# US 2015/0147243 A1 · Mixed multifunctional metal affinity surfaces for reducing aggregate content in protein preparations > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2015/0147243 A1 - Document ID: US20150147243A1 - Status: Published application - Kind: A1 - Application number: 14/555,165 - Filed: 2014-11-26 - Published: 2015-05-28 - Earliest claimed: 2012-05-31 - Assignees: Agency for Science, Technology and Research - Applicants: Agency for Science, Technology and Research - Inventors: Peter Gagnon - Web page: https://patentgrep.com/patent/US20150147243A1 ## CPC - B01D 15/36: involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion - C07K 16/32: against translation products of oncogenes - B01D 15/34: Size-selective separation, e.g. size-exclusion chromatography; Gel filtration; Permeation - B01D 15/362: {Cation-exchange} - B01D 15/363: {Anion-exchange} - B01D 15/3809: {of the antigen-antibody type, e.g. protein A, G or L chromatography} - B01D 15/3828: {Ligand exchange chromatography, e.g. complexation, chelation or metal interaction chromatography} - B01J 20/3202: {characterised by the carrier, support or substrate used for impregnation or coating} - B01J 20/3265: {with an organic functional group containing a metal, e.g. a metal affinity ligand} - B01J 20/3285: {Coating or impregnation layers comprising different type of functional groups or interactions, e.g. different ligands in various parts of the sorbent, mixed mode, dual zone, bimodal, multimodal, ionic or hydrophobic, cationic or anionic, hydrophilic or hydrophobic} - B01J 39/26: Cation exchangers for chromatographic processes - B01J 41/20: Anion exchangers for chromatographic processes - B01J 47/014: in which the adsorbent properties of the ion-exchanger are involved, e.g. recovery of proteins or other high-molecular compounds - B01J 47/04: Mixed-bed processes - C07K 1/165: {mixed-mode chromatography} - C07K 1/22: Affinity chromatography or related techniques based upon selective absorption processes - C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies - Y10T 428/2913: Rod, strand, filament or fiber - B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS - Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION ## Abstract Compositions for reducing the aggregate content of a protein preparation include a first substrate having a first surface-bound ligand possessing a metal affinity functionality and a second surface-bound ligand optionally provided on a second substrate and having an aggregate charge opposite to that of the metal affinity functionality of the first substrate, wherein the first surface-bound ligand and the second surface-bound ligand are positioned such that the protein preparation may contact both the first surface-bound ligand and the second surface-bound ligand simultaneously. ## Claims ### Claim 1 A composition of matter for reducing the aggregate content of a protein preparation, comprising a first substrate comprising a first surface-bound ligand possessing a metal affinity functionality and a second surface-bound ligand optionally provided on a second substrate and having an aggregate charge opposite to that of the metal affinity functionality of the first substrate, wherein the first surface-bound ligand and the second surface-bound ligand are positioned such that the protein preparation may contact both the first surface-bound ligand and the second surface-bound ligand simultaneously. ### Claim 2 (depends on 1) The composition of claim 1, wherein the first surface-bound ligand, the second surface-bound ligand, or both comprise a multidentate metal chelating moiety. ### Claim 3 (depends on 1) The composition of claim 1, wherein the second surface-bound ligand possesses metal affinity functionality. ### Claim 4 (depends on 1) The composition of claim 1, wherein the first surface-bound ligand has an aggregate charge which is electronegative. ### Claim 5 (depends on 4) The composition of claim 4, wherein the first substrate has additional chemical moieties bound to it, provided that an aggregate charge of the first substrate remains electronegative. ### Claim 6 (depends on 1) The composition of claim 1, wherein the first surface-bound ligand is electronegative and selected from the group consisting of iminodiacetic acid (2-(carboxymethylamino)acetic acid), ethylene glycol(aminoethylether)diacetic acid, nitriloacetic acid (2,2′,2″-nitrilotriacetic acid), aspartic acid, and glutamic acid. ### Claim 7 (depends on 1) The composition of claim 1, wherein the second surface-bound ligand is tris(2-aminoethyl)amine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, polypropylenimine tetraamine, or desferroxamine. ### Claim 8 (depends on 1) The composition of claim 1, wherein the first surface-bound ligand is iminodiacetic acid (2-(carboxymethylamino)acetic acid). ### Claim 9 (depends on 1) The composition of claim 1, wherein the first surface-bound ligand has an aggregate charge which is electropositive. ### Claim 10 (depends on 1) The composition of claim 1, wherein the first substrate has additional chemical moieties bound to it, provided that an aggregate charge of first substrate remains electropositive. ### Claim 11 (depends on 1) The composition of claim 1, wherein the first surface-bound ligand is tris(2-aminoethyl)amine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, polypropylenimine tetraamine, or desferroxamine. ### Claim 12 (depends on 1) The composition of claim 1, wherein at least one of the first substrate or optional second substrate has one or more further surface-bound ligands in addition to the first surface-bound ligand and the second surface-bound ligand, wherein the one or more further surface-bound ligands enhance the capacity of the composition to participate in hydrogen bonding, hydrophobic interactions, or pi-pi binding with a component of the protein preparation. ### Claim 13 (depends on 1) The composition of claim 1, wherein the first substrate, the second substrate, or both comprise more than one negatively charged metal chelating group in combination with one or more oppositely charged groups. ### Claim 14 (depends on 1) The composition of claim 1, wherein the first substrate, the second substrate, or both comprise more than one positively charged metal chelating group in combination with one or more oppositely charged groups. ### Claim 15 (depends on 1) The composition of claim 1, wherein the first surface-bound ligand and the second surface-bound ligand are covalently bound to the first substrate. ### Claim 16 (depends on 1) The composition of claim 1, wherein the first surface-bound ligand is covalently bound to the first substrate and the second surface-bound ligand covalently bound to the second substrate which is different from the first substrate. ### Claim 17 (depends on 1) The composition of claim 1 wherein the first substrate, second substrate, or both comprise particles. ### Claim 18 (depends on 17) The composition of claim 17, wherein the particles are non-porous. ### Claim 19 (depends on 17) The composition of claim 17, wherein the particles are porous. ### Claim 20 (depends on 17) The composition of claim 17, wherein the particles have a pore size large enough to permit entry of a protein in a protein preparation. ### Claim 21 (depends on 17) The composition of claim 17, wherein the particles have a pore size too small to permit entry of a protein in a protein preparation. ### Claim 22 (depends on 19) The composition of claim 19, wherein the particles have an average pore size between about 10 nm and about 100 nm. ### Claim 23 (depends on 19) The composition of claim 19, wherein the particles have an average pore size less than about 10 nm. ### Claim 24 (depends on 19) The composition of claim 19, wherein the particles have an average pore size more than about 100 nm. ### Claim 25 (depends on 1) The composition of claim 1, wherein the first substrate, second substrate, or both, comprise a membrane, or a monolith. ### Claim 26 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) The composition of any one of claims 1 to 12, wherein the first substrate, second substrate, or both comprise a solid or porous walled hollow fiber. ### Claim 27 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) An apparatus comprising the composition of any one of claims 1 to 26, wherein the apparatus is optionally configured for chromatography. ### Claim 28 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) The apparatus of claim 27, further configured to allow the protein preparation to subsequently contact, in any order, the composition according to any one of claims 1 to 26 and one or more further substrates comprising one or more further surface-bound ligands comprising a charge selected from the group consisting of electronegative, electropositive, neutral, and combinations thereof. ### Claim 29 (depends on 27, 28) The apparatus of any one of claim 27 or 28, wherein the apparatus is a chromatographic device packed with the first substrate, optional second substrate, one or more further substrates, and combinations thereof. ### Claim 30 (depends on 27, 28, 29) The apparatus of any one of claims 27 to 29, wherein a composition at an entrance of the apparatus is different from a composition at the exit of the apparatus. ### Claim 31 (depends on 27, 28, 29, 30) The apparatus of any one of claims 27 to 30, wherein a chemical surface of one or more components of the apparatus is substantially inert or of sufficiently low surface area as to make no significant contribution to the chemical functionality of the apparatus. ### Claim 32 (depends on 27, 28, 29, 30, 31) The apparatus of any one of claims 27 to 31, wherein the chemical surface of one or more components is configured to allow one or more of (1) create structural integrity, (2) direct flow of liquids therethrough, and (3) physically block, entrap, or entrain insoluble materials in a protein preparation to prevent them from interfering with the effective use of the device. ### Claim 33 (depends on 27, 28, 29, 30, 31, 32) The apparatus of any one of claims 27 to 32, wherein the apparatus comprises one or more porous membranes and at least one of the one or more porous membranes is the first substrate to which the first surface-bound ligand or the second surface-bound ligand is bound. ### Claim 34 (depends on 27, 28, 29, 30, 31, 32) The composition of any one of claims 27 to 32, wherein the first substrate, optional second substrate, or both comprise a permeable chopped fiber mat. ### Claim 35 (depends on 27, 28, 29, 30, 31, 32) The composition of any one of claims 27 to 32, wherein the first substrate, optional second substrate, or both comprise an ordered wound, woven, or non-woven fiber structure. ### Claim 36 (depends on 27, 28, 29, 30, 31, 32) The composition of any one of claims 27 to 32, wherein the first substrate, optional second substrate, or both comprise a combination of a permeable chopped fiber mat and an ordered wound, woven, or non-woven fiber structure. ### Claim 37 (depends on 27, 28, 29, 30, 31, 32) The apparatus of any one of claims 27 to 32, wherein the apparatus comprises porous reticular fibers, wherein the porous reticular fibers are hollow porous-walled fibers or non-porous fibers, and wherein the porous reticular fibers are the first substrate to which the first surface-bound ligand and the second surface-bound ligand are bound. ### Claim 38 (depends on 27, 28, 29, 30, 31, 32) The apparatus of any one of claims 27 to 32, wherein the apparatus comprises porous or non-porous particles sandwiched between porous membranes, or monoliths. ### Claim 39 (depends on 27, 28, 29, 30, 31, 32) The apparatus of any one of claims 27 to 32, wherein porous or non-porous particles are the first substrate to which either the first surface-bound ligand or the second surface-bound ligand is bound and a membrane or monolith is a second substrate to which the other of the first surface-bound ligand and the second surface-bound ligand is bound. ### Claim 40 (depends on 27, 28, 29, 30, 31, 32) The apparatus of any one of claims 27 to 32, wherein the apparatus comprises porous or non-porous particles sandwiched between a woven or amorphous fibrous filter, or embedded within a fibrous matrix. ### Claim 41 (depends on 27, 28, 29, 30, 31, 32) The apparatus of any one of claims 27 to 32, wherein particles comprise the first substrate or second substrate to which either the first surface-bound ligand or the second surface-bound ligand is bound and fibrous filters comprise the second substrate to which the other of the first surface-bound ligand and the second surface-bound ligand is bound. ### Claim 42 (depends on 27, 28, 29, 30, 31, 32) The apparatus of any one of claims 27 to 32, wherein the apparatus comprises porous or non-porous particles sandwiched between woven or crystalline frits. ### Claim 43 (depends on 27, 28, 29, 30, 31, 32) The apparatus of any one of claims 27 to 32, wherein the porous or non-porous particles are the first substrate to which either the first surface-bound ligand or the second surface-bound ligand is bound and the woven or crystalline frits are the second substrate to which the other of the first surface-bound ligand and the second surface-bound ligand is bound. ### Claim 44 (depends on 27, 28, 29, 30, 31, 32) The apparatus of any one of claims 27 to 32 wherein the apparatus comprises porous or non-porous particles embedded in a reticular polymer network. ### Claim 45 (depends on 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44) The apparatus of any one of claims 27 to 44, wherein the first substrate and second substrate comprise any combination of configurations as recited in claims 27-44.