Intravenously injectable immunoglobulin G (IGG) and method for producing same
First Claim
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1. A method for production of high purity IgG from animal plasma, or a fraction thereof, comprising:
- (1) separating IgG from said animal plasma or fraction thereof to produce partially purified IgG by ion exchange chromatography using a chromatographic media comprising a matrix material comprising a first surface reactive group--containing substrate selected from the group consisting of silica, polysaccharide, or polypeptide, said surface reactive group being selected from the group consisting of the hydroxy group of silica, the hydroxy group of polysaccharide, or the amino group of polypeptide, said first substrate covalently bonded to a first synthetic polymer, said first synthetic polymer comprising;
(a) a polymerizable compound containing an epoxy group capable of direct covalent coupling to said reactive group of said substrate; and
(b) one or more polymerizable compounds containing;
(i) an ionizable chemical group;
(ii) a chemical group capable of transformation to an ionizable group;
(2) separating high purity IgG from said partially purified IgG by affinity chromatography using a chromatographic media comprising a second matrix material comprising a second surface reactive group-containing substrate selected from the group consisting of silica, polysaccharide or polypeptide, said surface reactive group being selected from the group consisting of the hydroxy group of silica, the hydroxy group of polysaccharide, or the amino group of polypeptide, said second substrate covalently bonded to a second synthetic polymer, said second synthetic polymer comprising;
(a) a polymerizable compound containing an epoxy group capable of direct covalent coupling to said reactive group of said second substrate; and
(b) one or more polymerizable compounds containing a chemical group capable of causing the covalent coupling of said second synthetic polymer to an affinity ligand or a biologically active molecule.
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Abstract
A method for producing intravenously injectable IgG comprising a particulate separation step, an ion exchange separation step and an affinity separation step, and the substantially pure, intravenously injectable IgG produced by the method.
106 Citations
35 Claims
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1. A method for production of high purity IgG from animal plasma, or a fraction thereof, comprising:
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(1) separating IgG from said animal plasma or fraction thereof to produce partially purified IgG by ion exchange chromatography using a chromatographic media comprising a matrix material comprising a first surface reactive group--containing substrate selected from the group consisting of silica, polysaccharide, or polypeptide, said surface reactive group being selected from the group consisting of the hydroxy group of silica, the hydroxy group of polysaccharide, or the amino group of polypeptide, said first substrate covalently bonded to a first synthetic polymer, said first synthetic polymer comprising; (a) a polymerizable compound containing an epoxy group capable of direct covalent coupling to said reactive group of said substrate; and (b) one or more polymerizable compounds containing; (i) an ionizable chemical group; (ii) a chemical group capable of transformation to an ionizable group; (2) separating high purity IgG from said partially purified IgG by affinity chromatography using a chromatographic media comprising a second matrix material comprising a second surface reactive group-containing substrate selected from the group consisting of silica, polysaccharide or polypeptide, said surface reactive group being selected from the group consisting of the hydroxy group of silica, the hydroxy group of polysaccharide, or the amino group of polypeptide, said second substrate covalently bonded to a second synthetic polymer, said second synthetic polymer comprising; (a) a polymerizable compound containing an epoxy group capable of direct covalent coupling to said reactive group of said second substrate; and (b) one or more polymerizable compounds containing a chemical group capable of causing the covalent coupling of said second synthetic polymer to an affinity ligand or a biologically active molecule. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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23. A method for producing high purity IgG comprising:
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(1) diluting animal plasma to decrease the plasma solubility of lipids, lipimic colloids, euglobulins and other non-IgG components; (2) passing said diluted animal plasma through at least one separating column comprising a hollow cylinder and discs of solid stationary phase, said discs comprising at least one of activated carbon and at least one of fumed silica, to form a first filtrate containing IgG; (3) chromatographically separating high purity IgG from said first filtrate, said chromatographic separation comprising; (a) an ion-exchange chromatographic separation of said first filtrate to produce a second filtrate containing IgG free of proteins other than proteolytic enzymes; said ion-exchange chromatographic separation effected with an ion-exchange matrix; said ion-exchange matrix comprising a first surface reactive group-containing substrate selected from the group consisting of silica, polysaccharide or polypeptide, said surface reactive group being selected from the group consisting of the hydroxy group of silica, the hydroxy group of polysaccharide, or the amino group of polypeptide, said first substrate covalently bonded to a first synthetic polymer, said first synthetic polymer selected from a polymerizable compound containing an epoxy group capable of direct covalent coupling to said surface reactive group of said first substrate, said polymerizable compound selected from the group consisting of (i) homopolymers of a monomer capable of covalently bonding to said surface reactive groups of said first substrate and containing an ionic group or a group capable of transformation to an ionic group and, (ii) copolymers of a monomer capable of covalently bonding to said surface reactive groups of said first substrate and a monomer containing an ionic group or a group capable of being transformed into an ionic group; (b) an affinity chromatographic separation of said second filtrate to produce a third filtrate comprising essentially pure IgG; said affinity matrix comprising a second surface reactive group-containing substrate selected from the group consisting of silica, polysaccharide or polypeptide, said surface reactive group being selected from the group consisting of the hydroxy group of silica, the hydroxy group of polysaccharide, or the amino group of polypeptide, said second substrate covalently bonded to a second synthetic polymer, said second synthetic polymer selected from a polymerizable compound containing an epoxy group capable of direct covalent coupling to said surface reactive group of said second substrate, said polymerizable compound selected from the group consisting of (i) homopolymers of a monomer capable of covalently bonding to said surface reactive groups of said second substrate and containing a chemical group capable of coupling to an affinity ligand or a biologically active molecule; (ii) copolymers of a monomer capable of covalently bonding to said surface reactive groups of said second substrate and a monomer containing a chemical group capable of coupling to an affinity ligand or a biologically active molecule said ion-exchange matrix comprising a swellable fibrous matrix in sheet form, spirally wound and spaced apart; said ion-exchange matrix contained in a cylindrical housing with end caps having inlet and outlet orifices; said affinity matrix comprising a swellable, spirally wound, spaced apart fibrous matrix in sheet form, contained in a cylindrical housing with end caps having inlet and outlet orifices. - View Dependent Claims (24, 25, 26, 27, 28, 29, 30, 31)
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32. A continuous method for obtaining intravenously injectable IgG, in high yield, highly pure transferrin, and highly pure albumin from animal plasma comprising:
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(1) diluting said animal plasma to form diluted animal plasma; (2) filtering or adsorbing said diluted animal plasma to separate sparingly soluble and precipitated plasma components, whereby a first filtrate is formed; (3) passing said first filtrate through a first ion-exchange column, said first ion-exchange column containing an ion-exchange matrix materal comprising a first surface reactive group-containing substrate selected from the group consisting of silica, polysaccharide or polypeptide, said surface reactive group being selected from the group consisting of the hydroxy group of silica, the hydroxy group of polysaccharide, or the amino group of polypeptide, said substrate covalently bonded to a synthetic polymer, said synthetic polymer comprising; (a) a polymerizable compound containing an epoxy group capable of direct covalent coupling to said reactive group of said substrate; and (b) one or more polymerizable compounds containing; (i) an ionizable chemical group; (ii) a chemical group capable of transformation to an ionizable group, to form a first adsorbed fraction retained on said first ion-exchange column and a first unadsorbed fraction which has passed through said first ion-exchange column, said first unadsorbed fraction containing a predominance of the IgG contained in said animal plasma, said first adsorbed fraction containing some IgG, essentially all the transferrin, and essentially all the albumin; (4) eluting said first ion-exchange column to form a first eluate, said first eluate containing essentially all of the transferrin present in said animal plasma, and a portion of the albumin contained in said animal plasma; (5) further eluting said first ion-exchange column to form a second eluate containing the residual of the albumin originally adsorbed on said first ion-exchange column; (6) passing said first eluate from (4) through a second ion-exchange column, said second ion-exchange column containing a matrix material as in said first ion-exchange column, to form a second adsorbed fraction, said second adsorbed fraction containing essentially all of the albumin in said first eluate, and a second unadsorbed fraction, said second unadsorbed fraction containing essentially all of said transferrin in said first eluate; (7) passing said second unadsorbed fraction from (6) through a third ion-exchange column, said third ion-exchange column containing a matrix as in said first ion-exchange column, said third ion-exchange column equilibrated at pH 5.8, thereby forming a third unadsorbed fraction and a third adsorbed fraction, said third unadsorbed fraction being essentially pure transferrin, said third adsorbed fraction containing residual IgG; (8) eluting said second adsorbed fraction in said second ion-exchange column from (6) to form a third eluate containing the albumin from said second bound fraction; (9) combining said second eluate from (5) and said third eluate from (8) to form a combined eluate and passing said combined eluate through a fourth ion-exchange column, said fourth ion-exchange column containing a matrix material as in said first ion-exchange column, thereby forming a fourth unadsorbed fraction and a fourth adsorbed fraction, said fourth unadsorbed fraction being high purity albumin, said fourth adsorbed fraction containing small amounts of residual IgG; (10) recycling the residual IgG from (9) to said first ion-exchange cartridge by eluting said fourth adsorbed fraction; (11) recycling the residual IgG from (7) back through said first ion-exchange column by eluting said third adsorbed fraction, where it joins fresh diluted, filtered animal serum and the process repeats itself; (12) further purifying said first unadsorbed fraction from (3), whereby highly purified intravenously injectable IgG in high yield is obtained as a primary product and highly pure albumin and transferrin are obtained as by-product. - View Dependent Claims (33, 34, 35)
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Specification