title: Optimization of tetanus toxoid ammonium sulfate precipitation process using response surface methodology.
abstract: Tetanus toxoid (TTd) is a highly immunogenic, detoxified form of tetanus toxin, a causative agent of tetanus disease, produced by Clostridium tetani. Since tetanus disease cannot be eradicated but is easily prevented by vaccination, the need for the tetanus vaccine is permanent. The aim of this work was to investigate the possibility of optimizing TTd purification, i.e., ammonium sulfate precipitation process. The influence of the percentage of ammonium sulfate, starting amount of TTd, buffer type, pH, temperature, and starting purity of TTd on the purification process were investigated using optimal design for response surface models. Responses measured for evaluation of the ammonium sulfate precipitation process were TTd amount (Lf/mL) and total protein content. These two parameters were used to calculate purity (Lf/mgPN) and the yield of the process. Results indicate that citrate buffer, lower temperature, and lower starting amount of TTd result in higher purities of precipitates. Gel electrophoresis combined with matrix-assisted laser desorption ionization-mass spectrometric analysis of precipitates revealed that there are no inter-protein cross-links and that all contaminating proteins have pIs similar to TTd, so this is most probably the reason for the limited success of purification by precipitation.
authors.affiliation: a Centre for Research and Knowledge Transfer , University of Zagreb , Zagreb , Croatia.
authors.forename: Marija
authors.initials: M
authors.lastname: Brgles
authors.affiliation: b Faculty of Mechanical Engineering and Naval Architecture , University of Zagreb , Zagreb , Croatia.
authors.forename: Pero
authors.initials: P
authors.lastname: Prebeg
authors.affiliation: a Centre for Research and Knowledge Transfer , University of Zagreb , Zagreb , Croatia.
authors.forename: Tihana
authors.initials: T
authors.lastname: Kurtović
authors.affiliation: c Bacterial Vaccine Department, Institute of Immunology , Zagreb , Croatia.
authors.forename: Jelena
authors.initials: J
authors.lastname: Ranić
authors.affiliation: d Institute of Chemical Technologies and Analytics , Technische Universität Wien , Vienna , Austria.
authors.forename: Martina
authors.initials: M
authors.lastname: Marchetti-Deschmann
authors.affiliation: d Institute of Chemical Technologies and Analytics , Technische Universität Wien , Vienna , Austria.
authors.forename: Günter
authors.initials: G
authors.lastname: Allmaier
authors.affiliation: a Centre for Research and Knowledge Transfer , University of Zagreb , Zagreb , Croatia.
authors.forename: Beata
authors.initials: B
authors.lastname: Halassy
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D013745:Tetanus Toxoid / Q000096:biosynthesis / Q000737:chemistry*
chemical_list: D013745:Tetanus Toxoid; D000645:Ammonium Sulfate
keywords: Ammonium sulfate precipitation; design of experiments; mass spectrometry; radial immunodiffusion; tetanus toxoid; tetanus vaccine