Reduction in Immunological Manifestations of 6‐Aminopenicillanic Acid by Treatment with Water‐Insoluble Pronase

Penicilloylated proteins which are found as an impurity in 6‐aminopenicillanic acid can be exhaustively digested by pronase to yield amino acids and small peptides. This degradation converts the potent polyvalent antigens into a mixture of mostly monovalent haptens which are much less immunogenic and less capable of eliciting immune reactions in sensitized animals. The degradation of the protein impurity can be carried out in 6‐aminopenicillanic acid preparations, since even a large excess of 6‐aminopenicillanic acid does not appreciably inactivate or inhibit the enzyme. In order to avoid the contamination of 6‐aminopenicillanic acid with a proteolytic enzyme, pronase was converted into a water insoluble form by coupling it with bromoacetyl cellulose or carboxymethyl Sephadex. These insoluble derivatives of pronase were found to retain their activity and broad specificity. Thus, they can be readily removed from the medium upon completion of the impurity degradation, to be used repeatedly in a continuous process.