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

Prep Biochem Biotechnol. 2018;48(9):808-814. doi: 10.1080/10826068.2018.1509085. Epub 2018 Sep 28.


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.

Keywords: Depth filtration; filtration; microfiltration; precipitation; purification.

MeSH terms

  • Cell Culture Techniques / economics
  • Cell Culture Techniques / methods*
  • Chitosan / chemistry*
  • Clostridium tetani / metabolism
  • Costs and Cost Analysis
  • Filtration / methods
  • Flocculation
  • Tetanus Toxoid / biosynthesis
  • Tetanus Toxoid / isolation & purification*


  • Tetanus Toxoid
  • Chitosan