ABSTRACT
Se evaluó el uso de la tecnología de Flujo de Filtración Tan gencial (FFT), para obtener la toxina tetánica a partir de cultivos de la bacteria Clostridium tetani, usando el proceso de Micro filtración (MF), para eliminar el paquete celular y posteriormente, a partir del filtrado obtenido, concentrar y diafiltrar la Toxina Tetánica usando el proceso de Ultrafiltración (UF). Se determinaron las características de los filtros, condiciones de trabajo y el dimensionamiento de los equipos a adquirir para la nueva producción industrial de Toxina Tetánica. Se evaluaron el flujo, tiempo, rendimiento del proceso y las características del producto obtenido. Utilizando cultivos con Toxina Tetánica en un equipo de filtración de laboratorio, diseñado para producir el efecto de FFT. Se seleccionó las membranas tipo cassettes, formato Suspended Screen, porosidad 0,2μm, como las adecuadas para el proceso de MF, ya que mostraron un 100% de transmisión de la Toxina Tetánica, ausencia de restos celulares y flujo promedio de filtrado de 73.30 L/m2h. Así mismo, se seleccionaron las membranas tipo cassettes, formato Omega, porosidad 50 y 70 kDa, como las adecuadas para el proceso de UF, ya que mostraron 100% de recuperación de la toxina, ausencia de toxina en el filtrado y adecuados flujos de filtrado (106,7 y 104,4 L/m2h, respectivamente). Estos resultados permitieron dimensionar, considerando las variables a utilizar en la producción industrial (Volumen 650 a 950 litros, tiempo de procesos, 3 horas), el área de filtración de los equipos de MF y UF a adquirir, estimados en 20m2 y 5m2, respectivamente.
Tangential Flow Filtration (TFF) technology was evaluated to process tetanus toxin which is produced by Clostridium tetani bacterium. Microfiltration (MF) is used to retain cells while allowing passage of the toxin to the filtrate stream. The filtrate is co - llected and further processed by Ultrafiltration (UF) to concentrate the toxin and to maximize the wash of small species by a Dia filtration step. Both, MF and UF processes were evaluated to specify the filters and corresponding critical process parameters to scale-up the application. As part of the evaluation, flow ra te, processing time, yield and product attributes were characterized. The cell harvest containing the tetanus toxin was processed using a laboratory scale TFF system designed to product the TFF effect. The evaluation demonstrated that a cassette in sus pended screen format and membrane with 0.2μm pore is the right selection for the MF step. It showed 100% of toxin transmission without the presence of cellular debris and average process flux of 73.30 L/m2h. The UF step was conducted using the same system with cassettes in me dium screen format with pores of 50 and 70kDa. It showed 100% retention of the toxin with a process flux of 106,7 and 104,4L/m2h, respectively. To maximise product retention during UF, the 50 kDa membrane was selected. These results were used to scale-up the application to process the industrial volume of 650 a 950 liters in 3 hours of processing time. Membrane area sizing of MF and UF to be acquired is estimated in 20m2 and 5m2, respectively.
Subject(s)
Humans , Male , Female , Tetanus Toxin/analysis , Bacterial Infections/complications , Ultrafiltration/instrumentation , Proteins/metabolism , Cell Separation/methods , Microstraining , Public HealthABSTRACT
Cell-free extracts from 20 strains of Clostridium tetani isolated from soil samples, were tested for tetanus toxin production using an enzyme immunoassay. All the extracts were classified as positive for the toxin presence, and eight of them showed absorbance values corresponding to tetanus toxin concentrations between 3.2 and 88 ng/ml; thus, they fell within the linear absorbance range (0.135-0.317). All dilutions of toxin used to obtain the calibration curve (0.0071 to 1.1 ng) were lethal for mice.
Subject(s)
Animals , Mice , Rabbits , Antibodies, Bacterial/blood , Antigens, Bacterial/blood , Clostridium tetani/chemistry , Tetanus Toxin/analysis , Immunoenzyme Techniques/methods , Biological Assay , Soil Microbiology , Disease Models, Animal , Sheep , False Negative Reactions , Tetanus Toxin/biosynthesis , Tetanus Toxin/toxicity , Tetanus/etiology , Tetanus/prevention & controlABSTRACT
A sandwich ELISA for quantitation of tetanus toxin was devised using commercially available horse antitetanus antiserum and its HRP-conjugate. With commercial toxoid as a standard the assay had a lower sensitivity of 100 ng/ml and was linear up to 100 micrograms/ml. In terms of Lf equivalents the ELISA assay was linear from 0.02 to 20 Lf/ml. The Lf activity in 20 preparations of tetanus toxin as determined by ELISA was comparable to the activity measured by conventional flocculation tests (r = 0.987).