# US 5,453,493 A · Method of precipitating proteins from aqueous media with a polyhydroxy polycarboxylic acid > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,453,493 A - Document ID: US5453493A - Status: Granted - Kind: A - Application number: 08/207,274 - Filed: 1994-03-07 - Granted: 1995-09-26 - Earliest claimed: 1992-03-20 - Assignees: Affinity Technology, Inc. - Inventors: John Krupey - Web page: https://patentgrep.com/patent/US5453493A ## CPC - C08F 8/30: Introducing nitrogen atoms or nitrogen-containing groups - C08F 2810/20: leading to a crosslinking, either explicitly or inherently - C08F 2810/50: wherein the polymer is a copolymer and the modification is taking place only on one or more of the monomers present in minority - C08F: MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS ## Abstract There is provided water insoluble cross-linked polyhydroxy polycarboxylic acid having at least two strands each having a strand skeleton of the formula: ##STR1## wherein one carbonyl group of at least one maleoyl moiety thereof in each strand is covalently linked to a --HN.[(H).sub.p (CH).sub.z.(OH).sub.m ].NH-- moiety to provide the presence therein of at least one cross linking moiety of the formula: ##STR2## wherein R is hydrogen or lower alkylene or lower alkoxy of 1-4 carbon atoms, or phenyl, z is an integer of 1-4, p is 0 or an integer up to z-1, m is 1 or an integer up to z, wherein the ratio of cross-links to poly (alkylene carbonic acid) strands is between about 1 and about 200 to 2. There is disclosed a method of making such polyhydroxy polycarboxylic acid as well as methods of utilizing same to remove proteins from aqueous media containing same to provide a matrix. Methods are disclosed for recovering proteins from said matrix without denaturing them and methods for recovering the polyhydroxy polycarboxylic acid from said matrix. ## Claims ### Claim 1 A method of precipitating a protein from an aqueous medium containing the same which comprises adding thereto an effective amount of a water insoluble polyhydroxy polycarboxylic acid containing material selected from the group containing acids having at least two strands each having a strand skeleton of formula III: ##STR10## wherein one carbonyl group of at least one maleoyl moiety thereof in each strand is covalently linked to a --HN.[(H).sub.p (CH).sub.z.(OH).sub.m ].NH-- moiety to provide the presence therein of cross linking moieties of the formula: ##STR11## wherein R is hydrogen or lower alkylene or lower alkoxy of 1-4 carbon atoms, or phenyl, y is an integer up to m, z is an integer of 1-4, p is 0 or an integer up to z-1, m is 1 or an integer up to z, and the esters thereof with the anhydride corresponding to the skeleton of formula III wherein the ratio of cross-links to poly (alkylene carbonic acid) strands is between about 1 and about 200 to 2 to provide a protein/polyhydroxy polycarboxylic acid matrix. ### Claim 2 A method of precipitating a protein from an aqueous medium containing the same which comprises adding thereto an effective amount of a water insoluble polyhydroxy polycarboxylic acid having at least two strands each having a strand skeleton of formula III: ##STR12## wherein one carbonyl group of at least one maleoyl moiety thereof in each strand is covalently linked to a --HN.[(H).sub.p (CH).sub.z.(OH).sub.m ].NH-- moiety to provide the presence therein at least one cross linking moieties of the formula: ##STR13## wherein R is hydrogen or lower alkylene or lower alkoxy of 1-4 carbon atoms, or phenyl, z is an integer of 1-4, p is 0 or an integer up to z-1, m is 1 or an integer up to z, wherein the ratio of cross-links to poly (alkylene carbonic acid) strands is between about 1 and about 200 to 2 to provide a protein/polyhydroxy polycarboxylic acid matrix. ### Claim 3 (depends on 1) The method of claim 1 wherein the amount of polyhydroxy polycarboxylic acid containing material utilized, by weight, is at least equal to the amount of protein estimated to be contained in the aqueous medium containing same. ### Claim 4 (depends on 2) The method of claim 2 wherein the amount of polyhydroxy polycarboxylic acid utilized, by weight, is at least equal to the amount of protein estimated to be contained in the aqueous medium containing same. ### Claim 5 (depends on 1) The method of claim 1 wherein the polyhydroxy polycarboxylic acid containing material is utilized in an aqueous medium. ### Claim 6 (depends on 2) The method of claim 2 wherein the polyhydroxy polycarboxylic acid is utilized in an aqueous medium. ### Claim 7 (depends on 5) The method of claim 5 wherein the concentrations by weight of the protein and of polyhydroxy polycarboxylic acid containing material in their respective aqueous media have a ratio of between about 3:1 to about 1:3. ### Claim 8 (depends on 6) The method of claim 6 wherein the concentrations by weight of the protein and of polyhydroxy polycarboxylic acid in their respective aqueous media have a ratio of between about 3:1 to about 1:3. ### Claim 9 (depends on 7) The method of claim 7 wherein the concentrations by weight of the protein and of polyhydroxy polycarboxylic acid containing material in their respective aqueous media are substantially equal. ### Claim 10 (depends on 8) The method of claim 8 wherein the concentrations by weight of the protein and of polyhydroxy polycarboxylic acid in their respective aqueous media are substantially equal. ### Claim 11 (depends on 1) The method of claim 1 wherein R is hydrogen, and the pH of the polyhydroxy polycarboxylic acid containing medium is between about 3 to about 6.2 to provide a medium after mixture of the components which does not exceed about pH 6.5. ### Claim 12 (depends on 2) The method of claim 2 wherein R is hydrogen, and the pH of the polyhydroxy polycarboxylic acid containing medium is between about 3 to about 6.2 to provide a medium after mixture of the components which does not exceed about pH 6.5. ### Claim 13 (depends on 1) The method of claim 1 wherein R is phenyl, and the pH of the polyhydroxy polycarboxylic aid containing medium is between about 5.5 to about 7.5 to provide a medium after mixture of the components which does not exceed about pH 7.5. ### Claim 14 (depends on 2) The method of claim 2 wherein R is phenyl, and the pH of the polyhydroxy polycarboxylic aid containing medium is between about 5.5 to about 7.5 to provide a medium after mixture of the components which does not exceed about pH 7.5. ### Claim 15 (depends on 1) The method of claim 1 which comprises the further step of centrifuging the reaction mixture and recovering the matrix therefrom as a pellet. ### Claim 16 (depends on 2) The method of claim 2 which comprises the further step of centrifuging the reaction mixture and recovering the matrix therefrom as a pellet. ### Claim 17 (depends on 1) A method of separating the protein from the matrix of claim 1 without denaturing said protein which comprises treating said matrix with a buffer at about pH 8.6 to about 9.5. ### Claim 18 (depends on 2) A method of separating the protein from the matrix of claim 2 without denaturing said protein which comprises treating said matrix with a buffer at about pH 8.6 to about 9.5. ### Claim 19 (depends on 13) The method of claim 13 wherein there are used about 1 to about 5 volumes of buffer, at about pH 8.6 to about 9.5, per volume of pellet of the matrix. ### Claim 20 (depends on 15) The method of claim 15 wherein the buffer is an amino-2-hydroxy methyl-1, 3-propane diol (Tris) buffer. ### Claim 21 (depends on 1) A method of separating the polyhydroxy polycarboxylic acid containing material from the matrix of claim 1 to recover the same which comprises treating said matrix with an aqueous solution of sodium lauryl sulfate. ### Claim 22 (depends on 2) A method of separating the polyhydroxy polycarboxylic acid containing material from the matrix of claim 2 to recover the same which comprises treating said matrix with an aqueous solution of sodium lauryl sulfate. ### Claim 23 (depends on 17) The method of claim 17 which comprises the further step of centrifuging said matrix which has been treated with sodium lauryl sulfate solution and recovering the polyhydroxy polycarboxylic acid from the residual pellet thereof. ### Claim 24 (depends on 17) The method of claim 17 wherein there are used about 1 to about 3 volumes of sodium lauryl sulfate of a concentration of between about 0.5 and about 2% w/w per volume of matrix residual pellet, ### Claim 25 (depends on 18) The method of claim 18 which comprises the further step of washing the said recovered polyhydroxy polycarboxylic acid residue and resuspending same in phosphate buffered saline. ### Claim 26 (depends on 20) The method of claim 20 which comprises utilizing phosphate buffered saline at pH 7.1-7.5. ### Claim 27 (depends on 1) The method of claim 1 wherein the aqueous medium containing said protein further contains a nucleic acid or mixtures thereof. ### Claim 28 (depends on 2) The method of claim 2 wherein the aqueous medium containing said protein further contains a nucleic acid or mixtures thereof. ### Claim 29 (depends on 22) The method of claim 22 wherein the source of the nucleic acids or mixtures thereof is a cell lysate suspended in aqueous guanidinium thiocyanate.