Nucleotide Sugar Purification Using Membranes

Abstract

The invention provides methods of removing contaminants from a mixture of a desired product and contaminants by pH adjustments and molecular weight cut-offs. The contaminants include phosphate groups, magnesium sulfate, sodium pyruvate and tetrasodium pyrophosphate groups. The desired product includes nucleotide sugars, glycolipids, LnNT, sialyl lactose, and salts.

1 . A method of removing essentially all of a contaminant from a mixture comprising said contaminant and a desired product comprising a moiety with a structure selected from: 
       
         
           
           
               
               
           
         
       
       in which
 R 1  is selected from H, CH 2 OR 7 , COOR 7  or OR 7    
 wherein 
 R 7  is a member selected from H, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl; 
 R 2  is selected from H, OH, NH and a moiety that includes a nucleotide; 
 R 3 , R 4 , R 5 , R 6  and R 6′  are independently selected from H, substituted or unsubstituted alkyl, OR 9 , and NHC(O)R 10 ; 
 wherein
 R 9  and R 10  are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and sialic acid 
 
 the index d is 0 or 1; 
 with the proviso that at least one of R 3 , R 4 , R 5 , R 6 , and R 6′  includes the linker or linker-modifying group, said method comprising: 
 contacting said mixture with a first membrane for a length of time sufficient to allow essentially all of said contaminant to separate from said desired product 
 wherein
 said mixture has a pH such that said first membrane and said desired product have a net charge of the same sign and said contaminant has a net charge which is a member selected from neutral and a sign which is opposite of the sign of the net charge for the first membrane; and 
 said first membrane has a molecular weight cut-off that is greater than the molecular weight of said desired product, 
 
 
       thereby removing essentially all of the contaminant from the mixture. 
     
     
         2 . The method according to  claim 1 , wherein said desired product and said first membrane each have a net negative charge and said contaminant has a nct charge which is a member selected from neutral and a net positive charge. 
     
     
         3 . The method according to  claim 1 , wherein the contaminant is a member selected from phosphate, pyrophosphate, nucleotide monophosphate, nucleotide diphosphate, nucleotide triphosphate, sodium phosphate, manganese chloride, sodium pyruvate, GlcNAc, magnesium sulfate, tetrasodium pyrophosphate, lactose, benzoic acid, LNT-2, LNnT, sialic acid, cytidine, CMP, benzyl alcohol, CyLac, cylexin, cytilene and sodium chloride. 
     
     
         4 . The method according to  claim 1 , wherein said first membrane is contacted with at least about 500 mg of the desired product. 
     
     
         5 . The method according to  claim 1 , wherein the desired product is a member selected from a nucleotide sugar, glycolipid, sialylated ganglioside, LNnT, sialyl lactose and salts thereof. 
     
     
         6 . The method according to  claim 5 , wherein said nucleotide sugar is a member selected from CMP-Nan, GDP-Man, GDP-Fuc, UDP-Glc, UDP-Gal, UDP-GlcNAc, UDP-GalNAc, UDP-GlcA, UDP-IdoA, UDP-Xyl. 
     
     
         7 . The method according to  claim 5 , wherein the desired product includes a sugar moiety comprising a moiety having a structure which is a member selected from: 
       
         
           
           
               
               
           
         
         wherein
 L a  is a linker selected from a bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; 
 X 5 , R 16  and R 17  are independently selected from polymeric moieties and non-reactive groups; 
 X 2  and X 4  are independently selected linkage fragments joining polymeric moieties R 16  and R 17  to C; 
 m and n are integers independently selected from 0 to 5000; 
 A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , A 10  and A 11  are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —NA 12 A 13 , —OA 12  and —SiA 12 A 13    
 wherein
 A 12  and A 13  are members independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. 
 
 
       
     
     
         8 . The method according to  claim 5 , wherein, prior to said contacting, said method further comprises forming the mixture by contacting a nucleotide with a sugar and a nucleotide sugar synthetase capable of ligating said nucleotide and said sugar. 
     
     
         9 . The method according to  claim 5 , wherein, prior to said contacting, said method further comprises forming the mixture by subjecting a cell system to conditions whereby said cell system produces a nucleotide sugar. 
     
     
         10 . The method according to  claim 1 , wherein the mixture is not further purified prior to said contacting. 
     
     
         11 . The method according to  claim 1 , wherein the desired product is not further purified after said contacting. 
     
     
         12 . The method according to  claim 1 , wherein the desired product is further purified after said contacting. 
     
     
         13 . The method according to  claim 1 , wherein, prior to said contacting, said method further comprises contacting said mixture with a second membrane for a length of time sufficient to allow said desired product to pass through said second membrane and to allow molecules with a molecular weight greater than about 500 kDa to be retained in said second membrane. 
     
     
         14 . The method according to  claim 1 , wherein, prior to said contacting, said method further comprises contacting said mixture with a third membrane for a length of time sufficient to allow said desired product to pass through said third membrane and to allow molecules with a molecular weight greater than about 10 kDa to be retained in said third membrane. 
     
     
         15 . A method of purifying a desired product from a mixture comprising a phosphorus-containing contaminant and said desired product, said method comprising:
 contacting said mixture with a first membrane for a length of time sufficient to allow essentially all of said phosphorus-containing contaminant to pass through said first membrane   wherein
 said mixture has a pH such that said first membrane and said desired product have a net negative charge and said contaminant has a net charge which is a member selected from a neutral and a positive charge; and 
 said first membrane has a molecular weight cut-off that is greater than the molecular weight of said desired product, thereby purifying the desired product from the mixture. 
   
     
     
         16 . The method according to  claim 15 , wherein said first membrane is contacted with at least about 500 mg of the desired product. 
     
     
         17 . The method according to  claim 15 , wherein the desired product is a member selected from a nucleotide sugar, glycolipid, sialylated ganglioside, LNnT, sialyl lactose and salts thereof. 
     
     
         18 . The method according to  claim 17 , wherein said desired product is a nucleotide sugar and said nucleotide sugar is a member selected from CMP-Nan, GDP-Man, GDP-Fuc, UDP-Glc, UDP-Gal, UDP-GlcNAc, UDP-GalNAc, UDP-GlcA, UDP-IdoA and UDP-Xyl. 
     
     
         19 . The method according to  claim 17 , wherein said desired product includes a sugar moiety comprising a moiety having a structure which is a member selected from: 
       
         
           
           
               
               
           
         
         wherein
 L a  is a linker selected from a bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; 
 X 5 , R 16  and R 17  are independently selected from polymeric moieties and non-reactive groups; 
 X 2  and X 4  are independently selected linkage fragments joining polymeric moieties R 16  and R 17  to C; 
 m and n are integers independently selected from 0 to 5000; 
 A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , A 10  and A 11  are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —NA 12 A 13 , —OA 12  and —SiA 12 A 13    
 wherein
 A 12  and A 13  are members independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. 
 
 
       
     
     
         20 . The method according to  claim 15 , wherein said desired product comprises a moiety with a structure selected from: 
       
         
           
           
               
               
           
         
         in which
 R 1  is selected from H, CH 2 OR 7 , COOR 7  or OR 7    
 wherein 
 R 7  is a member selected from H, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl; 
 
         R 2  is selected from H, OH, NH and a moiety that includes a nucleotide; 
         R 3 , R 4 , R 5 , R 6  and R 6′  are independently selected from H, substituted or unsubstituted alkyl, OR 9 , and NHC(O)R 10    
         wherein
 R 9  and R 10  are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and sialic acid 
 
         the index d is 0 or 1;
 with the proviso that at least one of R 3 , R 4 , R 5 , R 6 , and R 6′  includes the linker or linker-modifying group. 
 
       
     
     
         21 . A composition of matter comprising a nucleotide sugar, produced by a process comprising:
 contacting a mixture with a first membrane for a length of time sufficient to allow essentially all of a contaminant to pass through said first membrane   wherein
 said mixture comprises said contaminant and said nucleotide sugar; 
 said mixture has a pH such that said first membrane and said desired product have a net charge of the same sign and said contaminant has a net charge which is a member selected from a neutral and a positive charge; 
 said first membrane has a molecular weight cut-off that is greater than the molecular weight of said nucleotide sugar; and 
 said composition of matter is essentially free of said contaminant. 
   
     
     
         22 . The composition according to  claim 21 , wherein said nucleotide sugar and said first membrane each have a net negative charge. 
     
     
         23 . The composition according to  claim 21 , wherein the contaminant is a member selected from phosphate, pyrophosphate, nucleotide monophosphate, nucleotide diphosphate, nucleotide triphosphate, sodium phosphate, manganese chloride, sodium pyruvate, GlcNAc, magnesium sulfate, tetrasodium pyrophosphate, lactose, benzoic acid, LNT-2, LNnT, sialic acid, cytidine, CMP, benzyl alcohol, CyLac, cylexin, cytilene and sodium chloride. 
     
     
         24 . The composition of matter according to  claim 21 , wherein said nucleotide sugar is a member selected from CMP-Nan, GDP-Man, GDP-Fuc, UDP-Glc, UDP-Gal, UDP-GlcNAc, UDP-GalNAc, UDP-GlcA, UDP-IdoA and UDP-Xyl. 
     
     
         25 . The composition according to  claim 21 , wherein the desired product includes a sugar moiety comprising a moiety having a structure which is a member selected from: 
       
         
           
           
               
               
           
         
         wherein
 L a  is a linker selected from a bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; 
 X 5 , R 16  and R 17  are independently selected from polymeric moieties and non-reactive groups; 
 X 2  and X 4  are independently selected linkage fragments joining polymeric moieties R 16  and R 17  to C; 
 m and n are integers independently selected from 0 to 5000; 
 A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , A 10  and A 11  are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —NA 12 A 13 , —OA 2  and —SiA 12 A 13    
 wherein
 A 12  and A 13  are members independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. 
 
 
       
     
     
         26 . The composition of matter according to  claim 21 , wherein said mixture is formed by contacting a nucleotide with a sugar and a nucleotide sugar synthetase capable of ligating said nucleotide to said sugar. 
     
     
         27 . The composition of matter according to  claim 21 , wherein said mixture is formed by subjecting a cell system to conditions whereby said cell system produces a nucleotide sugar. 
     
     
         28 . A method of removing essentially all of a contaminant from a mixture comprising said contaminant and a desired product, said method comprising:
 contacting said mixture with a first membrane for a length of time sufficient to allow essentially all of said contaminant to pass through said first membrane   wherein
 said mixture has a pH such that said first membrane and said desired product have a net charge of the same sign and said contaminant is at its isoelectric point; 
 said first membrane has a molecular weight cut-off that is greater than the molecular weight of said desired product; 
   
       thereby removing essentially all of the contaminant from the mixture. 
     
     
         29 . A method of purifying a nucleotide sugar from reactive components used to prepare said nucleotide sugar, said method comprising:
 a) contacting a reactive solution comprising said nucleotide sugar with a nanofiltration membrane, thereby removing a nucleotide monophosphate and a sugar from said reactive solution while retaining said nucleotide sugar in said reactive solution,   
       thereby forming a desalted nucleotide sugar solution. 
     
     
         30 . The method of  claim 28 , further comprising:
 b) passing said desalted nucleotide sugar solution over a charged media depth filter.