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Tetanus toxin production from Clostridium tetani, using a casein-based medium in a single-use bioreactor

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Abstract

Clostridium tetani, a spore-forming anaerobic bacterium, and a casein-based semisynthetic medium were used to produce tetanus toxin in this study. N-Z-Case TT (casein hydrolysate) solution and glucose stock media were mixed and autoclaved, which resulted in tetanus toxin expression. The toxin was expressed when the N-Z-Case TT solution reacted with the glucose stock at a high temperature, creating an adequate amount of Maillard reaction products (MRPs). After accumulating in C. tetani cells, tetanus toxin was secreted into the medium when cell lysis was induced by surface aeration. C. tetani was cultivated and tetanus toxin was expressed in a single-use bioreactor, which produced 80 Lf/mL of tetanus toxin in a medium with MRPs. While using the correct medium to induce tetanus toxin was important, other factors played a part in achieving the desired concentration of the toxin, including the medium processing and culture methods inside the bioreactor. Tetanus toxoid with a purity level greater than 2,500 Lf/mgPN was obtained by detoxifying and purifying the toxin recovered from the fermenter or single-use bioreactor. A single-use bioreactor could be used in a limited space without the need for constructing a large scale production facility, to produce the tetanus toxoid antigen for clinical trials.

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References

  1. Sheffield, J. S. and S. M. Ramin (2004) Tetanus in pregnancy. Am. J. Perinatol. 21: 173–182.

    Article  Google Scholar 

  2. Croxtall, J. D. and S. Dhillon (2012) Meningococcal quadrivalent (serogroups A, C, W135 and Y) tetanus toxoid conjugate vaccine (NimenrixTM). Drugs 72: 2407–2430.

    Article  CAS  Google Scholar 

  3. Mueller, J. H. and P. A. Miller (1954) Variable factors influencing the production of tetanus toxin. J. Bacteriol. 67: 271–277.

    CAS  Google Scholar 

  4. Latham, W. C., D. F. Bent, and L. Levine (1962) Tetanus toxin production in the absence of protein. Appl. Microbiol. 10: 146–152.

    CAS  Google Scholar 

  5. Demain, A. L., D. F. Gerson, and A. Fang (2005) Effective levels of tetanus toxin can be made in a production medium totally lacking both animal (e.g., brain heart infusion) and dairy proteins or digests (e.g., casein hydrolysates). Vaccine 23: 5420–5423.

    Article  CAS  Google Scholar 

  6. De Luca, M. M., H. D. Abeiro, J. A. Bernagozzi, and J. A. Basualdo (1997) Nitrogen-gas bubbling during the cultivation of Clostridium tetani produces a higher yield of tetanus toxin for the preparation of its toxoid. Microbiol. Immunol. 41: 161–163.

    Article  Google Scholar 

  7. Fratelli, F., T. J. Siquini, S. M. Prado, H. G. Higashi, A. Converti, and J. C. de Carvalho (2005) Effect of medium composition on the production of tetanus toxin by Clostridium tetani. Biotechnol. Prog. 21: 756–761.

    Article  CAS  Google Scholar 

  8. Fratelli, F., T. J. Siquini, M. E. de Abreu, H. G. Higashi, A. Converti, and J. C. de Carvalho (2010) Fed-batch production of tetanus toxin by Clostridium tetani. Biotechnol. Prog. 26: 88–92.

    CAS  Google Scholar 

  9. Eibl, R., S. Kaiser, R. Lombriser, and D. Eibl (2010) Disposable bioreactors: the current state-of-the-art and recommended applications in biotechnology. Appl. Microbiol. Biotechnol. 86: 41–49.

    Article  CAS  Google Scholar 

  10. Carabasa-Giribet, M. and A. Ibarz-Ribas (2000) Kinetics of colour development in aqueous glucose systems at high temperatures. J. Food Eng. 44: 181–189.

    Article  Google Scholar 

  11. Hepple, J. R. (1968) Large scale production of Clostridium tetani toxin. Chem. Ind. 21: 670–674.

    CAS  Google Scholar 

  12. Mellanby, J. (1968) The effect of glutamate on toxin production by Clostridium tetani. J. Gen. Microbiol. 54: 77–82.

    Article  CAS  Google Scholar 

  13. Baisier, W. M. and T. P. Labuza (1992) Maillard browning kinetics in a liquid model system. J. Agric. Food Chem. 40: 707–713.

    Article  CAS  Google Scholar 

  14. Thaysen-Andersen, M., S. B. Jorgensen, E. S. Wilhelmsen, J. W. Petersen, and P. Hojrup (2007) Investigation of the detoxification mechanism of formaldehyde-treated tetanus toxin. Vaccine 25: 2213–2227.

    Article  CAS  Google Scholar 

  15. Latham, W. C., C. B. Michelsen, and G. Edsall (1967) Preparative procedure for the purification of toxoids by gel filtration. Appl. Microbiol. 15: 616–621.

    CAS  Google Scholar 

  16. Levine, L. and J. L. Stone (1951) The purification of tetanus toxoid by ammonium sulfate fractionation. J. Immunol. 67: 235–242.

    CAS  Google Scholar 

  17. Lyng, J. (1990) Quantitative estimation of diphtheria and tetanus toxoids. 4. Toxoids as international reference materials defining Lf-units for diphtheria and tetanus toxoids. Biologic. 18: 11–17.

    Article  CAS  Google Scholar 

  18. Hiller, A., J. Plazin, and D. D. Van Slyke (1948) A study of conditions for Kjeldahl determination of nitrogen in proteins; description of methods with mercury as catalyst, and titrimetric and gasometric measurements of the ammonia formed. J. Biol. Chem. 176: 1401–1420.

    CAS  Google Scholar 

  19. Porfirio, Z., S. M. Prado, M. D. Vancetto, F. Fratelli, E. W. Alves, I. Raw, B. L. Fernandes, A. C. Camargo, and I. Lebrun (1997) Specific peptides of casein pancreatic digestion enhance the production of tetanus toxin. J. Appl. Microbiol. 83: 678–684.

    Article  CAS  Google Scholar 

  20. Vrany, B., Z. Hnatkova, and A. Lettl (1988) Production of toxic antigens in dialyzed cultures of microorganisms, Folia. Microbiol. 33: 148–154.

    CAS  Google Scholar 

  21. Brands, C. M. and M. A. van Boekel (2001) Reactions of monosaccharides during heating of sugar-casein systems: Building of a reaction network model. J. Agric. Food Chem. 49: 4667–4675.

    Article  CAS  Google Scholar 

  22. Kwak, E. J. and S. I. Lim (2004) The effect of sugar, amino acid, metal ion, and NaCl on model Maillard reaction under pH control. Amino Acids. 27: 85–90.

    Article  CAS  Google Scholar 

  23. Namiki, M. (1988) Chemistry of Maillard reactions: recent studies on the browning reaction mechanism and the development of antioxidants and mutagens. Adv. Food Res. 32: 115–184.

    Article  CAS  Google Scholar 

  24. Stojicevic, I., L. Dimitrijevic, N. Dovezenski, I. Zivkovic, V. Petrusic, E. Marinkovic, A. Inic-Kanada, and M. Stojanovic (2011) Tetanus toxoid purification: chromatographic procedures as an alternative to ammonium-sulphate precipitation. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 879: 2213–2219.

    Article  CAS  Google Scholar 

  25. Einarsson, H., T. Eklund, and I. F. Nes (1988) Inhibitory mechanisms of Maillard reaction products. Microbios. 53: 27–36.

    CAS  Google Scholar 

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Authors and Affiliations

  1. Boryung Biopharma Co., Ltd., Seoul, Korea

    Yong-Ju Chung, Mi-Young Jung, Jin-A Lee & Tae-Yeon Kim

  2. Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea

    Yong-Kyung Choe

  3. Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, Korea

    Yong-Ju Chung & Ik-Hwan Kim

Authors
  1. Yong-Ju Chung
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  2. Mi-Young Jung
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  3. Jin-A Lee
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  4. Tae-Yeon Kim
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  5. Yong-Kyung Choe
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  6. Ik-Hwan Kim
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Correspondence to Ik-Hwan Kim.

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Chung, YJ., Jung, MY., Lee, JA. et al. Tetanus toxin production from Clostridium tetani, using a casein-based medium in a single-use bioreactor. Biotechnol Bioproc E 21, 531–536 (2016). https://doi.org/10.1007/s12257-016-0355-6

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  • DOI: https://doi.org/10.1007/s12257-016-0355-6

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