New method for the large-scale preparation of diphtheria toxoid: purification of toxin.

Diphtheria toxin of high purity was prepared in batch cultures of 1 to 40 liters by procedures capable of processing 200-liter batches without modification. The procedure incorporates preliminary purification of the growth medium and, after deep fermentation or sulfate culture of Corynebacterium diphtheriae, both concentration and partial purification of the toxin by membrane ultrafiltration. Final purification is achieved by Sephadex G-100 gel filtration. Purities of 2,000 to 2,500 flocculation units per mg of protein nitrogen (260 to 410 flocculation units per unit of absorbance at 280 nm) were routinely obtained with only a 10% loss of toxin. The toxin appeared pure on immunoelectrophoresis and ultracentrifugation, and only minor amounts of lower-molecular-weight impurities were revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Significant advantages of the procedure are its rapidity and reproducibility and the fact that all stages are performed at 4 C in neutral isotonic buffer.