Abstract
The commercial production of purified tetanus toxoid mainly depends on the effective separation of the bacterial toxin and toxoid from large volumes of fermentation broth of Clostridium tetani (Harvard 49205) vaccine strain. Tangential flow or cross-flow filtration system was used as rapid drive in the processing of immunobiological assays of tetanus toxin. Tetanus toxoid was prepared by detoxifying the culture filtrates of C. tetani and further purified by ultrafiltration, salt fractionation and adsorption onto aluminium phosphate. Present study deals with the separation of tetanus toxins using a microporous membrane (0.22 µm) and concentration of tetanus toxoids using an ultrafiltration membrane (30 kDa, NMWL pore size) with operational variables like average trans-membrane pressure (ATP), cross flow rate, flux. Under the best conditions, >96% recovery was achieved. Additionally, potency control of 10 batches of tetanus toxoid, prepared from the filtered toxins/toxoid lots by microporous tangential flow filtration system, was evaluated by in vitro passive haemagglutination (PHA) assay and the results obtained in the in vitro PHA were compared with in vivo toxin neutralization (TN) test. An excellent correlation between in vitro test and in vivo TN test was observed by Spearman’s correlation coefficient. It reveals that the process development in which employing available equipment and the in vitro PHA is a promising alternative to the toxic TN test in the potency assay of tetanus vaccine. Key words: Tetanus toxin, Seitz filtration, tangential flow filtration, microfiltration module, flux, tetanus toxoid, immunogenicity test.
Highlights
Tetanus is an infectious bacterial disease caused by a highly toxinogenic strain of bacillus Clostridium tetani
This paper describes immunogenicity tests on the tetanus vaccines, prepared from the filtered toxin/toxoid lots by tangential flow filtration (TFF) system, the in vivo toxin neutralization (TN) test and in vitro passive haemagglutination assay (PHA), and results were compared by Spearman’s correlation coefficient
Modified Mueller Miller (MMM) medium was used for growing C. tetani (WHO, 1977)
Summary
Tetanus is an infectious bacterial disease caused by a highly toxinogenic strain of bacillus Clostridium tetani. Many manufacturer produce DTP group of vaccines for mass immunization program using large volume capacity bioreactors In these bioreactors, after the end of their incubation period, the concentration of toxin in supernatant usually reaches 80 to 120 flocculation units. The separation of bacterial cells, detoxification, concentration and sterile filtration of toxoids are some of the major unit operations in the processing of large volumes of fermentation media in the production of bacterial vaccines. The conventional depth filtration method normally used for the clarification of tetanus culture broth has been successfully replaced with that of microporous TFF system for large scale recovering of tetanus toxin at a commercial level and further use of a similar system for concentration of crude tetanus toxoid. This paper describes immunogenicity tests on the tetanus vaccines, prepared from the filtered toxin/toxoid lots by TFF system, the in vivo toxin neutralization (TN) test and in vitro passive haemagglutination assay (PHA), and results were compared by Spearman’s correlation coefficient
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