Hydrophobic interaction chromatography for purification of oligonucleotides

Abstract

The present invention is directed to a method of purifying oligonucleotides using hydrophobic interaction chromatography.

1 . A method for separating a target oligonucleotide from a mixture containing the target oligonucleotide and a product-related impurity, the method comprising the steps of:
 a) adding salt to the mixture;   b) contacting the diluted mixture with a hydrophobic adsorbent at a dynamic loading capacity of about 32 to about 78% of the capacity of the hydrophobic adsorbent;   c) washing the hydrophobic adsorbent with an aqueous salt solution;   d) eluting the target oligonucleotide with a eluting solution; and   e) collecting the eluent comprising the target oligonucleotide;   wherein the product-related impurity includes at least one n−1 impurity, thereby separating the target oligonucleotide from the product-related impurity.   
     
     
         2 . A method for separating a target oligonucleotide from a mixture containing the target thiolated oligonucleotide and a product-related impurity, the method comprising the steps of:
 a) adding salt to the mixture;   b) contacting the diluted mixture with a hydrophobic adsorbent at a dynamic loading capacity of about 40 to about 100% of the capacity of the hydrophobic adsorbent;   c) washing the hydrophobic adsorbent with an aqueous salt solution;   d) eluting the target oligonucleotide with a eluting solution; and   e) collecting the eluent comprising the target oligonucleotide;   
       wherein the product-related impurity includes at least one P═O impurity, thereby separating the target oligonucleotide from the product-related impurity. 
     
     
         3 . The method of  claim 1 , wherein the salt is added to the mixture as aqueous salt solution or the salt is dissolved directly into the mixture, and wherein the flow rate of the wash step is slower than the loading flow rate. 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the eluting solution is selected from water, an aqueous salt solution, ethylene glycol, or propylene glycol or mixtures thereof. 
     
     
         6 . The method of  claim 1 , wherein the elution collection is delayed such that is does not include the first 1-25%, 10-25% or 5-10% of the product elution peak or the last 1-25%, 5-10% or 10-25% of the product elution peak. 
     
     
         7 - 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein the hydrophobic adsorbent comprises phenyl, butyl or hexyl, and the hydrophobic adsorbent is packed at a bed height of at least 15 cm. 
     
     
         13 . The method of  claim 1 , wherein the salt includes any cation of NH 4   + , K +  or Na +  and any anion comprised of F − , [SO 4 ] −2 , [HPO 4 ] −2 , acetate or Cl −  or combinations thereof. 
     
     
         14 . The method of  claim 1 , wherein the salt is ammonium sulfate. 
     
     
         15 . (canceled) 
     
     
         16 . The method of  claim 1 , wherein the target oligonucleotide comprises 15 to 25 nucleotides. 
     
     
         17 . The method of  claim 1 , wherein the target oligonucleotide comprises nucleobases independently selected from the group consisting of adenine, guanine, thymine (5-methyl uracil), cytosine, hypoxanthine, xanthine, 7-methyl guanine, 5,6-dihydrouracil, 5-methylcytosine, 7-deaza purine and 5-hydroxymethylcytosine. 
     
     
         18 . (canceled) 
     
     
         19 . The method of  claim 1 , wherein the target oligonucleotide comprises sugar that is optionally substituted; two non-geminal ring atoms are bridged to form a bicyclic nucleic acid (BNA); or a ring oxygen atom of the sugar is replaced with S, N(R), or C(R 1 )(R) 2 , wherein R is H or C1-C12 alkyl and combinations of these. 
     
     
         20 . The method of  claim 19 , wherein the sugar is substituted at the 2′ position with O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , OCH 2 C(═O)N(H)CH 3 , and O(CH 2 ) n ON[CH 2 ) n CH 3 ] 2 , where n and m are independently from 1 to about 10. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 1 , wherein the target oligonucleotide comprises RNA only, DNA only, or a combination of RNA and DNA. 
     
     
         23 . (canceled) 
     
     
         24 . The method of  claim 1 , wherein the target oligonucleotide includes phosphodiesters (P═O), phosphorothioates (P═S), or a combination thereof. 
     
     
         25 . The method of  claim 1 , wherein the sequence of the target oligonucleotide is (SEQ ID NO: 1) or (SEQ ID NO: 2). 
     
     
         26 . (canceled) 
     
     
         27 . The method of  claim 1 , wherein the target oligonucleotide comprises 4,4′-dimethoxytrityl (DMT). 
     
     
         28 . The method of  claim 1 , wherein the target oligonucleotide does not comprise 4,4′-dimethoxytrityl (DMT). 
     
     
         29 . The method of  claim 1 , wherein the product related impurity further includes at least one P═O impurity. 
     
     
         30 . The method of  claim 2 , wherein the product related impurity further includes at least one n−1 impurity. 
     
     
         31 . The method of  claim 1 , wherein the product related impurity further includes at least one ABasic impurity or at least one CNEt impurity, or at least one N+1 impurity. 
     
     
         32 - 33 . (canceled)