Crustacean Proteases and Their Application in Debridement.

Digestive proteases from marine organisms have been poorly applied to biomedicine. Exceptions are trypsin and other digestive proteases from a few cold-adapted or temperate fish and crustacean species. These enzymes are more efficient than enzymes from microorganism and higher vertebrates that have been used traditionally. However, the biomedical potential of digestive proteases from warm environment species has received less research attention. This review aims to provide an overview of this unrealised biomedical potential, using the debridement application as a paradigm. Debridement is intended to remove nonviable, necrotic and contaminated tissue, as well as fibrin clots, and is a key step in wound treatment. We discuss the physiological role of enzymes in wound healing, the use of exogenous enzymes in debridement, and the limitations of cold-adapted enzymes such as their poor thermal stability. We show that digestive proteases from tropical crustaceans may have advantages over their cold-adapted counterparts for this and similar uses. Differences in thermal stability, auto-proteolytic stability, and susceptibility to proteinase inhibitors are discussed. Furthermore, it is proposed that the feeding behaviour of the source organism may direct the evaluation of enzymes for particular applications, as digestive proteases have evolved to fill a wide variety of feeding habitats, natural substrates, and environmental conditions. We encourage more research on the biomedical application of digestive enzymes from tropical marine crustaceans.

The clotting system in decapod crustaceans: History, current knowledge and what we need to know beyond the models.

Hemolymph coagulation is among the major arms of the humoral immune response in crustaceans. According to the current model, hemolymph clotting in decapod crustacean relies mostly on the polymerization of the plasmatic clotting protein (CP) which is directly promoted by calcium-depended transglutaminase (TGase) released from hemocytes upon microbial stimulus or injury. However, the type of hemocytes containing TGase, and hence how the TGase is released, might vary among species. Thus, we discourse here about possible mechanisms for clotting initiation. On the other hand, the initiation of coagulation reaction in the absence of microbial elicitors is poorly understood and seems to involve hemocytes lability, yet the mechanism remains unknown. A cellular clottable protein called coagulogen, different to the plasma CP, occurs in several species and could be related with the immune response, but the biological relevance of this protein is unknown. It is also demonstrated that the clotting response is actively involved in defense against pathogens. In addition, both TGase and the CP show pleiotropic functions, and although both proteins are relatively conserved, some of their physic-chemical properties vary significantly. The occurrence of differences in the clotting system in crustaceans is conceivable given the high number of species and their diverse ecology. Results from still non-studied decapods may provide explanation for some of the issues presented here from an evolutionary perspective.

Carbohydrates digestion and metabolism in the spiny lobster (Panulirus argus): biochemical indication for limited carbohydrate utilization.

As other spiny lobsters, Panulirus argus is supposed to use preferentially proteins and lipids in energy metabolism, while carbohydrates are well digested but poorly utilized. The aim of this study was to evaluate the effect of dietary carbohydrate level on digestion and metabolism in the spiny lobster P. argus. We used complementary methodologies such as post-feeding flux of nutrients and metabolites, as well as measurements of α-amylase expression and activity in the digestive tract. Lobsters readily digested and absorbed carbohydrates with a time-course that is dependent on their content in diet. Lobster showed higher levels of free glucose and stored glycogen in different tissues as the inclusion of wheat flour increased. Modifications in intermediary metabolism revealed a decrease in amino acids catabolism coupled with a higher use of free glucose as carbohydrates rise up to 20%. However, this effect seems to be limited by the metabolic capacity of lobsters to use more than 20% of carbohydrates in diets. Lobsters were not able to tightly regulate α-amylase expression according to dietary carbohydrate level but exhibited a marked difference in secretion of this enzyme into the gut. Results are discussed to highlight the limitations to increasing carbohydrate utilization by lobsters. Further growout trials are needed to link the presented metabolic profiles with phenotypic outcomes.

Panusin represents a new family of ß-defensin-like peptides in invertebrates.

Beta_defensin have been solely found in vertebrates until ß-defensin-like peptides were described as transcript isoforms in two species of Panulirus genus. They were considered as putative antimicrobials since their biological activity have not been demonstrated. Here we purified and characterized a defensin-like peptide from the hemocytes of spiny lobster P. argus, hereafter named panusin. Structurally, panusin presents a cysteine-stabilized α/ß motif, and is prone to form homodimers. Biological activity of panusin showed broad-spectrum antimicrobial activity, characterized for being strikingly salt-resistant. Panusin did not showed hemolytic activity but was demonstrated its binding capacity to different lipid membrane models, indicating amphipathicity of ß-sheet core as driving force for its antimicrobial activity. Panusin is considered a new kind of arthropod defensin which share structural and biological features with beta-defensin from vertebrates. The presence of beta-defensin like peptides in crustacean might suggest the emergence of the evolutionary relationship of ß-defensins from vertebrates.

Molecular, Biochemical, and Dietary Regulation Features of α-Amylase in a Carnivorous Crustacean, the Spiny Lobster Panulirus argus.

Alpha-amylases are ubiquitously distributed throughout microbials, plants and animals. It is widely accepted that omnivorous crustaceans have higher α-amylase activity and number of isoforms than carnivorous, but contradictory results have been obtained in some species, and carnivorous crustaceans have been less studied. In addition, the physiological meaning of α-amylase polymorphism in crustaceans is not well understood. In this work we studied α-amylase in a carnivorous lobster at the gene, transcript, and protein levels. It was showed that α-amylase isoenzyme composition (i.e., phenotype) in lobster determines carbohydrate digestion efficiency. Most frequent α-amylase phenotype has the lowest digestion efficiency, suggesting this is a favoured trait. We revealed that gene and intron loss have occurred in lobster α-amylase, thus lobsters express a single 1830 bp cDNA encoding a highly conserved protein with 513 amino acids. This protein gives rise to two isoenzymes in some individuals by glycosylation but not by limited proteolysis. Only the glycosylated isoenzyme could be purified by chromatography, with biochemical features similar to other animal amylases. High carbohydrate content in diet down-regulates α-amylase gene expression in lobster. However, high α-amylase activity occurs in lobster gastric juice irrespective of diet and was proposed to function as an early sensor of the carbohydrate content of diet to regulate further gene expression. We concluded that gene/isoenzyme simplicity, post-translational modifications and low Km, coupled with a tight regulation of gene expression, have arose during evolution of α-amylase in the carnivorous lobster to control excessive carbohydrate digestion in the presence of an active α-amylase.

Soluble ß-(1,3)-glucans enhance LPS-induced response in the monocyte activation test, but inhibit LPS-mediated febrile response in rabbits: Implications for pyrogenicity tests.

In the present study, we aimed to determine the influence of ß-(1,3)-d-glucans on the LPS-induced pro-inflammatory cytokine response in the Monocyte Activation Test (MAT) for pyrogens, and on the LPS-induced febrile response in the Rabbit Pyrogen Test (RPT), thus evaluating the resulting effect in the outcome of each test. It was found that ß-(1,3)-d-glucans elicited the production of pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α, also known as endogenous pyrogens, but not enough to classify them as pyrogenic according to MAT. The same ß-(1,3)-d-glucans samples were non-pyrogenic by RPT. However, ß-(1,3)-d-glucans significantly enhanced the LPS-induced pro-inflammatory cytokines response in MAT, insomuch that samples containing non-pyrogenic concentrations of LPS become pyrogenic. On the other hand, ß-(1,3)-d-glucans had no effect on sub-pyrogenic LPS doses in the RPT, but surprisingly, inhibited the LPS-induced febrile response of pyrogenic LPS concentrations. Thus, while ß-(1,3)-d-glucans could mask the LPS pyrogenic activity in the RPT, they exerted an overstimulation of pro-inflammatory cytokines in the MAT. Hence, MAT provides higher safety since it evidences an unwanted biological response, which is not completely controlled and is overlooked by the RPT.

Trypsin isozymes in the lobster Panulirus argus (Latreille, 1804): from molecules to physiology.

Trypsin enzymes have been studied in a wide variety of animal taxa due to their central role in protein digestion as well as in other important physiological and biotechnological processes. Crustacean trypsins exhibit a high number of isoforms. However, while differences in properties of isoenzymes are known to play important roles in regulating different physiological processes, there is little information on this aspect for decapod trypsins. The aim of this review is to integrate recent findings at the molecular level on trypsin enzymes of the spiny lobster Panulirus argus, into higher levels of organization (biochemical, organism) and to interpret those findings in relation to the feeding ecology of these crustaceans. Trypsin in lobster is a polymorphic enzyme, showing isoforms that differ in their biochemical features and catalytic efficiencies. Molecular studies suggest that polymorphism in lobster trypsins may be non-neutral. Trypsin isoenzymes are differentially regulated by dietary proteins, and it seems that some isoenzymes have undergone adaptive evolution coupled with a divergence in expression rate to increase fitness. This review highlights important but poorly studied issues in crustaceans in general, such as the relation among trypsin polymorphism, phenotypic (digestive) flexibility, digestion efficiency, and feeding ecology.

A holistic view of dietary carbohydrate utilization in lobster: digestion, postprandial nutrient flux, and metabolism.

Crustaceans exhibit a remarkable variation in their feeding habits and food type, but most knowledge on carbohydrate digestion and utilization in this group has come from research on few species. The aim of this study was to make an integrative analysis of dietary carbohydrate utilization in the spiny lobster Panulirus argus. We used complementary methodologies such as different assessments of digestibility, activity measurements of digestive and metabolic enzymes, and post-feeding flux of nutrients and metabolites. Several carbohydrates were well digested by the lobster, but maize starch was less digestible than all other starches studied, and its inclusion in diet affected protein digestibility. Most intense hydrolysis of carbohydrates in the gastric chamber of lobster occurred between 2-6 h after ingestion and afterwards free glucose increased in hemolymph. The inclusion of wheat in diet produced a slow clearance of glucose from the gastric fluid and a gradual increase in hemolymph glucose. More intense hydrolysis of protein in the gastric chamber occurred 6-12 h after ingestion and then amino acids tended to increase in hemolymph. Triglyceride concentration in hemolymph rose earlier in wheat-fed lobsters than in lobsters fed other carbohydrates, but it decreased the most 24 h later. Analyses of metabolite levels and activities of different metabolic enzymes revealed that intermolt lobsters had a low capacity to store and use glycogen, although it was slightly higher in wheat-fed lobsters. Lobsters fed maize and rice diets increased amino acid catabolism, while wheat-fed lobsters exhibited higher utilization of fatty acids. Multivariate analysis confirmed that the type of carbohydrate ingested had a profound effect on overall metabolism. Although we found no evidence of a protein-sparing effect of dietary carbohydrate, differences in the kinetics of their digestion and absorption impacted lobster metabolism determining the fate of other nutrients.

The trypsin inhibitor panulirin regulates the prophenoloxidase-activating system in the spiny lobster Panulirus argus.

The melanization reaction promoted by the prophenoloxidase-activating system is an essential defense response in invertebrates subjected to regulatory mechanisms that are still not fully understood. We report here the finding and characterization of a novel trypsin inhibitor, named panulirin, isolated from the hemocytes of the spiny lobster Panulirus argus with regulatory functions on the melanization cascade. Panulirin is a cationic peptide (pI 9.5) composed of 48 amino acid residues (5.3 kDa), with six cysteine residues forming disulfide bridges. Its primary sequence was determined by combining Edman degradation/N-terminal sequencing and electrospray ionization-MS/MS spectrometry. The low amino acid sequence similarity with known proteins indicates that it represents a new family of peptidase inhibitors. Panulirin is a competitive and reversible tight-binding inhibitor of trypsin (Ki = 8.6 nm) with a notable specificity because it does not inhibit serine peptidases such as subtilisin, elastase, chymotrypsin, thrombin, and plasmin. The removal of panulirin from the lobster hemocyte lysate leads to an increase in phenoloxidase response to LPS. Likewise, the addition of increasing concentrations of panulirin to a lobster hemocyte lysate, previously depleted of trypsin-inhibitory activity, decreased the phenoloxidase response to LPS in a concentration-dependent fashion. These results indicate that panulirin is implicated in the regulation of the melanization cascade in P. argus by inhibiting peptidase(s) in the pathway toward the activation of the prophenoloxidase enzyme.

Lobster (Panulirus argus) hepatopancreatic trypsin isoforms and their digestion efficiency.

It is well known that crustaceans exhibit several isoforms of trypsin in their digestive system. Although the number of known crustacean trypsin isoforms continues increasing, especially those derived from cDNA sequences, the role of particular isoenzymes in digestion remains unknown. Among invertebrates, significant advances in the understanding of the role of multiple trypsins have been made only in insects. Since it has been demonstrated that trypsin isoenzyme patterns (phenotypes) in lobster differ in digestion efficiency, we used this crustacean as a model for assessing the biochemical basis of such differences. We demonstrated that the trypsin isoform known to be present in all individuals of Panulirus argus has a high catalytic efficiency (k(cat)/K(m) ) and is the most reactive toward native proteinaceous substrates, whereas one of the isoforms present in less efficient individuals has a lower k(cat) and a lower k(cat)/K(m), and it is less competent at digesting native proteins. A fundamental question in biology is how genetic differences produce different physiological performances. This work is the first to demonstrate that trypsin phenotypic variation in crustacean protein digestion relies on the biochemical properties of the different isoforms. Results are relevant for understanding trypsin polymorphism and protein digestion in lobster.

Las B-glucanas: moléculas inmunomoduladoras contaminantes de productos farmacéuticos / B-glucans as immunomodulating moléculas polluting pharmaceuticals

Se realizó una búsqueda bibliográfica utilizando la base de datos Pubmed con énfasis en los artículos publicados en la última década. Como descriptores se utilizaron los siguientes: glucans, glucans recognition, glucans biological activitiy, glucans pharmaceuticals. Con la información disponible se realizó un análisis de los principales aspectos relacionados con el tema, que se exponen en el presente trabajo. Las b-(1®3)-glucanas son polímeros de glucosa que se encuentran mayoritariamente en la pared celular de hongos, levaduras y plantas. Se consideran patrones moleculares asociados a patógenos y son reconocidas por varios receptores, siendo la dectina-1 el principal receptor de reconocimiento de estas estructuras. Sus propiedades inmunomoduladoras han sido informadas por varios autores. Se ha demostrado que potencian y sinergizan la acción de ligandos de Toll like receptors sobre la liberación de citoquinas proinflamatorias, aunque también han mostrado un perfil antiinflamatorio, cuestión que depende en gran medida de sus características estructurales. Las b-(1®3)-glucanas son contaminantes importantes provenientes de los filtros de acetato de celulosa que se utilizan en la clarificación de parenterales hemoderivados, por tanto, es necesario estudiar las consecuencias de la presencia de estas moléculas inmunomoduladoras en inyectables. En esta revisión se resumen aspectos relacionados con el reconocimiento y actividad biológica de las b-(1®3)-glucanas y se profundiza en estudios relacionados con su presencia en hemoderivados como principal contaminante. Finalmente se destaca la utilidad de la Prueba de Activación de Monocitos en la detección de las b-(1®3)-glucanas en parenterales.

A literature review was made in Pubmed database, making emphasis on papers published in the last decade. The subject headings for this search were glucans, glucans recognition, glucans biological activitiy, glucans pharmaceuticals. On the basis of the available information, the main aspects related to this topic were analyzed and shown in this paper. b-(1®3)-glucans are glucose-derived polymers found mainly in the cellular wall of fungi, yeasts and plants. They are considered pathogens-associated molecular patterns that are ecognized by several receptors, being dectin-1 the key recognition receptor of these structures. Some authors have underlined their Immunomodulating properties. It has been demonstrated that they synergize and potentate the actions of Toll-like receptor ligands on the release of proinflammatory cytokines, though b-(1®3)-glucans have shown an antinflamatory profile which greatly depends on their structural characteristics. b-(1®3)-glucans are important pollutants stemming from cellulose depth filters used in clarification process of parenteral blood derivatives. For this reason, it is necessary to study the consequences of their presence in parenterals. This review summarized the main aspects related with the recognition and biological activities of b-(1®3)-glucans as well as it delved into studies on their presence in blood derivatives as main pollutant. Finally, the paper underlined the role of Monnocyte Activation Test to detect b-(1®3)-glucans in parenterals.

Las β-(1®3)-glucanas: moléculas inmunomoduladoras contaminantes de productos farmacéuticos / β-(1®3)-glucans as immunomodulating moléculas polluting pharmaceuticals

Se realizó una búsqueda bibliográfica utilizando la base de datos Pubmed con énfasis en los artículos publicados en la última década. Como descriptores se utilizaron los siguientes: glucans, glucans recognition, glucans biological activitiy, glucans pharmaceuticals. Con la información disponible se realizó un análisis de los principales aspectos relacionados con el tema, que se exponen en el presente trabajo. Las b-(1®3)-glucanas son polímeros de glucosa que se encuentran mayoritariamente en la pared celular de hongos, levaduras y plantas. Se consideran patrones moleculares asociados a patógenos y son reconocidas por varios receptores, siendo la dectina-1 el principal receptor de reconocimiento de estas estructuras. Sus propiedades inmunomoduladoras han sido informadas por varios autores. Se ha demostrado que potencian y sinergizan la acción de ligandos de Toll like receptors sobre la liberación de citoquinas proinflamatorias, aunque también han mostrado un perfil antiinflamatorio, cuestión que depende en gran medida de sus características estructurales. Las b-(1®3)-glucanas son contaminantes importantes provenientes de los filtros de acetato de celulosa que se utilizan en la clarificación de parenterales hemoderivados, por tanto, es necesario estudiar las consecuencias de la presencia de estas moléculas inmunomoduladoras en inyectables. En esta revisión se resumen aspectos relacionados con el reconocimiento y actividad biológica de las b-(1®3)-glucanas y se profundiza en estudios relacionados con su presencia en hemoderivados como principal contaminante. Finalmente se destaca la utilidad de la Prueba de Activación de Monocitos en la detección de las b-(1®3)-glucanas en parenterales.(AU)

A literature review was made in Pubmed database, making emphasis on papers published in the last decade. The subject headings for this search were glucans, glucans recognition, glucans biological activitiy, glucans pharmaceuticals. On the basis of the available information, the main aspects related to this topic were analyzed and shown in this paper. b-(1®3)-glucans are glucose-derived polymers found mainly in the cellular wall of fungi, yeasts and plants. They are considered pathogens-associated molecular patterns that are ecognized by several receptors, being dectin-1 the key recognition receptor of these structures. Some authors have underlined their Immunomodulating properties. It has been demonstrated that they synergize and potentate the actions of Toll-like receptor ligands on the release of proinflammatory cytokines, though b-(1®3)-glucans have shown an antinflamatory profile which greatly depends on their structural characteristics. b-(1®3)-glucans are important pollutants stemming from cellulose depth filters used in clarification process of parenteral blood derivatives. For this reason, it is necessary to study the consequences of their presence in parenterals. This review summarized the main aspects related with the recognition and biological activities of b-(1®3)-glucans as well as it delved into studies on their presence in blood derivatives as main pollutant. Finally, the paper underlined the role of Monnocyte Activation Test to detect b-(1®3)-glucans in parenterals.(AU)

Monocyte activation test (MAT) reliably detects pyrogens in parenteral formulations of human serum albumin.

Disadvantages of the regulatory pyrogen test to assure safety of the end-product Human Serum Albumin (HSA) for parenteral use call for the implementation of an alternative test. In the current study, 16 HSA batches were assayed for pyrogens in parallel with the Rabbit Pyrogen Test, conventional and endotoxin-specific LAL assay and monocyte activation test (MAT). It was found that all HSA batches were contaminated with (1,3)-beta-glucans, which interfere with the conventional LAL. Endotoxin-specific LAL was not suitable to test HSA due to unacceptable endotoxin recovery. Experiments combining polymyxin B and MAT demonstrated that pyrogenic batches were mainly contaminated with endotoxins. However, endotoxin-specific LAL failed to detect one of them. The contaminating (1,3)-beta-glucans enhanced the MAT/IL-6 response to endotoxin, but not that of MAT/IL-1beta. The endotoxin equivalent concentrations obtained using the IL-6 readout were usually higher than those using IL-1beta, probably owing to the direct induction of IL-6 release from monocytes by (1,3)-beta-glucans. The MAT correlates with the rabbit pyrogen test, providing a higher safety level for pyrogenicity testing of HSA and probably other therapeutic proteins.

Hemocyanin-derived phenoloxidase activity in the spiny lobster Panulirus argus (Latreille, 1804).

Hemocyanin and phenoloxidase belong to the type-3 copper protein family, sharing a similar active center whereas performing different roles. In this study, we demonstrated that purified hemocyanin (450 kDa) from the spiny lobster Panulirus argus shows phenoloxidase activity in vitro after treatment with trypsin, chymotrypsin and SDS (0.1% optimal concentration), but it is not activated by sodium perchlorate or isopropanol. The optimal pHs of the SDS-activated hemocyanin were 5.5 and 7.0. Hemocyanin from spiny lobster behaves as a catecholoxidase. Kinetic characterization using dopamine, L-DOPA and catechol shows that dopamine is the most specific substrate. Catechol and dopamine produced substrate inhibition above 16 and 2 mM respectively. Mechanism-based inhibition was also evidenced for the three substrates, being less significant for L-DOPA. SDS-activated phenoloxidase activity is produced by the hexameric hemocyanin. Zymographic analysis demonstrated that incubation of native hemocyanin with trypsin and chymotrypsin, produced bands of 170 and 190 kDa respectively, with intense phenoloxidase activity. Three polypeptide chains of 77, 80 and 89 kDa of hemocyanin monomers were identified by SDS-PAGE. Monomers did not show phenoloxidase activity induced by SDS or partial proteolysis.

Ensayo del lisado de amebocitos del Limulus (LAL) / Limulus amebocyte lysate (LAL) Assay

En los últimos años, los principales organismos reguladores de productos farmacéuticos (Farmacopeas) exigen cada vez más en sus monografías la aplicación del método del lisado de amebocitos de Limulus (LAL) para la liberación de pirógenos en productos terminados parenterales. El análisis de pirógenos constituye uno de los principales ensayos en el control de calidad de la fabricación de inyectables por su repercusión en la salud humana, puesto que la presencia y administración de los mismos, es capaz de provocar una serie de respuestas fisiológicas, en su mayoría de carácter perjudicial y en casos extremos, la muerte del paciente. Por las razones anteriores, existe un creciente interés en el conocimiento y dominio de estos métodos. El presente trabajo muestra una revisión bibliográfica del método del LAL, se tratan aspectos como su descubrimiento y estandarización, aparición en la industria farmacéutica y razones para su triunfo, y los basamentos de los principales métodos o variaciones comerciales del LAL (gelificación, turbidimétricos y cromogénicos) que se describen en las Farmacopeas

Ensayo del lisado de amebocitos del Limulus (LAL) / Limulus amebocyte lysate (LAL) Assay

En los últimos años, los principales organismos reguladores de productos farmacéuticos (Farmacopeas) exigen cada vez más en sus monografías la aplicación del método del lisado de amebocitos de Limulus (LAL) para la liberación de pirógenos en productos terminados parenterales. El análisis de pirógenos constituye uno de los principales ensayos en el control de calidad de la fabricación de inyectables por su repercusión en la salud humana, puesto que la presencia y administración de los mismos, es capaz de provocar una serie de respuestas fisiológicas, en su mayoría de carácter perjudicial y en casos extremos, la muerte del paciente. Por las razones anteriores, existe un creciente interés en el conocimiento y dominio de estos métodos. El presente trabajo muestra una revisión bibliográfica del método del LAL, se tratan aspectos como su descubrimiento y estandarización, aparición en la industria farmacéutica y razones para su triunfo, y los basamentos de los principales métodos o variaciones comerciales del LAL (gelificación, turbidimétricos y cromogénicos) que se describen en las Farmacopeas(AU)

Estandarización del ensayo del lisado de amebocitos de Limulus (LAL): método de gelificación / Standardization of the Limulus amebocyte lysate (LAL): gelification method

Entre las principales aplicaciones del método del lisado de amebocitos de Limulus (LAL) está el control de endotoxinas en producto final de drogas parenterales. Para la aplicación exitosa del ensayo se requiere del dominio de un conjunto de habilidades, por lo que usualmente se corre en laboratorios especializados que ya cuentan con una basta experiencia. Aunque ya es un método ampliamente establecido y conocido, la aplicación del ensayo puede ser laborioso, consumir tiempo y reactivos hasta la obtención de resultados correctos. En el presente trabajo se describe la estandarización del ensayo del LAL por el método de gelificación. Se evalúan varias estrategias con la finalidad de optimizar y/o reducir el tiempo total de ensayo, el empleo de materiales como puntas certificadas libres de endotoxinas, tubos de ensayo y la sustitución de agua libre de endotoxinas suministrada por los fabricantes de reactivo LAL por agua para inyección. El procedimiento estandarizado produce resultados válidos según los criterios de la Farmacopea de EE.UU(AU)

Validación de un ciclo de despirogenización por calor seco con el empleo del ensayo del lisado de amebocitos de limulus / Validation of a depyrogenation cycle by dry heat with the utilization of the limulus amoebocytes lysate assay

Para el cumplimiento de las buenas prácticas de laboratorio y de fabricación de parenterales, una de las exigencias es la validación de los ciclos de despirogenización que se emplean. En el presente trabajo se describe la validación de un ciclo de despirogenización por calor seco en un horno de convección. En la primera etapa se estudiaron características físicas del equipo como el tiempo requerido para alcanzar la temperatura establecida, su distribución, estabilidad, y la influencia de la carga en el patrón de calentamiento. Considerando los resultados de esta fase se estableció un proceso total de 7 h a 180 ºC. La segunda etapa consistió en la determinación del grado de despirogenización retando el proceso con bioindicadores de endotoxinas. La concentración de endotoxinas en los bioindicadores control y sometidos al proceso de despirogenización se cuantificó con el empleo del ensayo del lisado de amebocitos de limulus método de gelificación. La diferencia entre el contenido de endotoxinas en el control y los tratamientos fue de 2 400 U de endotoxinas, por lo que el proceso rindió una reducción logarítmica mínima de 4,6 log, la cual es mayor que el límite de 3 log establecido por la Farmacopea de los Estados Unidos

Validación de un ciclo de despirogenización por calor seco con el empleo del ensayo del lisado de amebocitos de limulus / Validation of a depyrogenation cycle by dry heat with the utilization of the limulus amoebocytes lysate assay

Para el cumplimiento de las buenas prácticas de laboratorio y de fabricación de parenterales, una de las exigencias es la validación de los ciclos de despirogenización que se emplean. En el presente trabajo se describe la validación de un ciclo de despirogenización por calor seco en un horno de convección. En la primera etapa se estudiaron características físicas del equipo como el tiempo requerido para alcanzar la temperatura establecida, su distribución, estabilidad, y la influencia de la carga en el patrón de calentamiento. Considerando los resultados de esta fase se estableció un proceso total de 7 h a 180 ºC. La segunda etapa consistió en la determinación del grado de despirogenización retando el proceso con bioindicadores de endotoxinas. La concentración de endotoxinas en los bioindicadores control y sometidos al proceso de despirogenización se cuantificó con el empleo del ensayo del lisado de amebocitos de limulus método de gelificación. La diferencia entre el contenido de endotoxinas en el control y los tratamientos fue de 2 400 U de endotoxinas, por lo que el proceso rindió una reducción logarítmica mínima de 4,6 log, la cual es mayor que el límite de 3 log establecido por la Farmacopea de los Estados Unidos(AU)