Patent ID: 8519108

Claim:
A method for preparing a purified anthrax protective antigen, comprising the steps of 1) adjusting the conductivity of a solution containing the anthrax protective antigen in the range of 13 to 15 mS/cm by adding NaCl, LiCl or CH 3 COONa to the solution, and subjecting the solution to ion exchange chromatography to obtain an eluted fraction containing the anthrax protective antigen, wherein the ion exchange chromatography employs an ion exchange resin selected from the group consisting of Q-Sepharose FF (quaternary ammonium), Q-Sepharose XL (quaternary amino), Macro-Prep High Q (quaternary ammonium), Macro-Prep 25 Q (quaternary ammonium), and Super Q-650M (quaternary ammonium); 2) adjusting the conductivity of the eluted fraction obtained in step 1) in the range of 1 to 5 mS/cm, and subjecting the resulting solution to ion exchange chromatography to obtain an eluted fraction containing the anthrax protective antigen, wherein the ion exchange chromatography employs an ion exchange resin selected from the group consisting of Q-Sepharose FF (quaternary ammonium), Q-Sepharose XL (quaternary amino), Macro-Prep High Q (quaternary ammonium), Macro-Prep 25 Q (quaternary ammonium), and Super Q-650M (quaternary ammonium); 3) adjusting the conductivity of the eluted fraction obtained in step 2) in the range of 170 to 175 mS/cm by adding (NH 4 ) 2 SO 4 , K 2 SO 4 , Na 2 HPO 4 or NaCl to the fraction, and subjecting the resulting solution to hydrophobic column chromatography to obtain an eluted fraction containing the anthrax protective antigen; 4) exchanging the solvent in the eluted fraction obtained in step 3) by ultrafiltration and dialysis to obtain an antigen solution having a conductivity in the range of 5 to 10 mS/cm; and 5) subjecting the antigen solution obtained in step 4) to hydroxyapatite chromatography to obtain a purified anthrax protective antigen.