With the aim to follow large scale production of recombinant E rns from Classical Swine Fever Virus, a monoclonal specific antibody was obtained and optimized it downstream lab scale purification. The anti-E rns gamma globulins were purified through Protein A and Protein G affinity chromatography from ascites fluid. Three binding factors were assessed (pH, flow rate and buffer) to define dynamic binding capacity. The best binding condition to Protein A (6.6 mg/mL of matrix) was in phosphate buffer, pH 8.0 and linear flow rate of 78cm/h with a purity of 90 %. For Protein G, the best arrangement of factors was phosphate buffer, pH 7.0 and linear flow 178cm/h with 6.45mg/mL of adsorbed antibody with a purity of 95 %. The nProtein-A Sepharose matrix allowed 1.21 times more recovery than the nProtein-G Sepharose matrix. However, the elutions obtained with the G-protein were more pure.

Introducción: los carbohidratos son las biomoléculas más abundantes en la naturaleza. Su importancia va desde aspectos nutricionales hasta aplicaciones industriales. Entre la inmensa variedad de carbohidratos se encuentran los fructooligosacáridos considerados prebióticos; los azúcares invertidos, de importancia industrial por su alto poder edulcorante; las dextranas, con diversas aplicaciones, pero cuya presencia en jugos de caña provoca afectaciones en la producción azucarera. Las investigaciones y aplicaciones relacionadas con carbohidratos requieren la obtención de datos fiables sobre las cinéticas de síntesis y degradación de los mismos. Uno de los métodos de cuantificación de los carbohidratos es mediante un equipo de cromatografía líquida de alta resolución (HPLC) acoplado a un detector de índice de refracción (IR). Objetivo: calificar un equipo de HPLC acoplado a un detector de índice de refracción (IR) para la validación del método de cuantificación de carbohidratos. Metodología: la calificación del HPLC-IR se realizó según la Guía para la calificación del equipamiento - Anexo 1 de 2011. Se evaluó la exactitud del flujo de la bomba, la precisión y linealidad del inyector manual, la exactitud y estabilidad de la temperatura del horno, así como la linealidad y relación señal/ruido en el detector IR. Resultados: este trabajo demostró que el sistema HPLC-IR se desempeña de manera consistente de acuerdo con las especificaciones del fabricante, de esta forma se aseguró el rendimiento correcto del equipo en condiciones reales de funcionamiento. Conclusión: la calificación satisfactoria del instrumento analítico permitió la validación del método de cuantificación de carbohidratos mediante esta tecnología.

To manufacturing the HCV UMELISA ® Test kit, a recombinant NS3 protein is used as part of the antigen mixture. This protein is obtained from Escherichia coli XL-1Blue transformed with pR2M6-NS3-His plasmid downstream trp promoter. Regulation of expression has been identified as the main cause that affects yield and reproducibility at the fermentation process. To improve protein expression and transcription regulation, cloning of NS3 sequence was performed downstream tac promoter. At the same time, three E. coli strains (XL 1-Blue, W3110 and C600) were used to assess the expression of the NS3 protein. The tac promoter improved the expression level at all the strains evaluated from 20% under trp until 30 % and a reduction of promoter leakage before induction was observed compared with trp promoter. Due to the insensibility to the high metabolic burden, C600 was settled as the strain to use for NS3 expression. The new construction was well recognized by a pool of positive sera and individual sera from infected humans in an indirect ELISA using the HCV UMELISA ® Test kit.

The sterilization process in biotechnological industry ensures safety on materials used during drugs and biological reagents production. The most frequently used technique is sterilization by means of pressurized steam. This paper describes the HG-80 autoclave qualification and tests for steam sterilization cycle performance qualification in order to demonstrate reliability and verify compliance with sanitary regulations. In a first step, it was evaluated whether the autoclave was able to control and maintain the programmed operating temperature, by determining the temperature distribution and behavior along the chamber. In the second stage, sterilization cycles were performed in triplicate of two loading patterns where the influence of the materials on the temperature profile was studied. Simultaneously, the biological indicators challenge was executed, which play an important role in the evaluation of the process effectiveness together with the F0 concept. The F values obtained at each studied point indicate the process provides sufficient lethality to eliminate the potentially resistant forms of microbial life present in the loading pattern materials. The results obtained show that the evaluated sterilization processes generated consistent and reproducible results according to the established specifications; therefore, the validated process is considered as follows.