-sulfate is effective.
• ligands suitable for the practice of the present invention include all ligands which may be immobilized by the invention affinity matrix and still maintain biological activity, such ligands being represented by, but not limited to, the following general classes: amino acids; avidinbiotins; carbohydrates; glutathiones; hydrophobic matrices; immunoglobulins; insoluble proteins; lectins; nucleotides; polyamino and polynucleic acids; and specialty ligands.
• Typical amino acids suitable as affinity ligands include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-lysine, L-methionine, L-phenylalanine, L-pro!ine, L-serine, L-threonine, L-thyroxine, D-tryptophan, L-tryptophan, L-tyrosine and L-valine.
• Typical avidin-biotin ligands include avidin, biotin, desthiobiotin, diaminobiotin, and 2-iminobiotin.
• Typical carbohydrates include the glucosamines, the glycopryranoses, the glactosamines, the fucosamines, the fucopyranosylamines, the galactosylamines, the glycopyranosides, and the like.
• Typical glutathione ligands include glutathione, hexylglutathione, and sulfobromophthalein-S-glutathione.
• Typical hydrophobic matrices include amino allyl, butyl, decyl, dodecyl, ethyl, hexyl, methyl, octyl, pentyl, phenyl and propyl.
• Typical imrnunoglobulins include the IgG's, including anti-goat IgG, anti-human IgG, anti-mouse IgG, anti-rabbit IgG, goat IgG, human IgG, rabbit IgG, and anti-glucose-6-phosphate dehydrogenase.
• Typical insoluble proteins include aprotinin, fetuin, gelatin, globin, glycophorin, hemoglobin, insulin, lactalbumin, parvalbumin, protamine, protein-A, protein-G, ribos-binding protein, and trypsin inhibitor.
• Typical lactins include Arachis hypogaea, concanavalin A, Dolichos biflorus. glycine max, Lens culinaris. Phytolacca americana. Pisum sativum, and the like.
• Typical nucleotides include the adenosine mono- and diphosphates, the cytidine di- and triphosphates, flavin mononucleotide, the guanosine mono-, di-, and triphosphates, and the uridine mono-, di-, and triphosphates.
• Typical polyamino and polynucleic acids include DNA, polyadenylic acid, polycytidylic acid, polylysine, polyriboadinylic, polyribocytidylic, polyriboguanylic, polyriboinosinic acid, and polyuridylic.
• the affinity matrices of the present invention involve the coupling of a ligand to the substrate-synthetic polymer matrix.
• a ligand which may be immobilized in the stationary phase and used to bind complementary molecules from a solute phase by affinity chromatography is contemplated.
• the affinity matrices of the invention may be configured within a device which allows radial or tangential flow of sample.
• radial flow is intended where the sample is passed through the affinity matrix.
• tangential flow is intended where the sample flows across the surface, but not through the affinity matrix.
• the composite material formed by copolymerization of vinyl monomers followed by covalently linking to the cellulosic substrate may be made in paper form of different thickness.
• the affinity matrix thus made may be die cut and installed in an appropriate holder, either discs, columns, cartridges or plates.
• Typical affinity matrices of this invention include those containing primary amino groups ("Type A”) formed through amination reaction of GMA and those containing aldehyde groups ("Type G”) formed through periodate oxidation of GMA as shown above. Where additional spacer arm length is desired, it is possible to utilize combinations of, for example, dialdehydes and diamines, alternately, and create spacer arms of any desired length.
• Type A matrices have free primary amino groups at the end of hydrophilic spacer arms (>10Ao). This is very convenient for small scale ligand immobilization and affinity purification of proteins, enzymes, antibodies, etc.
• Type A matrices comprise a composite material formed by covalent attachment of synthetic polymers to polysaccharide, i.e., cellulosic substrates.
• Amino functional groups are introduced into these matrices by covalent linkage of, for example, 1,6-diaminohexane, to activated composite material.
• the matrices thus formed are hydrophilic in nature and dimensionally stable due to properly controlled degree of cross-linking. Dimensional changes under pH and salt conditions are negligibly small.
• the matrix structure has sufficient rigidity to withstand high liquid flux for fast flow, yet is highly porous to facilitate mass transfer through the matrix where configured for radial flow.
• the free primary amino groups are capable of coupling ligands containing ayylazide, carboxyl and aldehyde groups.
• One embodiment of the present invention comprises the preligand intermediate with free primary amino groups.
• Aldehyde groups may be generated in the matrix by reaction with glutaraldehyde.
• aldehyde groups thus generated will take part in coupling reactions with ligands containing amino groups.
• Another embodiment contemplates the aldehyde-terminated intermediates. Glutaraldehyde Activation of Type A Matrices
• Type A matrix is activated by glutaraldehyde to make it suitable for immobilizing ligands containing amino groups, preferably primary aliphatic amino groups.
• Type A matrix is equilibrated by Buffer A at room temperature.
• the prepared glutaraldehyde solution (see A.4 above) is recirculated at room temperature for 6 hours.
• Amino functional ligand solutions are prepared in suitable buffers*, pH 6 to 8.5, containing NaCNBH 3 (i.e., 1 mg/ml in ligand solution) and recirculated through the matrix for 6-16 hours at room temperature or at 4oC.
• the uncoupled ligand is removed by washing with the same buffer until the baseline is obtained.
• Reagents are removed by washing with any desired buffer (pH 5 to 7.6) containing 0.25 to 0.5 M NaCl.
• the amount of ligand coupled to the matrix is calculated indirectly by measuring the amount of uncoupled ligand solution left in the buffer solution (Step C.2). Dialysis of the residual ligand solution is necessary to remove the reaction by-products which may interfere in the measurement.
• Type G matrix is very convenient for small scale ligand immobilization and affinity purification of proteins, enzymes, antibodies, etc.
• Type G matrices comprise a composite material, formed by covalent attachment of synthetic polymers to polysaccharide, i.e., cellulosic substrates. Functional groups capable of coupling amino functional ligands are introduced into the composite structure in the form of polymers. Typically, GMA-grafted cellulose is reacted with perchlorate to produce the hydroxylated intermediate. Further reaction with periodate produces the aldehyde termination.
• the matrices, thus formed are hydrophilic in nature and dimensionally stable due to properly controlled degree of cross-linking. Dimensional changes under pH and salt conditions are negligibly small.
• the matrix structure has sufficient rigidity to withstand high liquid flux for fast flow, yet is high porous to facilitate mass transfer through the matrix.
• the matrix has abundant vicinal hydroxy groups, which are readily available for periodate oxidation. Aldehyde groups, thus generated, take part in the coupling reaction with free primary amino groups, preferably aliphatic amino groups.
• Both low and high molecular weight ligands [L] lysine, p-aminobenzamidine, protein A, albumin, IgG, etc.
• a flow sheet representation of a typical affinity matrix preparation appears below.
• Buffers containing amino functionality e.g., Tris, ethanolamine, etc. should not be used.
• the affinity matrix is assembled with tubing and connected to a small laboratory pump.
• the matrix is washed with 100 ml (5 volumes) of deionized water. Freshly prepared aqueous solution of NaIO 4 (1.5%) is passed at 4-6 ml/min for 1/2 to 2 hours at room temperature. Maximum activation occurs when the oxidation is continued for 2 hours. 3.
• the matrix is washed with 200 ml (10 volumes) of deionized water to remove NaIO 4 .
• the conductivity and O.D. 280 of the eluate is checked to determine the removal of NaIO 4 .
• the matrix is equilibrated with approximately 200 ml of an appropriate phosphate buffer (as described in the Materials Section).
• a solution (10 mg/ml) of the ligand is prepared in equilibration buffer.
• the ligand solution is recirculated (4h-10h) at 4-5 ml/min in presence of NaCNBH 3 (final concentration 1 mg/ml in ligand solution*.
• a minimum of 10 mg NaCNBH 3 is necessary for those ligand solutions which are less than 10 ml.
• the uncoupled ligand is removed by washing with phosphate buffer until the O.D. stabilizes at baseline.
• the ligand coupled matrix may be stored at 4oC in 0.02% sodium azide with 0.9% NaCl or suitable preservative (pH 6.0-7.0). Measurement of Ligand Coupled
• the amount of ligand coupled to the matrix is calculated indirectly by measuring the uncoupled ligand left in the buffer solution (Step 6).
• the measurement is done by a suitable color reaction or U.V.
• macromolecules e.g., protein A, antibodies, etc.
• dialysis of the uncoupled ligand solution is necessary to remove the reaction by-products which may interfere in the measurement.
• the blood coagulation metabolism existing in the human body occurs with two opposing processes, a fibrin-forming one associated with the blood coagulation system, and a fibrin-removing process directed by the fibrinolysin system.
• the two systems remain practically inactive.
• plasmin is suddenly activated from inactive precursors by intrinsic blood factors and the system is unbalanced.
• Clinical study has shown that the blood of individuals who have died suddenly under stresses might be in liquid and incoagulable form. This condition is now known to be due to proteolytic dissolution of fibrin by plasmin which has been activated from plasminogen. Therefore, plasminogen must be removed from IgG to eliminate the fibrinolytic effect and to avoid fragmentation of IgG.
• Both plasminogen and plasmin behave as if they were gamma globulin.
• the isoelectric point of plasminogen has been estimated to be pH 5.6. It is probably a glycoprotein and contains small amounts of phosphorus.
• the molecular weight has been reported to be 143,000 or 84,000 by different investigators. The molecule behaves as if it were asymmetric in shape with an axial ratio of 9 to 16.
• the chemical properties of human plasmin are similar to those of plasminogen, but the molecular weight is slightly smaller due to splitting off of some molecules during activation.
• the conversion of plasminogen to plasmin involves change of shape from asymmetric to more compact spherical type.
• proteolytical enzymes from IgG is effected by passing the solution through an affinity matrix bonded with an enzyme inhibitor.
• an enzyme inhibitor Among all the potential inhibitors of the plasminogen system, the following three were found to be most effective;
• the inhibitor i.e. epislon-aminocaproic acid
• the inhibitor is bound to a cellulose-GMA matrix, utilizing a CNBr activation.
• CNBr activation since certain quaternary amines were found to exhibit plasmin activity and with plasminogen having isoelectric point 5.6, plasminogen removal by QAE and DEAEMA matrix occurs to a certain extent.
• Caseinolytic method Techniques developed to measure the concentration of plasminogen in human plasma depend on the proteolytic activity of plasmin. Plasminogen cannot be directly determined, but must be converted into plasmin through activation with urokinase and subsequently determining the plasmin formed from its proteolytic activity with certain substrates. Although the physiological substrate from plasmin is fibrinogen, synthetic substrate such as casein is preferred for better sensitivity and reproducibility.
• the principle of the caseinolytic method is to analyze the ability of the plasminogen in plasma to digest casein in a given time and be expressed as plasminogen activity.
• Such activity can be measured by the amount of tyrosine equivalent released from hydrolytic dissolution of casein due to the proteolytic effect of plasmin.
• a standard procedure was established by NHI (National Heart Institute) Committee on Thrombolytic Agents. 2. Affinity Matrix For Kallikrein Removal
• IgG made by the Cohn cold ethanol fractionation method is known to contain undesirable, deleterious exogenous activity such as prekallikrein activator (PKA) activity, activated clotting factor, and esterase activity.
• PKA prekallikrein activator
• Those exogenous activities have been reported on intravenous administration to cause hypotensive reaction in patients, Alving et al., New England J. Med. 299:66 (1978).
• PKA prekallikrein activator
• Those exogenous activities have been reported on intravenous administration to cause hypotensive reaction in patients, Alving et al., New England J. Med. 299:66 (1978).
• Kallikrein is also inhibited by synthetic compounds such as p-carboethoxy phenyl epsilon guanidine caproate. Benzamidine bound to cellulose-GMA is preferred as affinity ligand for kallikrein removal.
• Hydrophobic comonomers (b)(ii) are normally added to a copolymerization mixture in the presence of alcoholic solvents and/or surfactants. Washing is then carried out with alcohols.
• the preferred formation of self-supporting fibrous media from the modified polysaccharide materials of the invention can be carried out immediately after polymerization and polysaccharide modification.
• anchoring of affinity ligands or biomolecules may be carried out on the formed sheets themselves.
• the fibrous media is formed after anchoring of affinity ligands or biomolecules.
• the preferred method is to form the fibrous sheets after polysaccharide modification, and carry out further reactions, such as anchoring on the sheets.
• a self-supporting fibrous matrix using the modified polysaccharide of the invention can preferably be made by vacuum filtering an aqueous slurry of fibers and, if desired, additional resins and modified or unmodified particulate.
• This forms a sheet having uniformly high porosity, fine pore-size structure with excellent flow characteristics and substantial homogeneity with respect to fiber, resins and particulate.
• the vacuum filtration is performed on a foraminous surface, normally a woven wire mesh which, in practice, may vary from 50 mesh to 200 mesh, with mesh openings ranging from 280 micrometers to 70 micrometers, respectively. Finer meshes are unsuitable because of clogging problems and/or structural inadequacy.
• the sequence of adding the overall components to the slurry is relatively unimportant, provided that the slurry is subjected to controlled hydrodynamic shear forces during the mixing process.
• the slurry is normally prepared at, say, about 4% consistency and then diluted with additional water with a proper consistency required for vacuum filtering and sheet formation. This latter consistency will vary depending upon the type of equipment used to form the sheet.
• the slurry is cast onto a foraminous surface, vacuum filtered and dried in the conventional manner.
• the flat, dimensionally stable sheet can be of any desired thickness and is then cut to the appropriate dimensions for each type of application.
• the wet sheet is dried and then cut to proper size in order to form discs.
• These discs can be loaded onto an appropriately sized cylindrical column to form the desired medium.
• the disc and cylinder should preferably be in interference fit so that the disc can be pushed into the cylinder without distortion, but cannot fall under gravitational force allowing gaps between the discs and the cylinder.
• a pump can be used to pump solvent through the elements stacked in the column.
• the elements swell to form a substantially tight edge seal to the cylinder wall. Because the individual elements are dimensionally stable, the column is not sensitive to orientation or handling, a problem which is common with other chromatographic media, particularly of any gel type media. However, the media is equally suitable for use in sheet configuration.
• the stationary phase in sheet form, is used as the stationary phase in a chromatography column utilizing conventional column configuration.
• a chromatography column utilizing conventional column configuration.
• One such column is described in U.S. Patent No. 4,496,461 to Leeke et al., incorporated by reference herein in its entirety.
• the radial flow column which may be in cartridge form, generally designated 10, is comprised of a cylindrical stationary phase 12, and cylindrical tube 13, which form a cylindrical chamber 14 which acts as a housing for the stationary phase 12.
• the solid stationary phase 12 can be inserted into chamber 14 formed by a glass, metal or polymeric tube or cylinder 13 having a diameter somewhat larger than the external diameter of the stationary phase 12. Suitable fluid admission, collection and monitoring systems can also be employed with the column as in conventional analytical and preparative columns.
• the stationary phase 12 is positioned within the chamber 14 and preferably has a longitudinal axis 16 coaxial with the axis of the cylindrical chamber 14.
• a plurality of cartridges may be placed in a single housing in various configurations to effect parallel and/or series flow between the cartridges (not shown).
• the solid stationary phase has chromatographic functionality and is effective for affinity and reverse phase chromatographic separation.
• the stationary phase 12 is constructed of a swellable matrix 18, which may be hydrophilically swellable, in sheet form which is the active media for chromatographic separation.
• the matrix is fibrous.
• the chromatographic media in sheet form 18 is sandwiched between a single non-woven mesh 22 or plurality of mesh.
• the composite sheet of chromatography media 18 and mesh 22, e.g., non-woven, is spirally wound around a cylindrical core 24 having a longitudinal axis 16 to form a plurality of layers around the axis 16.
• the core 24 is provided with a plurality of longitudinal and axially oriented channels 21 for directing the liquid into circumferential channels 23 which are in fluid communication with core 24.
• the mesh 22 due to the openness and thickness thereof, acts as a spacer means between each layer of media 18 which permits the controlled swelling of the media and enhances the distribution of the sample flowing through the stationary phase 12.
• the cylindrical core 24 is provided with apertures 26 near the top thereof for the flow of sample from the circumferential channels 23 into the open interior of the core.
• the wound composite sheet 18 and 22 and core 24 are then capped by stationary phase end caps 32 and 34.
• the stationary phase end caps 32 and 34 of this subassembly are sealed by thermoplastic fusion to the core 24 and also to the ends of the composites 18 and 22.
• the subassembly, comprising 18, 22, 24, 32 and 34 is then slipped into chamber 14.
• the cylinder end cap 36 is then thermoplastically fused to the top edge 31 of cylinder 13.
• the fluid or sample 42 can thus flow radially from the outside through the solid stationary phase to the open channel 21 of core 24, since the interior and exterior are completely separated by the solid stationary phase and sealed off by stationary phase end caps 32 and 34.
• the preformed stationary phase end caps 32 and 34 are preferably applied to the cylindrical solid stationary phase 12 by heating an inside face of the thermoplastic stationary phase end cap to a temperature sufficient to soften a sufficient amount of the stationary phase end cap to form a thermoplastic seal with the ends of the core 24 and composite sheet 18 and 22. All of the edges are then embedded into the softened material. The softened material is then hardened, typically by ambient conditions, to form a thermoplastic sealing relationship between the sealing surface of the stationary phase end caps 32 and 34, the core 24 and the ends of the solid stationary phase 12 to form a leak-proof seal.
• Such method of applying stationary phase end caps are well known in the filtration art. See, for example, U.S. Serial No. 383,383 and U.S. Serial No.
• the stationary phase end caps can be molded integrally in situ onto the solid stationary phase.
• Stationary phase end caps of thermoplastic materials are preferred because of the ease of bonding, but it is also possible to use thermo-setting resins in a thermoplastic, fusible or heat-softenable stage of polymerization, until the bondings have been effected, after which the curing of the resin can be completed to produce a structure which can no longer be separated.
• Such a structure is autoclavable without danger of destroying the fluid tight seal, the solid stationary phase 12, and the stationary phase end caps 32 and 34.
• Thermoplastic resins whose softening point is sufficiently high so that they are not softened under sterilizing autoclaving conditions are preferred for biomedical use.
• Exemplary of the plastic materials which can be used are polyolefins.
• one preferred column 10 has a stationary phase end cap 34 on one end which does not open to the exterior of the subassembly 18, 22, 24, 32, and 34 but is closed off.
• This stationary phase end cap 34 can nest on the bottom end wall 44 of cylinder 13 while still permitting the flow of sample 42 into chamber 14 around the outside of stationary phase 12, or this lower stationary phase end cap 34 of the subassembly 18, 22, 24, 32 and 34 is in spaced apart relationship from the bottom end wall 44 of cylinder 13, thus permitting the flow of sample 42 into the chamber 14.
• the upper end of cartridge 40 has a cylinder end cap 36 which is in fluid communication with channels 21 of cylindrical core 24, thus permitting the flow of fluid from the outer periphery of cylindrical core 24 to the center of core 24 to the outside of cylinder end cap 36.
• the cylinder end cap 36 has molded thereon fitting 48 for fluid connection through a collection means (not shown).
• FIGs 4 through 10 depict another embodiment of the chromatography column of this invention, the embodiment wherein the column is in disc configuration, again wherein like character references indicate like parts.
• the column in disc configuration generally designated 110, comprises an inlet housing member 112, an outlet housing member 114, and a stationary phase 116.
• the inlet housing member 112 comprises a sample inlet means 118, baffle means 120, and sample distribution means 122.
• the sample inlet means 118 is in communication with the sample distribution means 122.
• the sample distribution means 122 comprises plural radial distribution channels or grooves 130 and plural concentric distribution channels 140, the radial distribution grooves 130 and concentric distribution channels 140 being in communication with each other and with inlet means 118.
• Radial distribution grooves 130 comprise distribution groove bottom portions lying in a plane represented by line P 1 in Fig. 10 and P 1 ' in Fig. 12, and distribution groove wall portions 134a and 134b.
• Concentric distribution channels 140 comprise concentric distribution channel bottom portions 142, concentric distribution channel wall portions 144a and 144b, and concentric distribution channel apex portions 146.
• the inlet housing member 112 may contain a venting means 150, the function and operation of which will be defined below.
• the venting means is in communication with a chamber 152.
• Chamber 152 is formed by inlet housing member 112 and outlet housing member 114 (see Figs. 4 and 10). Chamber 152 contains the stationary phase 116.
• the outlet housing member 114 comprises a sample collection means 154 and sample outlet means 156, sample collection means 154 being in communication with sample outlet means 156.
• Sample collection means 154 comprises plural radial collection grooves 160 and plural concentric collection channels 170.
• Radial collection grooves 160 and concentric collection channels 170 are in communication with each other and with sample outlet means 156.
• Radial collection grooves 160 comprise radial collection groove bottom portions lying in a plane represented by line P 2 in Fig. 4 and P 2 ' in Fig. 10, and radial groove wall portions 164a and 164b.
• Concentric collection channels 170 comprise concentric collection channel bottom portions 172, concentric collection channel side wall portions 174a and 174b and concentric collection channel apex portions 176.
• Stationary phase 116 has chromatographic functionality and is effective for chromatographic separation.
• the stationary phase 116 may comprise a plurality of layers of a matrix 180 in sheet form, having chromatographic functionality and being effective for chromatographic separation, and an optional spacer means 182 between each adjacent layer of swellable matrix 180.
• This configuration is best shown in Figure 9, a crosssectional view of one embodiment of the separation phase 10.
• the matrix may be a swellable matrix. In one preferred embodiment, the swellable matrix is fibrous.
• the matrix 180 may be hydrophilic swellable and comprises the active media for chromatographic separation.
• the optional spacer means 182 may be typically a woven or non-woven mesh similar to mesh 22 of Figures 2 and 3 above and is further described below.
• the mesh due to the openness and thickness thereof, acts as a spacer means between each layer of matrix 180 and permits the controlled expansion thereof without closing off the porous structure of the media, thereby enhancing the distribution of the sample flowing radially through the stationary phase 116.
• a manner of conforming the stationary phase 116 is to produce a "sandwich" of alternating layers of swellable matrix in sheet form and layers of spacer means, with the periphery of the sandwich compressed into a fluid tight configuration 184.
• the peripheral edges of alternating discs of swellable matrix 180 and spacer means 182 are joined.
• the matrix 180 contains or has bonded therein a thermoplastic polymeric material.
• spacer means 182 also is made of or contains thermoplastic polymeric materials. In this configuration, the edges may be optionally uniformly joined by appropriate means such as heat treating, sonic welding, etc.
• the fluid tight peripheral configuration 184 is itself contained in a fluid tight, hermetic seal formed by the mating edges 186 and 188 of, respectively, the inlet housing member 112 and the outlet housing member 114. In this manner, sample entering through inlet means 118 must pass through stationary phase 116 prior to exiting through outlet means 156.
• the disc configured chromatography column of Figures 4-10 is formed using conventional and well known fabrication techniques.
• the stationary phase 116 a preformed "sandwich" of alternating layers of swellable matrix and spacer means, with peripheral edges optionally sonically welded and configured as in Figure 9, may be placed in inlet housing 112 and outlet housing member 114 is placed thereover. Subsequently, the mating edges 186 of the inlet housing member 188 and of the outlet housing member 190 are joined to form an airtight and fluid tight seal.
• sonic welding the technique described in Branson Sonic Power Company, Danbury, Connecticut, Information Sheet PW-3, 1971, incorporated by reference herein, may be employed.
• Vent means 150 represents an optional configuration of the disc embodiment of the column. Its purpose is to allow air in the column to exit the column during use. Typically, vent means 150 is adapted to be sealed off when all air has been removed from the system. In an alternative embodiment, vent means 150 contains a hydrophobic media which will allow the passage of gases but not liquids, as disclosed in U.S. Patent 4,113,627, incorporated herein by reference.
• chamber 152 is radially outwardly expanding.
• radially outwardly expanding is meant that the volume at the interior chamber is less than the volume at the periphery of the chamber.
• the distance between distribution means 112 and collection means 114 at the interior, d 1 is less than the distance between distribution means 112 and collection means 114 at the periphery d 2 .
• the stationary phase 116 is hydrophilic swellable, sample solution on contact with separation phase 116 causes the separation phase to swell. As the separation phase swells, the pressure differential between the inlet and outlet sides of the separation media increases, thereby restricting sample flow-through.
• the pressure differential between the inlet and outlet sides of the stationary phase decreases towards the periphery, thereby maximizing utilization of the chromatographic separation function of the stationary phase and substantially increasing the adsorption capacity of a given unit.
• the above-described housing design may also be employed for a stationary phase which is not swellable.
• each succeeding concentric distribution channel 140 and concentric collection channel 170 increases from the interior to the periphery of the chromatographic column. In this manner, clogging of the channels by the swelling of the hydrophilic swellable stationary phase is vitiated, thereby promoting uniform distribution of sample and maximum utilization of column capacity.
• lines A, A', C and C' are lines which represent cross-sectional view of parallel planes which are perpendicular to the longitudinal axis L of the chromatography column.
• Lines B and B' respectively, represent crosssectional views of planes which are substantially tangent to the apices 146 and 176 of concentric distribution channels 140 and concentric collection channels 170.
• Planes B and B' form angles alpha and alpha' with planes A and A'.
• planes B and B', at angles alpha and alpha' to planes A and A', respectively, define a radially outwardly expanding chamber 152, which in turn defines the limits of expansion of stationary phase 116.
• the optimal configuration for the radially outwardly expanding embodiment is such that stationary phase 116 is just touching the most peripheral apices 146 and 176. If the stationary phase is swellable, then, in its maximally swelled status, it should just touch the most peripheral apices 146 and 176. It is to be understood that angles alpha and alpha' may be the same or different and may vary with the number of layers of swellable matrix and the particular matrix in use. Typically, alpha and alpha' are about 2-1/2o.
• Lines D and D' represent cross-sectional views of planes which contain concentric distribution channel bottom portions 142 and concentric collection channel bottom portions 172 and define angles beta and beta' with planes C and C'.
• planes D and D', at angles beta and beta' to planes C and C', respectively, define the slope of the increasing depth of channels 140 and 170.
• beta and beta' are typically each about 5o. However, these angles may be varied, both with respect to one another and absolutely.
• an optimal design for a radially outwardly expanding chamber is that where the distance d 2 is such as to allow the swellable stationary phase to swell to its maximum, but with no unused space left. In this manner, the pressure differential at the periphery is minimized, while at the same time reducing hold-up volume to its lower limit as well.
• This housing configuration permits as well the use of a single layer of matrix or a plurality of layers of matrix with no spacer means interposed between layers.
• the radially outwardly expanding chamber coacts with the thus configured stationary phase to uniformly distribute sample thereacross.
• the sheets which form the chromatographic media are formed by vacuum felting an aqueous slurry of fibers.
• the sheets may also be pressure felted or felted from a nonaqueous slurry.
• the sheet shows a uniform high porosity, with excellent flow characteristics, and is substantially homogeneous.
• the media can range in thicknesses from about 5 mils to about 150 mils (dry); however, thicker or even thinner media may be utilized provided the sheet can be spirally wound or layered to produce a column which can perform as described above.
• the chromatographic media used in the column be of uniform thickness throughout its length and width and that the media have a substantially uniform density throughout. It is preferred that the layer of media be substantially homogeneous with respect to itself; however, for certain applications and material, it is understood that non-homogeneous construction may be employed.
• the solid stationary phase Since the solid stationary phase is intended in use to effect separation by maintaining a substantial pressure differential across the solid stationary phase, it is essential that the solid stationary phase have a sufficient degree of compressive strength to withstand deformation under such loads as may be imposed upon it. Such compressive strength must not only exist in the media itself but in the spacer means (if present) and the internal core upon which the chromatography media, or solid stationary phase is compressed.
• an element of this invention is the spacer means between each layer of the media and/or the coaction of the chamber wall and the matrix.
• Such spacer means are not essential to this aspect of the present invention, and in one embodiment a chromatographic column may be prepared which does not contain such spacer means.
• the self supporting matrices of the present invention comprise a single and continuous column medium.
• Spacer means may be omitted advantageously in columns whose matrices do not undergo extensive swelling.
• the spacer means permits controlled expansion of the media and enhancement of the distribution of sample flowing through the stationary phase.
• the spacer means located between each layer of the swellable chromatographic media provides for the distribution movement of the sample as the sample passes through the solid stationary phase.
• the spacer means functions to uniformly control thickness and density of the chromatographic media during use.
• the spacer means can serve as a backing or support for the layer of chromatographic media. This latter aspect is particularly useful during the manufacturing phase.
• any spacer means used be composed of a material which is inert with respect to the chromatographic process.
• inert it is meant that the material does not adversely affect the function of the solid stationary phase.
• the spacer means comprises the mesh 22.
• the spacer means 182 may also comprise a mesh, or scrim and mesh.
• a scrim material can function to channel, to a certain extent, the sample flowing through the media and substantially evenly disperse the sample axially and circumferentially across the media.
• the mesh material provides spacing between the media to permit controlled expansion thereof to prevent the "cut-off" to flow therethrough by compression of the permeable media and also assists in distributing or channeling the sample flowing through the media.
• the mesh material is an open type of material having openings ranging, for general guidance, from 1/16 inch to 1/4 inch.
• the thickness of the optional spacer means i.e. the scrim and particularly the mesh material, and the pore size of each to be used may be readily determined by one skilled in the art by performing tests which vary these factors.
• Such factors as the openness and thickness of these spacer means are highly dependent on the type of media utilized, e.g. swellability, wettability, thickness, chemical composition, etc., the flow rate of the sample through the stationary phase, the surface area of the stationary phase, e.g. number of windings, thickness of media, diameter of stationary phase, etc. It is thus very difficult to clearly specify these variables, other than to say that these may be determined by either trial and error or more elaborate testing procedures to determine the optimum parameters.
• the preferred mesh material is polypropylene CONWED (Grade TD-620).
• the overall width of the stationary phase in accordance with the present invention can be infinite, the actual diameter being limited only by practical considerations such as space requirements. Since the diameter or width of the overall column can be increased without theoretical limitation, the sample size or amount of substance to be separated in the bed is not limited. Thus, the diameter can be increased to separate the desired amount of sample substance to be produced.
• the sample is driven through the stationary phase and separated into distinct chromatographic fractions by the chromatographic media.
• the optional spacer means induces and permits flow of this stream as it moves through the column and therefore provides for improved resolution and utilization of the media's potential capacity.
• the sample is preferably introduced at the bottom of the column flowing to the outer surface of the solid stationary phase and then flowing radially inward through the layers of chromatographic media and spacer means into the channels 21 of core tube 24 and is withdrawn centrally. It is apparent, from what has been set forth above, that the radial flow can also be caused to circulate in the opposite direction.
• sample is preferably introduced at the inlet 118, passes to distribution means 122, is substantially uniformly distributed over the surface of the stationary phase 116 by radial distribution grooves 132 and concentric distribution channels 130, and passes through radial collection grooves 140 and concentric collection channels 170 and exits through outlet 156.
• the chromatographic columns of this invention may be used for any of the well-known affinity and reverse phase chromatographic separations usually performed with conventional columns. Additionally, the columns of the present invention may be found useful in the areas where conventional columns are impractical.
• the columns of this invention can be used for separations in the analytical and preparative fields.
• the columns can be connected to all common types of chromatographic equipment.
• Several columns or cartridges of solid stationary phase can be connected in series or parallel.
• the columns can contain identical or different affinity or reverse phase chromatographic media and can be of identical or different length and/or diameter. See for example Daly et al., application Serial No. 611,662, filed May 18, 1984, incorporated by reference herein.
• the stationary phase of this invention produced unexpected results in that the flow of sample through the column is enhanced without destroying the adsorptive capacity of the media.
• protein and dye staining tests were performed it was found that the stationary phase of this invention provided even distribution of sample flow therethrough without an increase in pressure drop when compared to a stationary phase not utilizing the spacer means described herein.
• the stationary phases decrease total processing time and when used with the proper chromatographic media has excellent binding capacity.
• the stationary phases may be used with standard type pumps, gravity feed, or syringes, utilized, in their preferred mode, at from 1 to 50 p.s.i., and even under vacuum.
• the stationary phases of chromatographic media are totally enclosed and completely self-contained to ensure sterile conditions. Due to the fact that the solid stationary phase is manufactured in a factory and assembled therein, each is virtually identical to the other, does not vary as in previously known columns and eliminates the dependence upon packing expertise.
• the useful and effective life of the column is substantially improved.
• sheets of media may be stacked in the columns of Figure 1 in disc configuration, with or without spacer means.
• tangential flow of the blood across the affinity matrix is preferred.
• tangential flow or “tangential flow across the media” is intended that the fluid sample is directed across, but not through the chromatographic media.
• a typical tangential flow configuration is described in U.S. Patent No. 4,551,435 (1985) to Liberte, incorporated by reference herein.
• the difficulty in treating blood by flow through the media is due to the fact that blood comprises cells varying from 1 to 20 ⁇ M in size together with over 400 different proteins. As shown in Figure 14, when blood flows through a matrix 201, blood cells 219 tend to become caught on the surface of the matrix.
• Lysis of cell walls for example red blood cells, is associated with very high sheer stress, generally greater than 40,000 dynes/cm 2 for very short periods of time or about 3,000 dynes/cm 2 for times exceeding one second.
• the amount of sheer stress is also dependant on the surface area, surface properties of the matrix, adhesivity of blood components to the matrix, flow parameters and clotting mechanisms triggered by a particular matrix or wall. Hemolysis, or the liberation of free hemoglobin from the cell, is the primary indicator of red cell destruction.
• Cell lysis can be reduced by tangential flow of the blood across the surface of the matrix which gives rise to sheer stresses of less than 3,000 dynes/cm 2 .
• tangential flow of blood across the matrix 201 allows cells 219 to pass across the media and at the same time allows the impurities to be adsorbed by the matrix.
• Flow of blood in the system involves a shifting of molecular interactions between various components.
• any possible disturbance which may occur during the process of separation should be reduced as much as possible.
• Flow rate, pressure variation and viscosity change are the major parameters to be considered.
• plasma in blood functions as lubricant and diluent to keep cells apart and away from the walls of the device.
• a device for effecting the chromatographic separation of at least two components of a sample comprises:
• the matrix material within the tangential flow separation device may be in the form of sheets.
• one or more sheets of matrix material may be positioned in parallel fashion with means to distribute blood at one end and collect blood at the other.
• the sheets may be self supporting or adhered to a rigid supporting member.
• a plurality of parallel plates having a layer of matrix material in the form of a sheet theron may be stacked in a suitable housing with means to distribute and means to collect the sample.
• the layers may be separated by supporting means on the walls of said housing comprising grooves configured to accept the edge of said plates or may be separated by an array of filaments disposed parallel to one another and parallel to the diretion of sample flow to provide channels for sample flow tangential to the media.
• the size of the filaments and their spacing can be optimized to provide high efficiency separations with minimal pressure differentials ( ⁇ p) between the inlet and outlet of the device.
• the thickness of the filaments and their spacing can be determined by one of ordinary skill in the art using routine testing.
• the layers may be separated by two arrays of filaments disposed at oblique angles to one another.
• the two arrays of filaments are affixed at their crossover points.
• the means for distributing and collecting the sample may comprise one or more grooves on the interior of two sides of the housing which are in fluid communication with said channels and nozzles disposed on each side of the housing.
• the matrix may be configured in the form of a corrugated sheet which provide channels without the use of filaments or nets.
• the matrix material may be configured into hollow tubes through which a sample, e.g., blood, can flow tangentially to the surface of the matrix.
• a sample e.g., blood
• the matrix may form a layer of uniform thickness on the inner walls of an impermeable tube.
• This tube may then be coiled within a housing having means for introducing blood to the coil and means for collecting blood therefrom.
• the housing may also contain means for regulating the temperature of the coil such as heaters, fans, thermostats, thermally regulated fluids, and the like. See U.S. Patent No. 4,061,141 (1977) to Hyden et al., which is incorporated by reference herein.
• the matrix is in the form of one or more sheets spirally wound around a cyclindrical core.
• the separation device for effecting the chromatographic separation of at least two components of a sample flowing therethrough comprises:
• said first phase comprises the modified polysaccharide matrix material of the invention.
• said second phase comprises a means for supporting, separating and providing channels tangential to said core
• the device may further comprise a third stationary phase disposed between the first and second phases comprising an impermeable sheet.
• This impermeable sheet may comprise any impermeable material compatible with blood and the matrix material including, but not limited to polyvinylchloride (PVC), polyethylene, etc.
• the means for supporting and separating, and providing channels tangential to the cylindrical core may comprise two arrays of filaments including a first array spaced parallel to one another and to the axis of the cylindrical core and a second array of filaments spaced parallel to one another and perpendicular to the axis of said cylindrical core.
• the two arrays of filaments are affixed at their crossover points.
• the two arrays may be disposed perpendicular to one another or may be disposed at oblique angles, as long as one array is perpendicular to the axis of the cylindrical core.
• This type of screen or net provides a series of flow channels tangential to the cylindrical core.
• the individual filaments may be round, square, rectangular or triangular in cross section.
• the thickness and spacing of the filaments may be readily determined by one skilled in the art by performing tests which vary the dimensions. Such factors as the spacing and thickness of the filaments are highly dependent on the type of media utilized, e.g. swellability, wettability, thickness, chemical composition, etc., the flow rate of the sample tangential to the stationary phase, the surface area of the stationary phse, e.g. number of windings, thickness of media, etc. and the type of sample chromatographed, e.g. blood or fermentation media. It is very difficult to clearly specify these variables, but may be determined by trial and error or by more elaborate testing procedures.
• the first array of filaments are round and are 9.5 mil and the second array of filaments are round and 47 mil.
• the filaments of the first array may be disposed at an angle of about 49° to 90o relative to the second array of filaments. Most preferably, the first array is disposed at about a 90o angle to the second set.
• the filaments may be made of polyethylene, polypropylene, polystyrene, nylon, or a substantially non-deformable yet resilient elastomer or metal.
• the material employed should be one which separates adjoining layers of matrix material or impermeable sheete without deformation, one which may be easily fabricated, and one which is not deleteriously affected by contact with the sample or matrix.
• the filaments are constructed of polypropylene.
• the means for collecting the sample may comprise one or more grooves along the entire length of the cylindrical core. These grooves are in fluid communication with the tangential flow channels and a nozzle disposed on the end of the cylindrical housing.
• the means for distributing the sample comprises one or more grooves along the length of the cylindrical housing which are in fluid communication with the tangential flow channels and a nozzle disposed on the cylindrical housing.
• the means for collecting the sample may be used for distributing the sample and the means for distributing the sample may be used for collecting the sample.
• the device may comprise two sheets of matrix material 201 per winding so that the channel has a layer of matrix on each side.
• the two sheets may comprise two different ligands.
• one sheet of matrix 220 may absorb bacteria 221 or lipids preferentially and a second sheet of matrix 222 may comprise material which selectively adsorbs heparin.
• the tangential flow column which may be in cartridge form generally designated 206, is comprised of two layers spirally wound chromatographic media 201, spirally wound impermeable film 202 and spirally wound flow guidance web 203 around a cylindrical core 200.
• the housing 207 and outlet cap 208 contain the spirally wound device.
• the spirally wound media, impermeable film and web 203 are sealed on each end 209 by thermo- plastic fusion using techniques described above.
• the outlet cap 208 and housing are held together by a weld 210.
• the housing also contains inlet and outlet means 211 and 212 which may be protected with a protective cap 213.
• one layer each of chromatographic media 201, impermeable sheet 202 and flow guidance web 203 are wound around the cylindrical core 200.
• the sample flows tangential to the chromatographic media in the channels 205 and is collected by a series of grooves 214 in the cylindrical core 200 which are in fluid communication with the channels 205.
• the media, web and layer thickness 204 can be adjusted to achieve optimal flow rate, chromatographic efficiency and cell stability.
• the tangential flow cartridge having one layer of chromatographic media 201 is prepared by layering media 201 and impermeable film 202 onto the webbing 203, wherein one end 215 of the webbing 203 is not covered by media 201 or film 202.
• the opposite end of the media 216 has an additional layer of webbing 203 layer on top and extending beyond the layer of media 201.
• Figure 16 depicts a top plan view of the tangential flow cartridge having an impermeable sheet 202, a web as flow channel guidance
• the sample is delivered to the channels 205 defined by the web at the blood flow inlet 218.
• the affinity, reverse-phase, or bioactive materials of the invention can be used in any of the well known prior art processes of affinity or reverse-phase chromatography, or as supports for bioreactors.
• the materials obtained in the present invention have unique properties over materials used heretofore.
• a binary system formed by mixing modified polysaccharide, e.g., cellulose, with other types of polysaccharide, such as microcrystalline cellulose, and forming a fibrous sheet (without the addition of extra particulate material) has the advantage of lacking silica materials, which normally shows nonspecific adsorption of proteins.
• a ternary system formed from modified polysaccharide, modified or unmodified particulate and unmodified fibers has the advantage of potential maximization of swelling, rigidity and capacity obtainable through varying the multiple variables present in the system. Flow rates can be controlled by varying the ratio of organic to particulate (especially silica) components without significant loss of capacity.
• such a system shows advantages over prior art systems using non.modified celluloses in that, in many instances, no refined pulp is necessary, since the polymer linked on the polysaccharide will function as well as refined pulp in bridging particles to the fiber surfaces.
• the polymeric components in the present system may also function as binder resins; therefore, addition of resins to the slurry can, if desired, be eliminated.
• the materials of the present invention provide means of binding multifunctional groups per each polysaccharide molecule. As long as these functional groups are made accessible for anchoring, the preparation is no longer limited to high surface area materials. In protein separation and purifications, the key factor which ought to be avoided is possible damage to the protein molecules. In the present invention, this is avoided by using biocompatible materials such as polysaccharides with only limited amounts of organic polymers. The materials are swellable and provided for very little denaturation of protein. Nonspecific adsorption of biopolymers is decreased, since both acrylic and saccharide polymers show very low amounts thereof, and are hydrophilic in nature.
• the tangential flow cartridge may be used to separate or purify components of blood or fermentation media.
• the matrix may comprise a celluloseacrylate bi-component composite polymer with aldehyde groups extended on a spacer arm for coupling chemical or biological ligands for the separation of the components of a sample.
• biological ligands include, for example, anti-coagulant adsorption ligands which allow removal of citrate-phosphate-dextrose (CPD), a commonly used anticoagulant, or heparin from the blood.
• Ligand A low molecular weight amino compound comprising hexamethylenediamine
• Ligand B protamine
• Ligand C high molecular weight amino polymer comprising polyethyleneimine
• Another area of design flexibility and design control is in the possible adjustment of the length of the acrylic polymer carrying the various anchoring groups.
• the variability of the polymer length no only may eliminate steric hindrance due to solute or ligand accessibility, but also minimizes leakage of the ligand from the matrix.
• the polymer "arm” should not be too long to avoid potential internal hydrophilic interaction, such as folding back.
• An “arm” of about 5 to 20 atoms is generally ideal for attaching the bioligands.
• the materials can incorporate all of the synthetic methods developed in prior art commercial materials, such as Sephadex R or Sepharose R .
• the matrix is chemically and physically stable with minimum change of dimensional stability upon contact with salt and eluants.
• Component B glycidyl methacrylate
• Component C mono or di-saccharide
• Step 1 Coupl ing of C to B
• Component B (10%) polymerization Copolymer of 10% B by Redox catalyst & 90% BC
• Step 2 Component C is added to the polymer on a proper molar ratio such that 90% of the available epoxy groups will be reacted with C under acid or base catalytic conditions. The remaining 10% will be left available for coupling onto the cellulose surface afterwards.
• Examples 3 and 4 are directed to the formation of the pre-ligand matrix;
• Example 5 is directed to the coupling of the benzamidine ligand.
• the cellulose was dispersed in the 250 ml of D.I. H 2 O with agitation at 80oC and the glycidyl acrylate added to the reactor. Temperature and agitation were maintained, the APS, STS and HCl added, and the reaction permitted to proceed for one hour.
• the covalentlybonded cellulose-GA pre-ligandized matrix was removed, washed with 7 ⁇ 2 liters of deionized water and stored for further treatment (conversion to affinity ligand as in Example 5 below).
• the cellulose was dispersed in the D.I. H 2 O with agitation and heated to 80oC, with agitation.
• the glycidyl methacrylate, APS and STS were added to the reactor and the reaction permitted to proceed for one hour.
• the reaction was terminated and the covalently bound GMA-cellulose matrix removed, washed with 5 ⁇ 1.8 liters of D.I. H 2 O and stored for further processing (conversion to affinity ligand as in Example 5 below).
• the GMA-cellulose matrix of Example 4 was washed with five volumes of deionized H 2 O.
• the washed GMA-cellulose matrix was treated with a 1.5% aqueous solution of NaIO 4 at room temperature, five volumes of the NaIO 4 solution circulated through the matrix for 1 to 2 hours at room temperature.
• the resulting matrix, the epoxy groups now converted to aldehyde groups by the NaIO 4 solution was washed with 10 volumes of deionized water at 25oC and equilibrated with 0.01 M phosphate buffer at pH 7.8.
• Benzamidine in a concentration of mg/ml was circulated through the aldehyde-pendant matrix at a flow rate of 2 cc/min overnight at 4oC in the presence of NaCNBH 3 , concentration approximately 1 mg/ml.
• the coupling of benzamidine may be effected at room temperature by circulating the above benzamidine solution at room temperature for 8 hours. Following coupling, any uncoupled protein was removed from the matrix by washing with phosphate buffer at pH 7.8.
• any remaining aldehyde groups were deactivated by circulating glycine ethylester at pH 6.5 in the presence of NaCNBH 3 for four hours, the glycine ethylester produced by dissolving 1 g of glycine ethylester hydrochloride in 100 cc of deionized water, the pH adjusted to 6.5 by addition of sodium hydrroxide solution.
• n-Octyl acrylate and glycidyl methacrylate were well mixed before pouring into the reactor.
• reaction mixture was strongly agitated and the reaction temperature was raised to 82oC within 15 minutes.
• reaction mixture was strongly agitated, and the reaction temperature was raised to 80oC within 15 minutes.
• reaction mixture was cooled to 60oC, and then sodium iminodiacetate was charged into the reactor. Further reaction was continued for 26 hours.
• Protein A is a cell-wall protein which can be isolated from Staphylococcus aureus. Its rather unique reactivity directed to the F c region of IgG makes protein A a powerful bioligand for the selective removal of IgG from normal or immune sera. Immobilization of protein A to a solid support has been studied to isolate whole IgG or individual subclasses, to separate cells or to remove immuno-complex from plasma as a means of immunotherapy. (a) Materials and Methods
• Recombinant Protein A was purchased from Repligen with the following physicochemical properties. The purity is greater than 98% by TSK-3000 HPLC assayed at 214 nm and greater than 94% pure at 280 nm. A single major band is produced by SDS-PAGE at approximately 45,000 MW + 1,000. It has an extinction coefficient at 0.1% of 0.16+0.02 at 270 nm. The IgG binding activity is greater than 95% as determined by the percentage of total protein bound to an immobilized human IgG column when assayed by Lowry.
• the composite media prepared according to Example 4 was prepared by grafting to cellulose, with cellulose serving as solid support and GMA or GA polymers providing the chemical functional groups for coupling. Two types of affinity matrix were then prepared. Type A carried amino groups and used glutaraldehyde for protein coupling; Type G carried hydroxy groups requiring periodate oxidation for the formation of aldehyde as a means of protein coupling. PROTEIN A-COUPLING
• the amino groups were introduced to the matrix by covalently linking 1,6-diamino hexane to the glycidyl groups of the matrix.
• the total amino groups titrable from the potentiometric titration with 0.1 N hydrochloric acid was about 1.8 meq per gram of dry matrix.
• the matrix carrying amino groups was equilibrated with 0.1 M sodium phosphate, pH 7.3, as activation buffer, followed by recirculation of 10% glutaraldehyde in the same buffer at room temperature for 6 hours in the presence of 1.0 mg/ml sodium cyanoborohydride.
• Protein A solutions were prepared at 10 mg/ml in 0.1 M sodium phosphate pH 7.3, and recirculated in the presence of (1.0 mg/ml) sodium cyanogen borohydride for 16 hours at room temperature; the uncoupled ligand was removed by rinsing the matrix with buffer solution until the ligand was no longer detectable in the eluate. The excess amount of active groups was deactivated by recirculating 1% glycine ethylester hydrochloride in 0.1 M sodium phosphate pH 6.5 as blocking buffer for 4 hours. Then the matrix was rinsed with any desired buffer containing 0.25 M NaCl, followed by the equilibration buffer that was to be used when performing the affinity step.
• the amount of ligand coupled to the cartridge was determined by doing a Lowry protein assay of the dialyzed uncoupled ligand and subtracting this value from the amount of Protein A given.
• the cartridge was equilibrated with 50 mM NaP, pH 7.6 + 250mM NaCl.
• a human gammaglobulin (HGG) solution (Sigma G-4386), approx. 2.5 mg/ml in the above buffer, was passed through the cartridge either in a single pass or with recirculation.
• the unbound gammaglobulins were then washed from the matrix with the above buffer.
• the bound gammaglobulins were eluted from the matrix with a 0.2 M Gly-HCl pH 2.3 buffer.
• the amount of HGG bound was determined by OD 280 of the elution.
• the matrix was then re-equilibrated with the pH 7.6 buffer and it was ready for another sample.
• PROTEIN A AFFINITY MATRIX
• Goat anti-HGG was immobilized on type G matrices by forming aldehyde groups through periodate oxidation, with the results shown in Table V.
• Goat Anti-HGG was the immobilized antibody to interact with HGG as the antigen.
• the bound antigen were eluted by 0.2M glycine-HCl at pH2.3 from the column in purified form.
• Cartridges were washed well using above buffer and 0.2 M Gly-HCl, pH 2.3, alternately.
• Cartridges were deactivated with 1% glycine ethyl ester.
• Immobilization of glucoamylase has already been applied in the industrial production of fructose syrup from starch.
• the preparation used in the large-scale production of glucose was obtained by the covalent binding of enzyme by means of glutaraldehyde to porous inorganic materials containing primary amino groups.
• the silica type of inorganic material will affect the flavor and color of the beer due to the ionization of silica under low pH to silicic acid on solid surfaces.
• Glucoamylase ( ⁇ -1,4-glucan glucohydrolase, EC 3.2.1.3), purchased from Sigma Chemical Co., St. Louis, MO, is obtained from strains of Asperqillus oryzae.
• the enzyme has a molecular weight between 48,000 to 80,000 and usually has no subunit structure.
• the carbohydrate content of the enzyme ranges from 3% to 30% containing mainly mannose, but glycose, galactose, and, in some instances, glucosamine and xylose are also present.
• the carbohydrate structure is present as mono-, di-, tri-, and tetrasaccharide units linked O-glycosidically through mannose to the hyroxyl groups of serine and threonine.
• the isoelectric point is between 3.9 and 5.5, and has a pH optima of 5.0 to 5.5
• the enzymatic activity was determined by the kinetic determination of the amount of glucose released from starch versus time by glucoamylase.
• the glucose production was measured utilizing an assay system purchased from Sigma Chemical Company (Stock No. 15-10) and described in Sigma Technical Bulletin Co. 15 UV.
• the change in glucose production is measured as a change in optical density at 340 nm per minute on a Cary 210 spectrophotometer.
• the analysis is based upon the conversion of glucose to glucose6-phosphate by ATP in the presence of hexokinase, coupled with the subsequent reduction of NADP to NADPH.
• NADPH has a high absorbance at 340 nm (A 340 ) and NADP has no absorbance at this wavelength, the reaction can be followed by measuring the increase in A 340 .
• the increase in A 340 due to formation of NADPH is directly proportional to the amount of glucose present.
• One unit will liberate 1.0 mg of glucose from starch in 3 min at pH 4.5 at 55oC.
• Type A matrix prepared according to Examples 4 and 8 is a 12-atom spacer amino matrix which can be activated by glutaraldehyde to make it suitable for enzyme immobilization according to the mechanism shown above.
• the matrix was washed with 50 ml of deionized water
• the ligand (10 mg/ml in same buffer) was coupled overnight at room temperature in presence of *NaBH 4 (usually 1 mg/ml NaBH 4 is used);
• the amount of ligand coupled to the column was determined by nitrogen analysis.
• the activity of glucoamylase samples was determined using a 1% solution of Amylopectin (Sigma No. 1-8515) in 0.2M sodium acetate pH 4.5. To 1 ml of this substrate 0.25 ml of sample was added at Time 0 in a 55oC water bath. After 10.0 minutes, 1.25 ml of 0.2M potassium phosphate buffer pH 7.5 was added. A 40 ⁇ l aliquot of this solution was then added to 3.0 ml of reconstituted Sigma Glucose Assay No. 15-UV, and after a 5-minute incubation, the A 340 was recorded. Appropriate test blanks were subtracted from the absorbance reading to determine A 340 , and from the A 340 sample activity in units was calculated.
• PHMA is polyhyroxyl methacrylate prepared by acid hydrolysis of GMA to convert glycidyl into hydroxyl groups as demonstrated above.
• the conditions for activating PHMA for ligand coupling are as follows: a. wash cartridges with 2 liters H 2 O b. activated with 1.5% HalO 4 (in H 2 O) for 60 mins c. wash with H 2 O d. equilibrate cartridge with 0.1M borate buffer (pH 8.2) e. recirculate 42 mis of a 10 mg/ml solution of protamine in above buffer (Sigma No. P-4005 protamine, free base, from Salmon Sperm Grade IV) for 4 hrs at 15 mls/min f.
• wash cartridge with 0.1M borate buffer pH 8.2 (save wash to determine amount of coupled protamine) g. recirculate 100 mis of a 15% amine solution (either ethanolamine or diethylaminopropylamine) in the pH 8.2 buffer at 15 mls/min for approximately 20 hrs with 4 x 500 mg NaBH 4 additions h. wash cartridge with 1 liter solution of:
• This assay employs the metachromasia of azure A when heparin is added. It is useful for 0 to 10 units/ml and does not depend on the anticoagulant activity of heparin.
• the assay can be used in plasma, whole blood, and buffered saline solutions. The dye and sample are mixed together and then read immediately on a spectrophotometer at 620 nm. Controls include samples of known heparin concentration and a sample that contains no heparin. b. A Clotting Procedure for the Quantitative Determination of Heparin in Plasma, Sigma Kit #87 B
• activated factor X (Xa) is rapidly neutralized by its plasma inhibitor.
• the amount of factor Xa neutralized is proportional to the concentration of the heparin present.
• protamine coupled #1 - 12.98 mg protamine coupled; deactivator used was diethylaminopropylamine.
• PHMA media in 10 ml columns a. wash with 20 ml H 2 O b. 1.5% NaIO 4 , in H 2 O, passed through at 1 ml/min for 30 min c. wash with 20 ml H 2 O d. equilibrate with 0.1M Sodium Phosphate buffer, pH 7.5 e. 6 mis of a 5 mg/ml Polymyxin-B Sulfate solution in above buffer is recirculated overnight (0.5 mls/min) f. uncoupled Polymyxin-B Sulfate washed out with 0.1M Phosphate buffer (saved for determination of amount coupled) g. column then equilibrated with 0.1M Borate buffer, pH 8.0 h. 20 mis of a 1% ethanolamine solution recirculated with 4 ⁇
• the 10 ml column is the mini-column with a 10 mm diameter.
• Test used Whittaker Byproducts' QCL1000 Quantitative Chromogenic LAL assay.
• Gram-negative bacterial endotoxin catalyzes the activation of a proenzyme in the Limulus AmebocyteLysate (LAL).
• LAL Limulus AmebocyteLysate
• the initial rate of activation is determined by the concentration of endotoxin present.
• the activated enzyme catalyzes the splitting of p-nitroaniline (pNA) from a colorless substrate and the yellow color is measured photometrically at 405 nm after the reaction is stopped with acetic acid. After standards are run, the correlation between the absorbance and the endotoxin concentration is linear in the 10-200 pg/ml range.
• pNA p-nitroaniline
• the starting material is diluted with sterile saline to the proper concentration range and assayed to form a curve.
• the unknown samples diluted approximately the same way, are assayed against this curve.
• the pg/ml value obtained for the unknown, times the dilution equals the concentration of endotoxin in the unknown.
• test procedure A 10 ml cartridge
• test procedure B 530 ⁇ g/g media
• Protein A and Protein G Affinity Filters Configured for Radial Flow
• Protein A is a well-known staphylococcal-derived protein that interacts with Fc region of immunoglobulins, especially immunoglobuli n G .
• Uses of Protein A include the detection and puri fi cati on of antibodies and immune complexes, bulk production of immunoglobulin fractions, and monoclonal antibodies, and as a potential for treatment of certain cancers and autoimmune diseases via extracorporeal plasma filtration.
• Protein G is an immunoglobulin binding bacterial cell wall protein isolated from group G streptococci; it has recently been cloned and expressed in bacterial cell systems and hence is available in sufficient quantity to fully investigate its properties. Protein G is analogous to Protein A in that it too binds to the Fc region of immunoglobulins but is reported to be a more general IgG binding reagent since it will bind to certain animal IgGs and human IgG subclasses that interact poorly or not at all with Protein A.
• the Type III receptors (Protein G) are thought to have broader applications in the immunological arena than the Type I Fc Receptor (Protein A).
• Recombinant Protein A was purchased from Repligen Corporation (Cambridge, MA).
• Protein G (Type GXIlTM) was supplied by Genex Corporation (Gaithersburg, MD)D Both proteins were immobilized to the new matrix as described in Tables X and XI.
• the affinity support used was activated to contain aldehyde groups at the end of an 18-atom spacer arm.
• the extended arm support was selected to prevent steric problems that sometimes occur when macromolecules are immobilized and used to purify equally large macromolecu!es. All standard chemicals used in the immobilization or evaluation steps were reagent grade and purchased from typical laboratory suppliers. Purified bovine and human IgG fractions (Fraction II, III) were purchased from Sigma Chemical Co. (St. Louis, MO).
• Human serum was obtained from the American Red Cross (Farmington, CT) and was partially purified by ion exchange.
• Polyclonal rabbit antisera to human albumin and goat antisera to human IgG were purchased from Cone BioProducts (Seguin, TX) and were also partially purified by ion exchange before use.
• an affinity purified fraction of goat anti-human IgG was used.
• Laboratory peristaltic pumps (Rainin) were used during the coupling and testing of the affinity supports in order to achieve the flow rates required.
• On-line UV monitors (Gilson) were used to monitor the dynamic events. Protein measurements were routinely made by O.D. 280 and verified by Lowry.
• the Protein A column coupled at 100% efficiency and contained 5 mg of immobilized ligand, whereas the Protein G column coupled at 88% efficiency and contained 4.4 mg of immobilized ligand.
• the test solutions were adjusted (by dilution) so as to offer 50 mg (+/- 5 mg) total IgG to each column.
• Protein A and Protein G columns were tested simultaneously with the same starting pool of test sample to avoid the possibility of variation in the starting sample.
• the ratio provided above is defined as the interaction ratio between total eluted IgG and immobilized ligand (i.e., eluted IgG/bound ligand).
• Bovine IgG (pure) 25.19 2.03 44.44 4.20
• the test solutions were adjusted (by dilution) so as to offer 125 mg (+/- 5 mg) total IgG to each column.
• Protein A and Protein G columns were tested simultaneously with the same starting pool of test sample to avoid the possibility of variation in the starting sample.
• the Ratio reported above Eluted IgG/bound ligand.
• the matrix composition and coupling methods are compatible with both ligands.
• Protein A was immobilized to hexamethylenediamine derivatized matrix configured in a 250 mL (housing volume) tangential flow cartridge containing 13.8 gm of matrix.
• the cartridge was first equilibrated with 0.1 M borate buffer, pH 8.2 (about 2000 mL) then treated with 7 mL of a 10 mg/ml solution of protein A, pH 8.2, was recirculated through the cartridge overnight (21 hrs).
• the flow rate was about 100 mL/min for the first 2.5 hrs, 50 mL/min for the next 1.5 hrs and then 25 mL/min for the remaining time.
• Sodium borohydride (4 ⁇ 250 mg each) was then added at 30 minute intervals. After about 1-1/2 hours, the solution was allowed to recirculated at 4oC overnight. Then, four portions of sodium borohydride (400 mg) were added (at 45 min. intervals) and the solution allowed to recirculate for about four hours at room temperature.
• H ⁇ G ws dissolved in 0.05 M phosphate buffer (5 mg/ml), pH 7.6 containing 0.25 M NaCl and recirculated at flow rates of 25, 100, and 140 mL/min for 60 minutes.
• Table XII the capacity of the column was only slightly higher at 100 mL/min flow (208 mg) than at 50 mL/min (194 mg) and at 25 ml/min (195 mg).
• Experimental media was placed in a test tube. Buffered saline, human plasma, and whole blood were made to contain 10 or 20 units/ml heparin (Sigma Heparin from Porcine Intestinal micosa H-7005; activity 150-180 USPK1 U (units)/mg). Test fluid was left in contact with media for 30 min, then samples were centrifuged for approximately 5 min at low speed, just until media was pelleted.
• heparin Sigma Heparin from Porcine Intestinal micosa H-7005; activity 150-180 USPK1 U (units)/mg
• the high molecular weight polymer of ligand C (polyethyleneimine ligand) is the strongest heparin adsorbent. 100% of heparin removal is achievable by contacting 10 ml of blood containing 20 U/ml with 250 cm 2 media for 1 hour. Approximately 20% of the 29 mg citrate existing in the blood was also found to be adsorbed by the same media.
• a tangential flow cartridge was constructed based on the "250 size" cartridge as shown in Figure 11.
• Webbing material as first wrapped around the core twice to prevent the media from blanking off the core. Then, the media and impermeable film are wrapped together with the webbing material until a diameter of 2.600 inches was obtained. The media and film were then cut and a 4-inch piece of webbing was inserted 2 inches between the media and the preceding wrap of webbing. Finally, the subassembly is held together with elastic bands and then capped with polypropylene end caps.
• the 250 mL size cartridge (housing volume) contains 14.4 grams of matrix with a total surface area of 180 cm 2 .
• the tangential flow cartridge was thoroughly flushed with saline solution at 50 ml/min.
• the filtered blood was fraction-collected at the last 10 ml per every 100 ml fraction. Heparin concentration in each fraction was analyzed by the colorimetric assay and plotted as shown in Figure 23.
• Figure 23 shows that approximately 70% of the heparin was removed. In a separate experiment (data not shown), 80% of the heparin was removed by passing two units of blood through an 800 size cartridge.
• Table XV the analysis of blood components for various volumes of filtered blood for the 250 mL size tangential flow cartridge is depicted.
• Table XVI shows the analysis of blood components for various volumes of blood using the 800 mL size tangential flow cartridge.
• Blood Component 0 100 200 300 400 500 Hct ml/100 ml 35.0 28.3 33.5 34.0 35.0 28.0 Hb gm/d.l 10.9 9.5 10.8 11.0 10.6 8.9 Hb in plasma 15.15 4.34 9.61 11.16 9.3 7.13 RBC ⁇ 10 6 / ⁇ l 3.59 3.04 3.49 3.59 3.43 2.56

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