The present invention provides a method of purifying diphtheria toxin comprising (1) fermenting a microorganism strain capable of producing diphtheria toxin using media supplemented with a mixture of glycerol, nitrogen salts & trace elements whereby the addition of glucose maintains microorganism growth effective to support diphtheria toxin production ( 2) adding a stabilizer mixture of atleast one Sugar,atleast one detergent & atleast one protease inhibitor during purification and (3) operating two chromatographic separation steps wherein ion exchange chromatography is followed by hydrophobic interaction chromatography hydrophobic matrix, and eluting a fraction containing the toxin having purity of more than 99%.
