OPTIMIZATION OF CULTURE PROCESS AND PURIFICATION OF DIPHTHERIA TOXOID BY STEP REDUCTION

Diphtheria is an acute vaccine-controlled respiratory disease caused by the gram-positive bacterium Corynebacterium diphtheria. Since the preparation, purification, and detoxification of the vaccine against this disease involves many and complex steps, the present study aimed reduction of time of various stages of toxoid purification.

For this purpose, three factors were first optimized for the concentration of iron in the culture medium, incubation temperature and rotation speed to produce more toxins. In each case, the experiment was carried out in triplicate and Lf and Kf values were tested to evaluate toxin production. After toxin inactivation and production of the toxoid, step reductions were performed to purify the toxoid. For this, ultrafiltration, precipitation, dialysis and chromatography with Sephadex G-25 column are used. In step reduction conditions, dialysis, chromatography or both of them were removed in the first, second and third cases, respectively. The results of each method were evaluated by SDS-PAGE and the quantitative data was analyzed using Minitab 18 (p‹0.05).

The results for optimizing the culture medium showed that highest amounts of toxin were produced at an iron concentration of 1 mg/l, a temperature of 35 °C, and a rotation speed of 200 rpm(p‹0.05). In the reduction experiments related to toxoid purification steps, the best results were obtained in a series of purified samples with ultrafiltration, precipitation and column chromatography steps.

Since time and cost are two key factors in industrial production, toxoid production for the diphtheria vaccine can be produced at a lower cost and in less time by eliminating the two-week dialysis phase.