In order to reduce the toxicity of Clostridium perfringens fermentation broths used in vaccine preparation, we developed two-phase aqueous systems for removal of toxin-activating proteases. Removal of the proteases inhibits the conversion of protoxin to active toxin. In order to establish the conditions under which the phase separation occurs, binodal curves, formed by poly(ethylene glycol) (PEG) and sodium citrate, were investigated at different values of pH and PEG molar mass. A 2(4)-experimental design was used to evaluate the influence of PEG molar mass and concentration, citrate concentration and pH on protease partition coefficient, removal.factor and protease removal yield. It has been found that simultaneous increase in PEG molar mass and decrease in citrate concentration remarkably improved the removal factor, whereas the protease removal yield showed an opposite trend. The best conditions for the system under consideration (removal factor of 2.69 and yield of 116%) were obtained at pH 8.0 using PEG molar mass of 8000 g mol(-1) and concentrations of PEG and citrate of 24 and 15%, respectively.