LARGE-SCALE PRODUCTION AND PURIFICATION OF CLINICAL GRADE PSEUDOMONAS-AERUGINOSA EXOTOXIN-A FROM ESCHERICHIA-COLI

Immunotoxin therapy for cancer utilizes hybrid proteins composed of a cell targeting moiety which is a monoclonal antibody reactive with the cancer cell surface, and a cytotoxic agent which is either a bacterial or plant toxin, to specifically attack cancer cells. The immunotoxin B3LysPE38, composed of the modified Pseudomonas aeruginosa exotoxin A (LysPE38) chemically linked to a murine monoclonal antibody (B3), was effective in killing various forms of cancer in mice. To test the therapeutic value in human, a phase I clinical study was designed in which 0.1-1.0 mg of B3LysPE38/Kg of body weight would be administered to each patient. Early estimates of the expected enrolled patient population for this study indicated a need for gram amounts of highly purified immunotoxin. Therefore, a fermentation and purification procedure was developed to isolate 10 grams of LysPE38 (> 99% pure) with clearance levels of pyrogen at 10E.U. and bacterial DNA at 6pg per mg of LysPE38. The high density fermentation process was based on an adaptive control strategy and the purification process was composed of ion exchange and hydrophobic interaction column chromatography.