Method for anion-exchange adsorption and anion-exchangers

Abstract

A method for the removal of a substance carrying a negative charge and being present in an aqueous liquid (I). The method comprises the steps of: (i) contacting the liquid with a matrix carrying a plurality of ligands comprising a positively charged structure and a hydrophobic structure, and (ii) desorbing the substance. The characterizing feature is that (I) each of said ligands together with a spacer has the formula: --SP---[Ar—R 1 —N + (R 2 R 3 R 4 )]  where (A) [Ar—R 1 —N + (R 2 R 3 R 4 )] represents a ligand a) Ar is an aromatic ring, b) R 1 is [(L) n R′ 1 ] m where n and m are integers selected amongst zero or 1; L is amino nitrogen, ether oxygen or thioether sulphur; R′ 1 is a linker selected among 1) hydrocarbon groups; 2) —C(═NH)—; c) R 2-4 are selected among hydrogen and alkyls; (B) SP is a spacer providing a carbon or a heteroatom directly attached to Ar—R 1 —N + (R 2 R 3 R 4 ); (C)--- represents that SP replaces a hydrogen in (Ar—R 1 —N + (R 2 R 3 R 4 ); (D)-- represents binding to the matrix; and (II) desorption. There is also described (a) anion-exchangerrs having high breakthrough capacities, (b) a screening method and (c) a desalting protocol.

1 . A method for the removal of a substance carrying a negative charge and being present in an aqueous liquid (I), said method comprising the steps of 
 (i) contacting the liquid with a matrix carrying a plurality of ligands comprising a positively charged structure (anion-exchanger) and a hydrophobic structure under conditions permitting binding between the ligands and the substance, and    (ii) desorbing said substance from said matrix,    characterized in that    (I) each of said ligand plus a spacer has the formula:    --SP---[Ar—R 1 —N + (R 2 R 3 R 4 )]    where 
 (A) [Ar—R 1 —N + (R 2 R 3 R 4 )] represents a ligand in which 
 a) Ar is an aromatic ring,  
 b) R 1  is [(L) n R′ 1 ] m  where 
 n and m are integers selected amongst zero or 1;  
 L is an amino nitrogen, an ether oxygen or a thioether sulphur;  
 R′ 1  is a bivalent linker group selected among  
  1) linear, branched or cyclic hydrocarbon groups;  
  2) —C(═NH)—;  
 
 c) R 2-4  are selected among hydrogen and lower alkyls;  
 
 (B) SP is a spacer providing a carbon, a nitrogen, a sulphur or an oxygen directly attached to Ar—R 1 —N + (R 2 R 3 R 4 );  
 (C)--- represents that the spacer is replacing a hydrogen in (Ar—R 1 —N + (R 2 R 3 R 4 );  
 (D)-- represents binding to the matrix; and  
   (II) desorption in step (ii) is carried out under anion-exchange conditions when the substance is a serine protease and in particularly when R′ 1 =—C(═NH)—.    
     
     
         2 . The method of  claim 1 , characterized in that anion-exchanger (1) is capable of 
 (a) binding to the substance of interest in an aqueous reference liquid (II) under anion-exchange condition at an ionic strength corresponding to 0.3 M NaCl and,    (b) permitting a maximal break through capacity in the pH interval 2-12 for the substance ≧200%, such as ≧300% or ≧500% or ≧1000%, of the maximal break through capacity in the pH-interval 2-12 of the substance for Q-Sepharose Fast Flow (Amersham Pharmacia Biotech, Uppsala, Sweden),    said anion-exchangers having essentially the same ligand density and break through capacities being determined under the same conditions.    
     
     
         3 . The method of any of claims  1 - 2 , characterized in that m=1 and R′ 1  is a bivalent linker group selected among linear, branched or cyclic hydrocarbon groups that may be substituted and/or have a carbon chan that is interrupted by ether oxygen, thioether sulphur or amino nitrogen.  
     
     
         4 . The method according to any of claims  1 - 3 , characterized in that the matrix with its plurality of ligands has a pKa≦12 and/or is a primary or secondary nitrogen.  
     
     
         5 . The method of any of claims  1 - 4 , characterized in that at least one of Ar, SP, R′ 1  and R 2-4 , comprises one or more electron acceptor-donor atoms or groups at a distance of 1-7 atoms from the positive nitrogen in —N + (R 2 R 3 R 4 ), preferably said acceptor-donor atoms or groups participating in hydrogen-bonding, and with the proviso that for Ar this atoms or groups are not sp 2 -carbons in an aromatic structure.  
     
     
         6 . The method of any of claims  5 , characterized in that said 
 (i) electron donor-acceptor interaction is hydrogen bonding and/or    (ii) donor atoms/groups are selected among: 
 (a) oxygen with a free pair of electrons, such as in hydroxy, ethers, carbonyls, and esters (—O— and —CO—O—) and amides,  
 (b) sulphur with a free electron pair, such as in thioether (—S—),  
 (c) nitrogen with a free pair of electron, such as in amines, amides including sulphone amides,  
 (d) halogen (fluorine, chlorine, bromine and iodine), and  
 (e) sp- and sp 2 -hybridised carbons; and/or  
   (iii) acceptor groups are selected amongst groups that consists of a electron-deficient atom such as hydrogen and/or an electronegative atom.    
     
     
         7 . The method of any of claims  5 - 6 , characterized in that at least one of said one or more hydrogen-bonding atoms is present as a branch group in SP or as a part of the chain in SP extending from the base matrix to the ligand.  
     
     
         8 . The method according to any of claims  1 - 7 , characterized in that SP contains 
 (a) a carbon atom with preference for a carbonyl carbon or an sp 3 -hybridised carbon; or    (b) a nitrogen atom with preference for an amino or an amido nitrogen; or    (c) a sulphur atom with preference for a thioether sulphur atom; or    (d) an oxygen, with preference for an ether oxygen atom;    which is directly attached to the ligand Ar—R 1 —N + (R 2 R 3 R 4 ), with the proviso that items (b)-(d) only apply when the spacer binds to Ar or R 1 .    
     
     
         9 . The method of any of claims  1 - 2 , characterized in that n=0, m=1, R′ 1 =—C(═NH)—, R 2-4 =hydrogen, Ar=p-C 6 H 4 —, SP is attached to Ar via a secondary amino nitrogen, such as —NH—.  
     
     
         10 . The method of any of claims  1 - 9 , characterized in that the ionic strength during the adsorption/binding step (i) is larger or equal with the ionic strength of 0.25 M NaCl water solution.  
     
     
         11 . The method of any of claims  1 - 10 , characterized in that the pH of aqueous liquid (I) is ≦pKa+2, such as ≦pKa+1, of the anion-exchanger or of an anion-exchanger ligand present in the anion-exchanger.  
     
     
         12 . The method of any of claims  1 - 11 , characterized in that the pH of aqueous liquid (II) is different from the pH of aqueous liquid (I) in order to decrease the negative charge of the substance.  
     
     
         13 . The method of any of claims  1 - 12 , characterized in that the polarity of aqueous liquid (II) is lower than the polarity of aqueous liquid (I).  
     
     
         14 . The method of any of claims  1 - 13 , characterized in that a structural analogue of Ar—R 1 —N + (R 2 R 3 R 4 ) is present in aqueous liquid (II) in a larger concentration than in aqueous liquid (I).  
     
     
         15 . An anion-exchanger (1) comprising a plurality of anion-exchange ligands each of which is attached via a spacer to a hydrophilic base matrix, characterized in that 
 (a) the ligands plus their spacers comply with the formula:    --SP---[Ar—R 1 —N + (R 2 R 3 R 4 )]    where the symbols have the same meaning as in any of claims  1 - 10 , and    (b) the anion-exchanger (1) has a maximal breakthrough capacity in the pH-interval 2-13 for at least one reference proteins selected amongst ovalbumin, conalbumin, bovine serum albumin, β-lactglobulin,α-lactalbumin, lyzozyme, IgG, soybean trypsin inhibitor (STI) which is ≧200%, such as ≧300% or ≧500% or ≧1000% of the maximal breakthrough capacity in the pH-interval 2-12 obtained for a Q-exchanger (—CH 2 CH(OH)CH 2 N + (CH 3 ) 3 ) (anion-exchanger 2), the support matrix, degree of substitution, counter-ion and running conditions being the same for anion-exchanger (1) and anion-exchanger (2).    
     
     
         16 . The anion-exchanger of  claim 15 , characterized in that the relative break-through capacity is measured under anion-exchanger condition.  
     
     
         17 . A method for testing (screening) the appropriateness of one or more anion-exchangers for removing a substance from a liquid, said method comprising the steps: 
 (a) providing a library which comprises 
 (i) one or more anion-exchangers to be tested (exchangers 1, 2, 3, 4 . . . n; n=an integer >0) each of which anion-exchangers differs with respect to kind of ligand (ligands 1, 2, 3, 4, . . . n), and  
 (ii) a reference anion-exchanger having a reference ligand, the support matrix etc being essentially the same in the exchangers 1, 2, 3, 4 . . . n and in the reference anion-exchanger;  
   (b) determining the maximal breakthrough capacity in the pH-interval 2-12 of exchanger 1 for the substance at a predetermined condition;    (c) determining the maximal breakthrough capacity in the pH-interval 2-12 of the reference anion-exchanger for the substance at the same condition as in step (b);    (d) concluding with the aid of the relation between the maximal breakthrough capacities obtained in steps (b) and (c), if anion-exchanger 1 is appropriate to use for removing the substance; and    (e) repeating, if necessary, steps (b)-(c) for at least one of the exchangers 2, 3, 4 . . . n.    
     
     
         18 . The method of  claim 17 , characterized in that the steps (b) and (c) are carried out under anion-exchanger conditions.  
     
     
         19 . A method for removing salt from a negatively charged substance, preferably amphoteric, when present in a solution (liquid (I)), which method comprises the steps of: 
 (i) contacting liquid (I) liquid with an anion-exchanger (1) that comprises a base matrix carrying a plurality of ligands in which there is a positively charged nitrogen under conditions permitting binding between the anion-exchangerr and the substance,    (ii) desorbing said substance from said anion-exchanger by the use of a liquid (liquid (II)).    characterized in:    (A) selecting anion-exchanger (1) among anion-exchangers that are 
 (a) capable of binding the substance of interest in an aqueous reference liquid at an ionic strength corresponding to 0.25 M NaCl; and  
 (b) permitting a maximal breakthrough capacity in the pH interval 2-12 for the substance ≧200%, such as ≧300% or ≧500% or ≧1000%, of the breakthrough capacity of the substance for Q-Sepharose Fast Flow (anion-exchanger 2, Amersham Pharmacia Biotech, Uppsala, Sweden), said anion-exchangers having essentially the same ligand density and the breakthrough capacities being determined under the same conditions;  
   (B) adjusting the pH of liquid (II) in step (ii) by the use of an acid-base pair to a value that means a lower net positive charge on-the anion-exchanger and/or a lower net negative or positive charge on the substance thereby enabling elution at a lowered ionic strength compared to liquid (I).    
     
     
         20 . The method of  claim 19 , characterized in that at least one member of the acid-base pair buffer has a vapour pressure that is higher than the substance.  
     
     
         21 . The method of any of claims  19 - 20 , characterized in that the substance in the liquid of low salt content obtained in step (ii) is ionized in a mass spectrometer.