Removal of porphyrins from diphtheria toxoid by selective adsorption on Ecteola-cellulose.

The occurrence of porphyrins in diphtheria toxin is well known (for references, see A. M. Pappenheimer, J. Biol. Chem. 167:251, 1947). In toxoid, the porphyrins remain as red pigments which, unless removed before the addition of alum, impart a red color to the final alum-precipitated toxoid. Removal of the porphyrins as a first step was examined by L. B. Holt (Developments in Diphtheria Prophylaxis, Heinemann, London, 1950), who found that a specially prepared and aged Mg(OH)2 was the most satisfactory adsorbent. Recently, the possibility that ion-exchange celluloses might be effective in adsorbing pigments from toxins was suggested by A. G. M. Marr and R. W. Patterson (Australian J. Sci. 23:131, 1960). These celluloses have the advantage that they are commercially available in a stable form. Their use in selective batchwise adsorption of the porphyrins from crude diphtheria toxoid was therefore investigated. Crude toxoid was prepared by use of stationary cultures of Corynebacterium diphtheriae P.W.8 grown on Norlin's modification of Mueller and Miller's medium and detoxified with 0.5% Formalin at 30 C. Protein content was based on Kjeldahl nitrogen estimation or ultraviolet absorption after precipitation with trichloroacetic acid. Porphyrins were estimated by the ether extraction method of Pappenheimer. Flocculating activity (Lf/ml) was determined by Ramon's method. In preliminary experiments, Ecteola-cellulose adsorbed the red pigment satisfactorily when used at a concentration of 8.75 mg/ml for 30 min at room temperature (approximately 20 C). A range of anion-exchange celluloses were therefore prepared in the chloride form and compared by use of these conditions. Cellulose was included as a control. Ecteola-cellulose was the most specific in adsorption of the porphyrins without undue loss of flocculating activity (Table 1). When only one-fifth the amount of adsorbent was used, the protein adsorption on the celluloses was reduced, but Ecteola-cellulose remained the most specific. After testing the effect of the concentration of Ecteola-cellulose with several batches of toxoid, we selected a concentration of 8.75 mg/ml as the best for further use since it usually removed 90% or more of the porphyrin with a reduction of not more than 7% in the Lf/ml.