Reduction of endotoxin in polysialic acids

Abstract

The present invention relates to process for reducing the endotoxin content of a sample of fermentation broth containing polysialic acid and endotoxin comprising the sequential steps: (i) adding to the sample a base having a pKa of at least 12 to form a basic solution having a pH of at least 12, incubating the solution for a pre-determined time at a pre-determined temperature; and (ii) recovery of PSA, suitably by (iii) passing the sample through an anion-exchange column whereby polysialic acid is absorbed on the ion exchange resin; (iv) washing the column with one washing buffer, whereby polysialic acid remains absorbed on the ion exchange resin; and (v) eluting the polysialic acid from the column using an elution buffer to provide a product solution of polysialic acid having reduced endotoxin content.

1 . A process for reducing the endotoxin content of a sample containing polysialic acid (PSA) and endotoxin comprising the steps of:
 (i) adding to the sample a base having a pKa of at least 12 to form a basic solution having a pH of at least 12,   (ii) incubating the solution for a predetermined time at a pre-determined temperature; and   (iii) recovering polysialic acid having reduced endotoxin content.   
     
     
         2 . The process of  claim 1  wherein the base has a pKa of at least 13. 
     
     
         3 . The process of  claim 1  wherein the pH of the said basic solution is at least 13. 
     
     
         4 . The process of  claim 1  wherein the base is NaOH, KOH, Ca(OH) 2  or LiOH. 
     
     
         5 . The process of  claim 4  wherein the base is 2N NaOH. 
     
     
         6 . The process of  claim 1 , wherein the pre-determined temperature is in the range 0 to 60° C. 
     
     
         7 . The process of  claim 1  in which step (iii) includes the following sequential substeps:
 (a) passing the sample through an anion-exchange column whereby polysialic acid is adsorbed on the ion exchange resin; 
 (b) washing the column with a washing buffer, whereby polysialic acid remains adsorbed on the ion exchange resin; and 
 (c) eluting the polysialic acid from the column using an elution buffer to provide a product solution of polysialic acid having reduced endotoxin content. 
 
     
     
         8 . The process of  claim 7 , wherein the sample, after step (ii), is neutralised before performing step (iii). 
     
     
         9 . The process of  claim 7 , wherein in step (b), the column is washed in a first wash buffer of low ionic strength to elute endotoxin from the column and wherein the elution buffer used in step (c) has relatively higher ionic strength. 
     
     
         10 . The process of  claim 9 , wherein the washing buffer and/or the elution buffer contains volatile base. 
     
     
         11 . The process of  claim 9 , wherein a second wash buffer comprises NaCl at a concentration of at least 0.2M. 
     
     
         12 . The process of  claim 7 , wherein in step (b) the column is washed more than once. 
     
     
         13 . The process of  claim 7 , wherein steps (i) and (iii) are repeated on the product solution. 
     
     
         14 . The process of  claim 1  comprising at least one further step in which the endotoxin level of polysialic acid is reduced by purification carried out either before step (i), between steps (ii) and (iii) or after step (iii) wherein said step is selected from ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, size exclusion chromatography and combinations thereof. 
     
     
         15 . The process of  claim 14 , wherein hydrophobic interaction chromatography is carried out before step (i). 
     
     
         16 . The process of  claim 1  wherein the sample is incubated with a surfactant, a chelating agent, an organic solvent, an oxidant or a peroxidase either before step (i), between steps (ii) and (iii) or after step (iii). 
     
     
         17 . The process of  claim 1 , further comprising obtaining said sample by fermenting E. coli to produce a fermentation broth containing the PSA and endotoxin, and optionally removing protein, lipid, nucleic acid and/or nutrients, to form said sample. 
     
     
         18 . The process of  claim 1  wherein the endotoxin content of the PSA in the product solution is no more than 25 EU/mg of PSA, measured by the LAL test. 
     
     
         19 . A sample of fermentation broth having reduced endotoxin content obtainable by a process according to  claim 1 . 
     
     
         20 . A sample of polysialic acid (PSA) comprising an endotoxin content suitable for veterinary or human administration. 
     
     
         21 . A sample according to  claim 20 , having an endotoxin content of no more than 25 EU/mg of PSA. 
     
     
         22 . A sample according to  claim 20 , wherein the PSA is a poly(2,8-linked sialic acid), a poly(2,9-linked sialic acid), or alternating 2,8-2, 9-linked polysialic acid, preferably poly (2,8-linked sialic acid). 
     
     
         23 . A sample according to  claim 22 , wherein the PSA is colominic acid (CA), or an oxidised, reduced, aminated and/or hydrazide derivative thereof. 
     
     
         24 . The process of  claim 1 , wherein the predetermined time ranges from 5 minutes to 24 hours. 
     
     
         25 . The process of  claim 24 , wherein the predetermined time ranges from 30 minutes to 6 hours. 
     
     
         26 . The process of  claim 1  wherein the sample contains polysialic acid-protein conjugate and reduced endotoxin content. 
     
     
         27 . The process of  claim 26  wherein the reduced endotoxin content is no more than 5 EU/mg. 
     
     
         28 . The process of  claim 27  wherein the reduced endotoxin content is no more than 0.5 EU/mg. 
     
     
         29 . A process for preparing a polysialic acid-biological molecule conjugate, comprising reducing the endotoxin content of a sample containing polysialic acid according to the process of  claim 1  and conjugating the polysialic acid obtained with a biological molecule. 
     
     
         30 . The process of  claim 29 , wherein the biological molecule is a protein.