Methods for use of mixed multifunctional surfaces for reducing aggregate content in protein preparations

Abstract

Methods for reduction of aggregate levels in antibody and other protein preparations comprising the steps of treatment with an organic multivalent cation (e.g., ethacridine, chlorhexidine, or polyethylenimine) and treatment with a composition having a combination of surfaces bearing electronegative chemical moieties and surfaces bearing electropositive chemical moieties.

1 . A method of reducing the aggregate content of a sample containing a desired protein, wherein the method comprises the steps of: (i) providing a first component which is a first solid substrate having an electronegative surface: (ii) providing a second component which is a second solid substrate having an electropositive surface; (iii) contacting the sample with the first and second components, where the first and second components are configured such that the sample may contact both components simultaneously, where the operating conditions substantially prevent the binding of the desired protein to the first or second components; and (iv) separating a resulting sample containing the desired protein with a reduced aggregate content from the first and second components. 
     
     
         2 . The method of  claim 1  wherein the first and second components are combined prior to contacting the sample. 
     
     
         3 . The methods of  claims 1 - 2  wherein the sample is contacted with a soluble organic multivalent cation of mixed chemical character prior to contacting the first substrate having an electronegative surface. 
     
     
         4 . The method of  claim 3  wherein the sample is incubated with the soluble organic multivalent cation of mixed chemical character for an appropriate period of time prior to contacting the first and second components. 
     
     
         5 . The methods of  claims 1 - 4  wherein the sample is incubated with the first and second components for an appropriate period of time. 
     
     
         6 . The methods of  claims 3 - 5  wherein the organic multivalent cation having mixed chemical character is removed from the sample in the step of separating the sample with a reduced aggregate content from the first and second components to the extent that the organic multivalent cation having mixed chemical character is associated with a substrate of the first or second component. 
     
     
         7 . The method of  claims 1 - 6  wherein the first substrate is particulate. 
     
     
         8 . The method of  claims 1 - 6  wherein the second substrate is particulate. 
     
     
         9 . The method of  claims 7 - 8  wherein the particulate substrate or substrates is each provided as a plurality of particles. 
     
     
         10 . The method of  claim 7 - 9  wherein the particles of the first substrate, the second substrate or both are non-porous. 
     
     
         11 . The method of  claim 7 - 9  wherein the particles of the first substrate, the second substrate or both are porous. 
     
     
         12 . The method of  claim 11  wherein the pore size of the porous particles is large enough to permit entry of a protein in a protein preparation. 
     
     
         13 . The method of  claim 11  wherein the pore size of the porous particles is too small to permit entry of a protein in a protein preparation. 
     
     
         14 . The method of  claim 11  wherein the average pore size of the porous particles is between about 10 nm and about 100 nm. 
     
     
         15 . The methods of  claims 7 - 14  wherein the particles are sandwiched between porous membranes or monoliths. 
     
     
         16 . The methods of  claims 7 - 14  wherein the particles are sandwiched between woven or amorphous fibrous filters. 
     
     
         17 . The methods of  claims 7 - 14  wherein the particles are sandwiched between crystalline frits. 
     
     
         18 . The methods of  claims 7 - 14  wherein the particles are embedded in a reticular polymer network. 
     
     
         19 . The method of  claims 3 - 18  wherein the conductivity of the sample is at a sufficiently high level to substantially avoid precipitation of the desired protein from the sample while contacting the soluble organic multivalent cation of mixed chemical character. 
     
     
         20 . The method of  claim 19  wherein the conductivity of the sample is greater than 20 mS/cm. 
     
     
         21 . The method of  claim 20  wherein the conductivity of the sample is greater than 30 mS/cm. 
     
     
         22 . The method of  claim 20  wherein the conductivity of the sample is greater than about 40 mS/cm. 
     
     
         23 . The method of  claim 20  wherein the conductivity of the sample is greater than about 100 mS/cm. 
     
     
         24 . The method of  claim 19  wherein the conductivity of the sample is at least 5%, 10%, 20%, 50%, 100% or 200% higher than the conductivity sufficient to substantially avoid precipitation of the desired protein from the sample while contacting the soluble organic multivalent cation of mixed chemical character. 
     
     
         25 . The method of  claims 1 - 24  wherein the aggregates comprise homo-aggregates of the desired protein. 
     
     
         26 . The method of  claim 25  wherein the presence of homo-aggregates of the desired protein is substantially eliminated. 
     
     
         27 . The method of  claims 1 - 26  wherein the aggregates comprise hetero-aggregates of the desired protein and a contaminant. 
     
     
         28 . The method of  claim 27  wherein the hetero-aggregates are of substantially the same hydrodynamic size as the desired protein. 
     
     
         29 . The method of  claim 27  wherein the contaminant is a nucleic acid, nucleotide, endotoxin, metal ion, protein, lipid, or cell culture media component. 
     
     
         30 . The method of  claims 27 - 29  wherein the presence of hetero-aggregates of the desired protein and a contaminant is substantially eliminated. 
     
     
         31 . The method of  claims 1 - 30  wherein the desired protein is an antibody or antibody fragment. 
     
     
         32 . The method of  claims 1 - 30  wherein the desired protein is a recombinant protein. 
     
     
         33 . The method of  claims 1 - 30  wherein the desired protein is a clotting factor. 
     
     
         34 . The method of  claim 33  wherein the clotting factor is Factor VIII. 
     
     
         35 . The method of  claim 32  wherein the desired protein is a growth hormone. 
     
     
         36 . The method of  claim 31  wherein the sample is a cell culture harvest, a cell culture supernatant, an antibody-containing solution derived from a cell culture, or an antibody-containing solution from a previous stage of protein purification. 
     
     
         37 . The method of  claim 31  wherein the cultured cells are mammalian cells, a bacterial cells, or a yeast cells. 
     
     
         38 . The method of  claim 35  wherein the sample is an antibody-containing solution from a previous stage of protein purification. 
     
     
         39 . The method of  claim 38  wherein the sample is an eluate from a chromatography column. 
     
     
         40 . The method of  claims 1 - 39 , wherein the sample may be unpurified, at an intermediate level of purity, or highly purified. 
     
     
         41 . The method of  claims 1 - 40 , wherein the sample is contacted with the first and second components by flowing the preparation through the first and second components. 
     
     
         42 . The method of  claims 1 - 41  wherein the electronegativity of the surface of the first component is conferred through one or more kinds of complex chemical moieties that embody more than one chemical functionality, or through a combination of simple and complex chemical groups mixed on the surface, or a combination of surfaces of differing chemical composition. 
     
     
         43 . The method of  claims 1 - 42 , wherein the electronegativity of the surface of the first component is conferred in part by a moiety from the group consisting of iminodiacetic acid, ethylene glycol(aminoethylether)diacetic acid, nitriloacetic acid, aspartic acid, glutamic acid, a carboxylic acid, sulfurous acid, sulfonate, and phosphoric acid. 
     
     
         44 . The method of  claims 1 - 41  wherein the electropositivity of the surface of the second component is conferred through one or more kinds of complex chemical groups that embody more than one chemical functionality, or through a combination of simple and complex chemical groups mixed on the surface, or a combination of surfaces of differing chemical composition. 
     
     
         45 . The method of  claims 1 - 44 , wherein the electropositivity of the surface of the second component is conferred in part by a moiety from the group consisting of tris(2-aminoethyl)amine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, polypropylenimine tetramine, PAMAM dendrimer (ethylenediamine core), deferoxamine, a primary amino group, a secondary amino group, a tertiary amino group, and a quaternary amino group. 
     
     
         46 . The method of  claims 1 - 45  wherein the electronegativity of the surface of the first component is conferred in part by iminodiacetic acid and the electropositivity of the surface of the second component is conferred in part by tris(2-aminoethyl)amine. 
     
     
         47 . The method of  claims 1 - 46  wherein the first component has a surface-bound chemical moiety possessing metal affinity functionality. 
     
     
         48 . The method of  claims 1 - 46  wherein the electropositive surface of the second component includes a surface-bound chemical moiety possessing metal affinity functionality. 
     
     
         49 . The method of  claims 47 - 48  wherein at least one of the substrates has one or more chemical moieties in addition to the surface-bound chemical moiety possessing metal affinity functionality wherein such additional chemical moieties enhance the capacity of one or more of the components to participate in hydrogen bonding, hydrophobic interactions, or pi-pi binding with a protein of the protein preparation. 
     
     
         50 . The method of  claims 47 - 48  wherein the surface-bound chemical moiety possessing metal affinity functionality is a multidentate metal chelating moiety. 
     
     
         51 . The method of  claims 1 - 50  wherein the electropositive surface of the first component includes a surface-bound chemical moiety possessing metal affinity functionality, the electronegative surface of the second component includes a surface-bound chemical moiety possessing metal affinity functionality, and another surface includes a surface-bound chemical moiety possessing an elevated hydrophobic functionality. 
     
     
         52 . The method of  claims 3 - 50  wherein the organic multivalent cation of mixed chemical character is selected from the group consisting of ethacridine, 9-aminoacridine (aminacrine), 3,6 acridinediamine (proflavin), acrisorcin, acrizane (phenacridane), acridine orange, quinacrine, acricide, acridone, acridine-9-carboxylic acid, acranil (1-[(6-chloro-2-methoxy-9-acridinyl)amino]-3-(diethylamino)-2-propanol dihydrochloride), phenosafranin, phenoxazine, phenothiazine, acriflavine (3,6-diamino-10-methylacridinium, chloride and 3,6-acridineidiamine), polyethyleneimine, chlorhexidine, and poly-amino acids. 
     
     
         53 . The method of  claim 51  wherein the organic multivalent cation of mixed chemical character is ethacridine, polyethylenimine or chlorhexidine or a salt thereof. 
     
     
         54 . The method of  claim 52  wherein the organic multivalent cation of mixed chemical character is ethacridine or a salt thereof. 
     
     
         55 . The method of  claims 51 - 53  wherein the organic multivalent cation of mixed chemical character is present in an amount between approximately 0.01% and approximately 0.05%. 
     
     
         56 . The method of  claims 51 - 53  wherein the organic multivalent cation of mixed chemical character is present in an amount less than approximately 0.01%. 
     
     
         57 . The method of  claims 51 - 53  wherein the organic multivalent cation of mixed chemical character is present in an amount less than approximately 0.005%. 
     
     
         58 . The method of  claims 51 - 53  wherein the organic multivalent cation of mixed chemical character is present in an amount less than approximately 0.001%. 
     
     
         59 . The method of  claims 51 - 53  wherein the organic multivalent cation of mixed chemical character is present in an amount between approximately 0.020 and approximately 0.025%. 
     
     
         60 . The method of  claims 3 - 50  wherein the sample is treated with more than one organic multivalent cation from the group consisting of polyethyleneimine, polyallyamine, ethacridine, and chlorhexidine and salts thereof prior to the step of contacting the sample with the first and second components. 
     
     
         61 . The method of  claim 60  wherein the organic multivalent cations used to treat the sample prior to the step of contacting the sample with the first and second components are provided in a concentration of less than 1%. 
     
     
         62 . The method of  claim 60  wherein the organic multivalent cations used to treat the sample prior to the step of contacting the sample with the first and second components are provided in a concentration between approximately 0.01% and approximately 0.05%. 
     
     
         63 . The method of  claim 60  wherein the organic multivalent cations used to treat the sample prior to the step of contacting the sample with the first and second components are provided in a concentration less than approximately 0.01%. 
     
     
         64 . The method of  claim 60  wherein the organic multivalent cations used to treat the sample prior to the step of contacting the sample with the first and second components are provided in a concentration less than approximately 0.005%. 
     
     
         65 . The method of  claim 60  wherein the organic multivalent cations used to treat the sample prior to the step of contacting the sample with the first and second components are provided in a concentration less than approximately 0.001%. 
     
     
         66 . The method of  claim 60  wherein the organic multivalent cations used to treat the sample prior to the step of contacting the sample with the first and second components are provided in a concentration between approximately 0.020 and approximately 0.025%. 
     
     
         67 . The method of  claims 1 - 66 , wherein the sample is additionally contacted with a soluble organic modulator selected from the group consisting of nonionic organic polymers, organic solvents, surfactants, and ureides, prior to the step of contacting the sample with the first and second components. 
     
     
         68 . The method of  claims 1 - 67  wherein the sample is additionally contacted with an antiviral agent, prior to the step of contacting the sample with the first and second components. 
     
     
         69 . The method of  claim 68  wherein the antiviral agent is a non-multivalent organic cation. 
     
     
         70 . The method of  claim 68  wherein the antiviral agent is selected from the group consisting of benzalkonium chloride, methylene blue and tri (n-butyl) phosphate. 
     
     
         71 . The method of  claim 65 - 70  wherein the antiviral agent is present in an amount less than approximately 1% (w/v). 
     
     
         72 . The method of  claim 68  wherein the antiviral agent is present in an amount less than approximately 0.1% (w/v). 
     
     
         73 . The method of  claim 72  wherein the antiviral agent is present in an amount less than approximately 0.01% (w/v). 
     
     
         74 . The method of  claim 73  wherein the antiviral agent is present in an amount less than approximately 0.001% (w/v). 
     
     
         75 . The method of  claims 1 - 74  comprising the additional steps of, prior to the step of contacting the sample with the first and second components, contacting the sample with a ureide in an amount sufficient for the ureide to be supersaturated in the protein preparation, and separating the supernatant containing the desired protein from the solid or undissolved portions of the sample. 
     
     
         76 . The method of  claim 75  wherein the step of contacting the sample with the ureide occurs prior to the step of contacting the sample with the soluble organic multivalent cation of mixed chemical character. 
     
     
         77 . The method of  claim 75  wherein the step of contacting the sample with the ureide occurs substantially simultaneously with the step of contacting the sample with the soluble organic multivalent cation of mixed chemical character. 
     
     
         78 . The method of  claim 75  wherein the step of contacting the sample with the ureide occurs after the step of contacting the sample with the soluble organic multivalent cation of mixed chemical character. 
     
     
         79 . The method of  claims 75 - 78  wherein the ureide is selected from the group consisting of urea, uric acid, hydantoin, allantoin, alcloxa, aldioxa, hemocane, ureidohydantoin, 5-ureidohydantoin, glyoxylureide, glyoxylic acid diureide, 2,5-dioxo-4-imidazolidinyl urea, and purines. 
     
     
         80 . The method of  claim 79  wherein the ureide is allantoin. 
     
     
         81 . The method of  claim 79  wherein the ureide is uric acid. 
     
     
         82 . The method of  claim 80  wherein the allantoin is present in an amount greater than 0.5% (w/v). 
     
     
         83 . The method of claim of  claim 82  wherein the allantoin is present in an amount greater than approximately 1% (w/v). 
     
     
         84 . The method of  claim 81  wherein the uric acid is present in an amount greater than 0.0025% (w/v). 
     
     
         85 . The method of  claim 83  wherein the allantoin is present in an amount greater than approximately 2% (w/v). 
     
     
         86 . The method of  claim 83  wherein the allantoin is present in an amount greater than approximately 5% (w/v). 
     
     
         87 . The method of  claim 83  wherein the allantoin is present in an amount greater than approximately 10% (w/v). 
     
     
         88 . The method of  claim 67  wherein the step of contacting the sample with the organic modulator occurs prior to the step of contacting the sample with the soluble organic multivalent cation of mixed chemical character. 
     
     
         89 . The method of  claim 67  wherein the step of contacting the sample with the organic modulator occurs substantially simultaneously with the step of contacting the sample with the soluble organic multivalent cation of mixed chemical character. 
     
     
         90 . The method of  claim 67  wherein the step of contacting the sample with the organic modulator occurs after the step of contacting the sample with the soluble organic multivalent cation of mixed chemical character. 
     
     
         91 . The method of  claims 88 - 90  wherein the organic modulator is a nonionic organic polymer selected from the group consisting of glycerol, polyethylene glycol, polypropylene glycol and polybutylene glycol. 
     
     
         92 . The method of  claim 91  wherein the nonionic organic polymer has an average molecular weight of approximately 500 D or less. 
     
     
         93 . The method of  claims 88 - 90  wherein the organic modulator is an organic solvent selected from the group consisting of ethylene glycol, propylene glycol, butylene glycol, dimethylsulfoxide, ethanol, isopropanol, and phenoxyethanol. 
     
     
         94 . The method of  claim 88 - 93  wherein the organic modulator is provided at a concentration of approximately 1% (w/v) or greater 
     
     
         95 . The method of  claims 88 - 90  wherein the organic modulator is a surfactant selected from the group consisting of Tween, triton, CHAPS, CHAPSO and octyl glucoside. 
     
     
         96 . The method of  claim 95  wherein the surfactant is provided at a concentration of approximately 1% (w/v) or less. 
     
     
         97 . The method of  claim 95  wherein the surfactant is provided at a concentration of approximately 0.1% (w/v) or less. 
     
     
         98 . The method of  claim 91 - 93  wherein the organic modulator is a ureide provided in a sub saturating amount. 
     
     
         99 . The method of  claim 98  wherein the ureide is selected from the group consisting of urea, hydantoin, and allantoin. 
     
     
         100 . A kit providing for the convenient practice of a method of any of  claims 1 - 99 .