Procedures for large-scale production and purification ofClostridium botulinumC1 toxin for preparation of toxoid

Fortified cooked meat medium containing calcium carbonate (CaCO3-FCM) supported toxin production of a strain of Clostridium botulinum type C to a level of 2 × 106 mouse i.p. LD50/ml. C1 toxin was purified by sequential steps of acid precipitation from 5-fold diluted culture supernatant in the presence of RNA, 2nd acid precipitation by dialysis, removal of RNA by protamine treatment, removal of excess protamine and bufferisation by ultrafiltration through Amicon PM-30 membrane, sulphopropyl-Sephadex chromatography, and Sephadex G-200 gel filtration. By these procedures, 25 mg or more of highly purified C1 toxin was constantly obtained from a lot of 600-ml culture.