Aplicación del análisis de riesgo a la producción de proteínas recombinantes expresadas en Escherichia coli / The risk analysis used to manufacture recombinant proteins in E coli

En este trabajo se aplicó el análisis de riesgo, empleando la metodología de análisis de modos y efectos de fallas a los procesos de fermentación que utilizan la bacteria Esherichia coli como hospedero, para obtener proteínas recombinantes con fines terapéuticos, vacunales o diagnósticos. Se realizó el análisis del tipo y probabilidad de ocurrencia de las fallas en el proceso fermentativo, la evaluación del impacto en la calidad del mismo y la probabilidad de detección de dichas fallas. Se evaluó la severidad, probabilidad de ocurrencia y probabilidad de detección y se calculó el número de probabilidades de riesgo. Además, se emplearon técnicas utilizadas para el aseguramiento de la calidad como: tormenta de ideas y diagrama causa-efecto. Se concluye que las causas potenciales que tienen mayor influencia en las fallas de un proceso fermentativo de E coli recombinante son: la inadecuada manipulación durante la inoculación, la presencia de fagos y el personal no calificado. Se proponen acciones a tomar para minimizar el riesgo(AU)

In this paper a risk analysis management is applied using the Failure Mode Effects Analysis to the fermentation processes that use E coli as a host, to produce recombinant proteins with therapeutic, vaccinate or diagnostic aims. The analysis of the type and probability of occurrence of failures in the fermentation process, the evaluation of the impact in the quality of the product and the probability of detection of these failures are carried out. The severity, occurrence probability and detection probability are evaluated and the risk priority number is calculated. Techniques used in Quality assurance as brainstorming and Ishikawa diagram were used. The potential causes that have higher influence in the failures of a fermentation process of recombinant E coli are: inadequate handling during inoculation, presence of phages and unqualified personnel. Actions to minimize the risks are proposed(AU)

Application of a risk analysis method to different technologies for producing a monoclonal antibody employed in hepatitis B vaccine manufacturing.

CB.Hep-1 monoclonal antibody (mAb) is used for a recombinant Hepatitis B vaccine manufacturing, which is included in a worldwide vaccination program against Hepatitis B disease. The use of this mAb as immunoligand has been addressed into one of the most efficient steps of active pharmaceutical ingredient purification process. Regarding this, Quality Risk Management (QRM) provides an excellent framework for the risk management use in pharmaceutical manufacturing and quality decision-making applications. Consequently, this study sought applying a prospective risk analysis methodology Failure Mode Effects Analysis (FMEA) as QRM tool for analyzing different CB.Hep-1 mAb manufacturing technologies. As main conclusions FMEA was successfully used to assess risks associated with potential problems in CB.Hep-1 mAb manufacturing processes. The severity and occurrence of risks analysis evidenced that the percentage of very high severe risks ranged 31.0-38.7% of all risks and the huge majority of risks have a very low occurrence level (61.9-83.3%) in all assessed technologies. Finally, additive Risk Priority Number, was descending ordered as follow: transgenic plants (2636), ascites (2577), transgenic animals (2046) and hollow fiber bioreactors (1654), which also corroborated that in vitro technology, should be the technology of choice for CB.Hep-1 mAb manufacturing in terms of risks and mAb molecule quality.

Comparison of different ligand densities in immunoaffinity chromatography of the plantibody HB-01 coupled to Sepharose CL-4B to purify the rHBsAg.

This paper evaluates the immunopurification behavior of a plantibody HBsAg specific plantibody coupled to Sepharose CL-4B at different ligand densities. Results show no significant differences in the adsorption and elution capacities, and rHBsAg recovery of immunosorbents at 3.43, 4.45, and 5.31 mg/mL of ligand densities compared to its mouse-derived mAb counterpart consistently used in the rHBsAg purification process. Therefore, plantibody ligand densities higher than 3.43 mg/mL do not improve the immunopurification behavior of this immunosorbent, but increase the antibody consumption and the Hepatitis B vaccine cost. Immunosorbent of 2.23 mg/mL of ligand density demonstrated a poor performance. The IgG leached detectable level never exceeded the approved limit (3 ng IgG/microg rHBsAg). Values close to this limit were only observed at the ligand density of 5.31 and 2.27 mg/mL. In the case of the ligand density of 2.23 mg/mL the IgG leached value was high (2.90 ng IgG/microg rHBsAg) due to a low level of eluted antigen. In conclusion, it supports feasibility of using this plantibody at 3.43 mg/mL of ligand density for large-scale immunopurification of rHBsAg for human use, avoiding the biosafety and ethical concerns of the massive use of animals for this purpose.

An integral approach towards a practical application for a plant-made monoclonal antibody in vaccine purification.

The use of transgenic plants for the production of pharmaceutical compounds has received increasing attention in the last few years. However, many technological and regulatory issues regarding the practical exploitation of this alternative system of production remain to be solved; a situation that explains the lack of commercial products derived from such a system. This paper reports the expression in transgenic plants and cells of a single-chain antibody variable-region fragment (scFv) and a mouse monoclonal antibody to the hepatitis B virus surface antigen (HBsAg). The large-scale purification of the scFv from plants and its use for immunopurification of HBsAg are also described, together with elements concerning regulatory issues and technologies for compliance with good manufacturing and agricultural practices.

Large-scale purification of an antibody directed against hepatitis B surface antigen from transgenic tobacco plants.

The application of bioengineering to plants for production of biological products for human and animal use has expanded in recent years. The reasons for this expansion are several and include advances in the technology for novel production systems and the need for very large quantities of therapeutic proteins. The process of growing pharmaceutical proteins in plants, extracting, and purifying is a hard task considering the lack of available information concerning these topics. In this work, a recombinant murine monoclonal antibody specific for the hepatitis B surface antigen, expressed in stably transformed transgenic Nicotiana tabacum plants, was purified by means of a recombinant protein A Streamline chromatography as the main purification step. The antibody expression level varied with the age of the plants and the number of harvests from 40 to 15microg/ml and the maximum process yield was about 25mg of plantibody/kg of biomass. Protein A Streamline chromatography was successfully used in the purification process yielding a recovery of about 60% and a plantibody SDS-PAGE purity of over 90% but unexpectedly, previous clarification steps could not be totally avoided. The amino acid sequence recognized by this affinity purified plantibody was similar to its murine counterpart verifying the potentiality of plants to replace animals or bioreactors for large-scale production of this monoclonal antibody.

Niveles de resistencia a insecticidas y sus mecanismos en una cepa de Aedes aegypti de Santiago de Cuba / Resistance levels to insecticides and their mechanisms in a stump of Aedes aegypti of Santiago from Cuba

Producto del más reciente brote de dengue en el municipio Santiago de Cuba, se estudió una cepa de este vector para determinar sus niveles de susceptibilidad y/o resistencia a insecticidas organofosforados y piretroides. Los resultados de los bioensayos mostraron bajos niveles de resistencia a fentión, malatión y deltametrina, se obtuvieron moderados para temefos, metil-pirimifos y cipermetrina, y altos para clorpirifos. Según los resultados obtenidos, en el uso del sinergista S,S,S tributil fosfotritiado, se demostró que las enzimas esterasas juegan una función importante en la resistencia a temefos y clorpirifos. Utilizando el sinergista piperonil butóxido se demostró que las enzimas oxidasas de función múltiple no intervienen en la resistencia a ninguno de los insecticidas evaluados. Se realizaron las técnicas bioquímicas para la detección de los mecanismos de resistencia mediado por enzimas esterasas, glutatión-s-transferasa (GST) y acetilcolinesterasas, (AchE) en Aedes aegypti y quedó demostrado, de acuerdo con los altos valores de frecuencia observados para cada uno de los mecanismos, que las enzimas esterasas y GST intervienen en la resistencia a insecticidas, no resultando así para la AchE. Sin embargo, por primera vez en Aedes aegypti, se encontró la presencia del gen de la AchE, aunque a baja frecuencia. Mediante electroforesis en gel de poliacrilamida se observó una banda fuertemente teñida con un valor de movilidad relativa de 0,779; la cual se nombró A4, que no se observó en la cepa de referencia, y que pudiera estar asociada con la resistencia a organofosforados, hecho que queda por demostrar en futuros trabajos(AU)

Niveles de resistencia a insecticidas y sus mecanismos en una cepa de Aedes aegypti de Santiago de Cuba / Levels of insecticide resistance and its mechanisms in a strain of Aedes aegypti of Santiago de Cuba

As a result of the most recent dengue outbreak in Santiago de Cuba province, a strain of this vector was studied to determine the levels of sensitivity and/or resistance to organophosphate and pyrethoid insecticides. The results of bioassays showed low levels of resistance to fention, malathion and deltametrine, moderate levels of resistance to temephos, metyl-pirimifos and cipermetrine and high levels of resistance to chlorpirifios. According to the results obtained from the use of S.S.S. phosphotrithiate trybutil synergist, it was shown that esterases play an important role in resistance to temephos and chlorpirifos. Piperonyl butoxide synergist disclosed that multifunction oxidases were not involved in the resistance to any of the evaluated insecticides. Biochemical techniques were applied to detect esterase-, glutathione-S-transferase- and acetylcholineaterase-mediated resistance mechanisms of Aedes aegypti. In accordance with the high frequency values observed in each of the mechanisms, it was proved that esterases and glutathione-S-transferase were involved in the insecticide resistance but acetylcholinesterases were not. However, acetylcholinesterase gen was found in Aedes aegypti for the first time though at low frequency. The polyacrylamide-gel electrophoresis made it possible to observe a well-stained band with a relative mobility value of 0.779; this band was called A4 it was not observed in the reference strain and may be associated to organophosphate resistance which remains to be proved in future research.