An effective, simple and low-cost pretreatment for culture clarification in tetanus toxoid production (original) (raw)
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International Journal of Biotechnology and Molecular Biology Research, 2013
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.
The most effective way to control tetanus (caused by Clostridium tetani) is immunization with tetanus toxoid vaccine. However, the production of tetanus vaccine is a complex process including growth of bacteria, harvesting of toxin, and then conversion of that toxin into potent toxoid vaccine. During production, purification of vaccine either at toxin stage or at toxoid stage is essential. In the present study, effect of purification using ultrafiltration at toxin stage was evaluated on four commercial batches of tetanus vaccine in terms of antigenic content (lf/ml), purity in terms of protein nitrogen, antigenic potential by minimum lethal dose and potential efficacy by maximal toxic value. The standard reference methods as per the WHO manual for the production and quality control of tetanus vaccine were followed. A significant increase in the antigenic content, purity, antigenic potential, and potential efficacy of all four batches was observed after ultrafiltration. The results indicate that ultrafiltration before detoxification is an effective method of tetanus toxin purification. It simultaneously increase the quality of the toxin along with the removal of contaminating agents, which otherwise results in adverse effects during use.
Biotechnology and Bioprocess Engineering, 2016
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.
Standardization of process for increased production of pure and potent tetanus toxin
Journal of Microbiology and Infectious Diseases, 2013
Objectives: The aim of the study was to increase the yield of tetanus toxin in short time fermentor cultivation and also to produce pure and potent tetanus toxin replacing initial nitrogen source (N.Z Case) with papain digest broth in the modified Mueller Miller medium (MMMM). Methods: A fermentor, using a vibromixer and optimum supply of sterile air to the headspace of the fermentor to flush out the accumulated gases was used. The MMMM containing initial N.Z Case was replaced with papain digest broth was used successfully. Results: It was found that under optimal conditions of temperature, vibromixing, surface aeration, and an alkaline pH favored toxin release. Furthermore, to enhance the production volume, fermentor culture is more suitable. The tetanus toxin was produced with good Limes flocculation (Lf) titre and high antigenic purity. A significant increase in the tetanus toxin yield in short time cultivation (about 5 to 6 days against 8 days) was noticed even with MMMM containing papain digest broth instead of N.Z.Case. Conclusions: For large-scale production of purified and potent (antigenic purity) tetanus toxin, the use of fermentor technology can be utilized under optimal conditions. The production medium using indigenously available ingredients containing high level of aminonitrogen as in the case of PDM can be substituted in place of N.Z Case, which is being imported and expensive, in addition to lot-lot variation.
Effect of Medium Composition on the Production of Tetanus Toxin by Clostridium tetani
Biotechnology Progress, 2008
The tetanus toxin is a neurotoxin synthesized by the bacillus Clostridium tetani that, after detoxification with formaldehyde, still exhibits antigenic and immunologic properties, hence its denomination of tetanus toxoid. Such a neurotoxin is produced by cultivation of the microorganism in vegetative form on a relatively complex specific medium containing glucose and peptone. The simultaneous effects of the starting levels of glucose (G 0 ) and N-Z Case TT (NZ 0 ) as carbon and nitrogen sources, respectively, on the production of tetanus toxin have been investigated in this work in static cultivations by means of a five-level star-shaped experimental design and evaluated by response surface methodology (RSM) for optimization purposes. The highest final average yield of tetanus toxin (72 L f /mL), achieved at G 0 ) 9.7 g/L and NZ 0 ) 43.5 g/L, was 80% higher than that obtained with standard cultivations (G 0 ) 8.0 g/L and NZ 0 ) 25.0 g/L).
International Journal of Green Nanotechnology, 2011
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Microbiology and Immunology, 1997
We investigated the effect of exposing cultures of Clostridium tetani to nitrogen (N2) gas on the recovery of tetanus toxin to be processed for the preparation of its toxoid. N2 was bubbled through nine 10liter cultures during the growth of the bacteria, while nine parallel control incubations were maintained without bubbling. We found that treatment of the C. tetani anaerobes with an inert gas in this manner during cultivation produced a highly significant increase in the yield of tetanus toxin from them in comparison with the standard procedure.
Chitosan–HPMC-blended microspheres as a vaccine carrier for the delivery of tetanus toxoid
Artificial Cells, Nanomedicine, and Biotechnology, 2014
The purpose of this research was to develop a suitable and alternate adjuvant for the tetanus toxoid (TT) vaccine that induces long term immunity after a single-dose immunization. In our study, the preformulation studies were carried out by using different ratios (7/3, 8/2, and 9/1) of chitosan-hydroxypropyl methylcellulose (HPMC)-blended empty microspheres. Moreover, TT was stabilized with heparin (at heparin concentrations of 1%, 2%, 3%, and 4% w/v) and encapsulated in ideal chitosan -HPMC (CHBMS) microspheres, by the water-in-oil-in-water (W/O/W) multiple emulsion method. The vaccine entrapment and the in vitro release efficiency of the CHBMS was evaluated for a period of 90 days. The release of antigens from the microspheres was determined by ELISA. Antigen integrity was investigated by SDS-PAGE. From the optimization studies, it was found that a chitosan/HPMC ratio of 8/2 produced a good yield, with microspheres that were spherical, regular and uniformly-sized. In the CHBMS, a heparin concentration of 3% w/v resulted in well-sustained antigen delivery for a period of 90 days. It was found that the characteristics of initial release could be observed in 2 days, followed by a constant release, and an almost 100% complete release in 90 days. From the in vitro release characteristics, the ideal batch of CHBMS (3% w/v heparin) was evaluated for in vivo studies by the antibody induction method. The antibody levels were measured for different combinations for the period of 9 months, and finally, with a second booster dose after 1 year. In conclusion, it was observed that CHBMS (combination-1) resulted in the antibody level of 4.5 IU/mL of guinea pig serum, and the level was 3.5 IU/mL for the Central Research Institute's alum-adsorbed tetanus toxoid (CRITT) (combination 2), after 1 year, with a second booster dose. This novel approach of using CHBMS may have potential advantages for single-step immunization with vaccines.