Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration

Abstract

The invention provides methods for purifying carbohydrates, including oligosaccharides, nucleotide sugars, and related compounds, by use of ultrafiltration, nanofiltration and/or reverse osmosis. The carbohydrates are purified away from undesired contaminants such as compounds present in reaction mixtures following enzymatic synthesis or degradation of oligosaccharides.

What is claimed is:  
     
         1 . A method of purifying a carbohydrate compound from a feed solution containing a contaminant, the method comprising contacting the feed solution with a nanofiltration or reverse osmosis membrane under conditions such that the membrane retains the carbohydrate compound while a majority of the contaminant passes through the membrane, wherein the carbohydrate compound is selected from the group consisting of: 
 sialyl lactosides;    sialic acid;    LnNT;    LNT-2;    NeuAcα(2→3)Galβ(1→4)(Fucβ1→3)Glc(R 1 )β1-OR 2 , wherein R 1  is OH or NAc; R 2  is a hydrogen, an alkoxy, a saccharide, an oligosaccharide or an aglycon group having at least one carbon atom; and    Galα(1→3)Galβ(1→4)Glc(R 1 )β-O—R 3 , wherein wherein R 1  is OH or NAc; R 3  is —(CH 2 ) n —COX, with X═OH, OR 4 , —NHNH 2 , R 4  being a hydrogen, a saccharide, an oligosaccharide or an aglycon group having at least one carbon atom, and n=an integer from 2 to 18.    
     
     
         2 . The method of  claim 1 , wherein the carbohydrate compound is selected from the group consisting of a sialyl lactoside, LnNT, LNT-2, and Galα1,3Galβ1,4GlcNAcβ1-O—(CH 2 ) 5 —COOH.  
     
     
         3 . The method of  claim 2 , wherein the carbohydrate compound is selected from the group consisting of a sialyl(α2,3)-lactoside and a sialyl(α2,6)-lactoside.  
     
     
         4 . The method according to  claim 2 , wherein the membrane comprises polyamide or polybenzamide.  
     
     
         5 . The method according to  claim 4 , wherein the membrane is a membrane selected from the group consisting of a YK, a GE (G-5), and an MX07.  
     
     
         6 . The method of  claim 1 , wherein the carbohydrate compound is NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,4Galβ1-OEt.  
     
     
         7 . The method according to  claim 6 , wherein the membrane is a membrane selected from the group consisting of a YK, a GE (G-5), a GH (G-10), an HL, and an MX07.  
     
     
         8 . The method of  claim 1 , wherein the feed solution is an enzymatic reaction mixture used for synthesis of the carbohydrate compound.  
     
     
         9 . The method of  claim 1 , wherein the ability of the membrane to retain the carbohydrate compound while passing the contaminant is optimized by adjusting the pH of the feed solution.  
     
     
         10 . A method of purifying a carbohydrate compound from a feed solution containing a contaminant, the method comprising contacting the feed solution with a nanofiltration or reverse osmosis membrane under conditions such that the membrane retains the carbohydrate compound while a majority of the contaminant passes through the membrane, wherein the carbohydrate compound has the formula  
       
         
           
           
               
               
           
         
       
       wherein Z is hydrogen, C 1 -C 6  acyl or  
       
         
           
           
               
               
           
         
         Y is selected from the group consisting of C(O), SO 2 , HNC(O), OC(O) and SC(O);  
         R 1  is selected from the group consisting of an aryl, a substituted aryl and a phenyl C 1 -C 3  alkylene group, wherein said aryl substitutent is selected from the group consisting of a halo, trifuloromethyl, nitro, C 1 -C 18 , alkyl, C 1 -C 18  alkoxy, amino, mono-C 1 -C 18  alkylamino, di-C 1 -C 18  alkylamino, benzylamino, C 1 -C 18  alkylbenzylamino, C 1 -C 18  thioaklyl and C 1 -C 18  alkyl carboxamido groups, or  
         R 1 Y is allyloxycarbonyl or chloroacetyl;  
         R 2  is selected from the group consisting of monosaccharide (including β1,3Gal-OR, where R═H, alkyl, aryl or acyl), disaccharide, hydrogen, C 1 -C 18  straight chain, branched chain or cyclic hydrocarbyl, C 1 -C 6  alkyl, 3-(3,4,5-trimethoxyphenyl)propyl, C 1 -C 5  alkylene ω-carboxylate, ω-trisubstituted silyl C 2 -C 4  alkylene wherein said ω-trisubstituted silyl is a silyl group having three substituents independently selected from the group consisting of C 1 -C 4  alkyl, phenyl,  
         or OR 2  together form a C 1 -C 18  straight chain, branched chain or cyclic hydrocarbyl carbamate;  
         R 3  is hydrogen or C 1 -C 6  acyl;  
         R 4  is hydrogen, C 1 -C 6  alkyl or benzyl;  
         R 5  is selected from the group consisting of hydrogen, benzyl, methoxybenzyl, dimethoxybenzyl and C 1 -C 6  acyl;  
         R 7  is methyl or hydroxymethyl; and  
         X is selected from the group consisting of C 1 -C 6  acyloxy, C 2 -C 6  hydroxylacyloxy, hydroxy, halo and azido.  
       
     
     
         11 . The method of  claim 10 , wherein the carbohydrate compound has a formula NeuAcα(2→3)Galβ(1→4)GlcN(R 1 )β-OR 2  or NeuAcα(2→3)Galβ(1→4)GlcN(R 1 )β(1→3)Galβ-OR 2 , wherein R 1  is alkyl or acyl from 1-18 carbons, 5,6,7,8-tetrahydro-2-naphthamido; benzamido; 2-naphthamido; 4-aminobenzamido; or 4-nitrobenzamido, and R 2  is a hydrogen, a saccharide, an oligosaccharide or an aglycon group having at least one carbon atom.  
     
     
         12 . The method of  claim 10 , wherein the carbohydrate compound has a formula NeuAcα(2→3)Galβ(1→4)(Fucα1→3)GlcN(R 1 )β-OR 2  or NeuAcα(2→3)Galβ(1→4) (Fucα1→3)GlcN(R 1 )β(1→3)Galβ-OR 2 , wherein R 1  is alkyl or acyl from 1-18 carbons, 5,6,7,8-tetrahydro-2-naphthamido; benzamido; 2-naphthamido; 4-aminobenzamido; or 4-nitrobenzamido, and R 2  is a hydrogen, a saccharide, an oligosaccharide or an aglycon group having at least one carbon atom.  
     
     
         13 . A method of purifying a carbohydrate compound from a feed solution comprising an enzymatic reaction mixture used to synthesize the carbohydrate compound, the method comprising the steps of: 
 removing any proteins present in the feed solution by contacting the feed solution with an ultrafiltration membrane so that proteins are retained the membrane while the carbohydrate compound passes through the membrane as a permeate; and    contacting the permeate from the ultrafiltration step with a nanofiltration or reverse osmosis membrane under conditions such that the nanofiltration or reverse osmosis membrane retains the carbohydrate compound while a majority of an undesired contaminant passes through the membrane.    
     
     
         14 . A method of purifying a carbohydrate compound from a feed solution containing a contaminant, the method comprising contacting the feed solution with a nanofiltration or reverse osmosis membrane under conditions such that the membrane retains the carbohydrate compound while a majority of the contaminant passes through the membrane, wherein the carbohydrate compound is a nucleotide or a nucleotide sugar.  
     
     
         15 . The method of  claim 14 , wherein the nucleotide sugar is selected from the group consisting of GDP-fucose, GDP-mannose, CMP-NeuAc, UDP-glucose, UDP-galactose, and UDP-N-acetylgalactosamine.  
     
     
         16 . The method of  claim 14 , wherein the carbohydrate compound is a nucleotide or nucleoside.  
     
     
         17 . A method for removal of a contaminant from a feed solution comprising a carbohydrate of interest, the method comprising contacting the feed solution with a first side of a nanofiltration or reverse osmosis membrane having rejection coefficients for the carbohydrate and the contaminant such as to retain the carbohydrate while allowing the contaminant to pass through the membrane, wherein the feed solution comprises an enzymatic reaction mixture used to synthesize the carbohydrate.  
     
     
         18 . The method according to  claim 17 , wherein the enzymatic reaction comprises enzymatic degradation of an oligosaccharide or polysaccharide.  
     
     
         19 . The method according to  claim 17 , wherein the membrane separates the solution into a retentate portion and a permeate portion, the retentate portion comprising a lower concentration of the contaminant relative to the contaminant concentration in the feed solution.  
     
     
         20 . The method according to  claim 17 , wherein the pH of the feed solution is selected so as to adjust rejection coefficients of the membrane for the carbohydrate compound and the contaminant to obtain increased removal of the contaminant from the feed solution and/or increased retention of the carbohydrate compound.  
     
     
         21 . The method according to  claim 20 , wherein the contaminant is an anion and the pH of the feed solution is between about 1 and about 7.  
     
     
         22 . The method according to  claim 20 , wherein the contaminant is a cation and the pH of the feed solution is between about 7 and 14.  
     
     
         23 . The method according to  claim 20 , wherein the pH of the feed solution is selected so as to make the net surface charge of the nanofiltration membrane positive.  
     
     
         24 . The method according to  claim 20 , wherein the pH of the feed solution is selected so as to make the net surface charge of the nanofiltration membrane negative.  
     
     
         25 . The method according to  claim 20 , wherein the pH of the feed solution is selected so as to make the net surface charge of the nanofiltration membrane neutral.  
     
     
         26 . The method according to  claim 17 , wherein the carbohydrate is an oligosaccharide or polysaccharide.  
     
     
         27 . The method according to  claim 17 , wherein the carbohydrate is sialylated.  
     
     
         28 . The method according to  claim 17 , wherein the contaminant comprises one or more compounds selected from the group consisting of nucleotide sugars, unreacted sugars, inorganic ions, pyruvate, phosphate, phosphoenolpyruvate, nucleotides, nucleosides, and proteins.  
     
     
         29 . The method according to  claim 17 , wherein proteins are removed from the mixture prior to nanofiltration.  
     
     
         30 . The method according to  claim 17 , which further comprises an ion exchange purification step.