An effective, simple and low-cost pretreatment for culture clarification in tetanus toxoid production.

Chemically inactivated tetanus toxin (tetanus toxoid, TT), purified from cultures of a virulent Clostridium tetani strain, is the active pharmaceutical ingredient of anti-tetanus vaccines. Culture clarification for TT production and is usually performed by filtration-based techniques. Final clarification of the culture supernatant is achieved by passage through 0.2 µm pore size filtering membranes. Large particles removal (primary clarification) before final filtration (secondary clarification) reduces costs of the overall clarification process. With this aim, chitosan-induced particle aggregation was assessed as an alternative for primary clarification. Three chitosan variants were tested with similar results. Optimal clarification of culture supernatant was achieved by the addition of 8 mg chitosan per l of culture. Extrapolation analysis of filter sizing results indicate that 100 l of chitosan-treated supernatant can be finally filtered with a 0.6 m2 normal filtration cartridge of 0.45 + 0.2 µm pore size. The clarified material is compatible with current standard downstream processing techniques for TT purification. Thus, chitosan-induced particle aggregation is a suitable operation for primary clarification.

Use of sephacryl high resolution (HR) in tetanus anatoxin purification.

The tetanus purified anatoxin is used in the preparation of the tetanus toxoid and multiple vaccines (dT, DT and DTP), all of them strictly following specifications established by the WHO with a minimum antigenic purity equal to 1,000 Lf/mgPN. Aiming to establish more sensitive and accurate methods for purification, samples from four different lots of tetanus anatoxin were submitted to gel filtration in twenty independent trials using the Sephacryl S-100 HR and S-200 HR resins. The Authors were careful to optimize their parameters of performance as to sample volume, elution and selectivity flow for tetanus anatoxin purification, allowing their use in industrial scale. The Sephacryl S-100 HR resin presented the best selectivity, that is, the best separation, allowing a greater linear-flow and, consequently, the best purity index. Satisfactory results were also achieved with the Sephacryl S-200 HR resin after optimization of chromatographic parameters for elution flow and volume of the sample applied. The good results of purification obtained, as well as the high chemical stability, have pointed out both the Sephacryl S-100 HR and S-200 HR resins as equally efficient for industrial production.