A Method for Purifying Diphtheria Toxoid and Combining it with Protamine

Abstract

Discussion and Summary The use of protamine offers a means of separating bacillary protein and therefore removes a major protein component of crude diphtheria toxoid. No specific and constant formula, in terms of quantity of protamine or with respect to ionic strength can be given which will apply equally well to crude toxoids made in broths of varying composition. It is necessary to determine optimal conditions for each lot. It is desirable to employ toxoids that contain only minimal amounts of protein derivatives precipitated by trichloracetic acid at pH 4.0. Using such preparations, this step precipitates toxoid of high purity, once the bacillary protein is removed. On further treatment with Norite, preparations containing 2000 to 2100 Lf/mgN were obtained. Several additional steps have been employed between the removal of bacillary protein and the addition of trichloracetic acid when the crude toxoid contained larger amounts of protein derivatives originating in the production broth. The chemical steps employed in preparing toxoid of this degree of purity do not appear to have resulted in noticeable changes in the stability of the toxoid, since flocculation has occurred fairly promptly with solutions in phosphate buffer held at 2 C for approximately 2 years and solutions held at 37 C for 4 weeks showed only 10 to 20 per cent loss in Lf. Precipitation of purified toxoid by protamine, and adsorption of the complex on a small quantity of aluminum hydroxide, gave a product which, when suspended in phosphate buffer and warmed at 37 C for 2 weeks, lost only 5 to 10 per cent of its Lf value; when warmed for 4 weeks the loss was 20 per cent. The absence of change in the toxoid molecule as a result of the procedures used is further indicated by the fact that guinea pigs have produced 4 units of antitoxin following injection of the protamine-precipitated product. The high degree of purity of the toxoid obtained is reflected in the absence of disturbing reactions following injection of the protamine-toxoid into human adults. It seems possible that even the occasional individual who is sensitive to toxoid as such will experience less discomfort if given the protamine-precipitated material, since this would be expected to reduce the rate of release of toxoid at the site of the injection. Some data are presented dealing with electrophoretic, and sedimentation-measurements of the purified toxoid.