Method for the production of improved chymopapain

A process for the purification of crude chymopapain to produce a purified chymopapain of reduced toxicity comprising:

• (a) contacting an aqueous buffered solution of crude chymopapain with a weakly acidic cationic exchanger comprising a column of carboxymethyl substituted cross-linked agarose gel, said exchanger having been previously equilibrated with aqueous buffer solution having a pH of between about 6.5 and 7.5;
• (b) eluting the chymopapain retained on the exchanger with a similar aqueous buffer solution having a pH in the same pH as the buffer used for equilibrating the carboxymethyl substituted agarose gel, but having a linearly increasing ionic concentration of a compatible, non-reactive, water-soluble, pharmaceutically acceptable, neutral inorganic salt with respect to eluent volume;
• (c) collecting and discarding a first series of fractions of eluent from said exchanger containing an initial protein component from crude chymopapain until the molarity of the eluent with respect to said soluble salt increases to and reaches the range of 0.25 to 0.4;
• (d) continuously collecting and retaining a further series of fractions of chymopapain eluted from the exchanger comprising two proteolytically active chymopapain components at increasing soluble salt concentrations greater than about 0.25 to 0.4 molar, until substantially all of the said absorbed cymopapain is recovered;
• (e) treating said retained fractions containing proteolytically active components of the chymopapain to remove the dissolved ionic inorganic salts, and buffer components;
• and
• (f) lyophilizing the essentially salt-free chymopapain solution to produce a dry, purified proteolytically active chymopapain essentially free of proteolytically inactive or toxic components.