ABSTRACT
Las endotoxinas bacterianas son lipopolisacáridos (LPS) localizados exclusivamente en la membrana externa de las bacterias gramnegativas, su ingreso al organismo a través de productos parenterales puede causar fiebre, taquicardia, aumento de presión sanguínea y en algunos casos ocasiona la muerte. Existen normativas internacionales acerca del límite de endotoxina para productos farmacéuticos inyectables, tal como las inmunoglobulinas (IgG), que pueden estar expuestas a contaminación durante el proceso de producción y por lo tanto es necesario realizar pruebas para la determinación de endotoxinas bacterianas. El método del lisado de amebocitos de Limulus (LAL) es una de ellas.Este método se fundamenta en la reacción del LAL, el cual es un extracto de células sanguíneas que interaccionan con endotoxinas, activando la cascada del proceso de coagulación y originando la formación de la coagulina. En diversas ocasiones algunas proteínas intervienen en la activación o desactivación de esta cascada, bien sea potenciando o inhibiendo la formación del coágulo. En el caso de la potenciación, el calentamiento es uno de los métodos recomendados por la farmacopea estadounidense (USP) para eliminar interferencias, puesto que desnaturaliza las proteínas que causan la potenciación sin pérdida de endotoxinas. En este trabajo se validó la determinación de endotoxinas bacterianas en IgG mediante el método LAL, el cual es un método rápido y de fácilejecución, por lo que puede implementarse como ensayo de rutina en control de calidad y por ende nos permite agilizar las Liberaciones de Lotes de estos productos.
Bacterial endotoxins are lipopolysaccharides (LPS) located exclusively in the outer membrane of gram-negative bacteria, their entry into the body through parenteral products can cause fever, tachycardia, increased blood pressure and in some cases cause death. There are international standards for endotoxin limit for injectable pharmaceuticals, such as immunoglobulins (IgG), which may be exposed to contamination during the production process and therefore it is necessary to test for determination of bacterial endotoxins. The method of the Limulus amebocyte lysate (LAL) is one of them. This method is based on the LAL reaction, which is an extract of blood cells which interact with endotoxin, triggering the cascade of the coagulation process and causing the formation of coagulin. On several occasions some proteins involved in the activation or deactivation of this waterfall, either by enhancing or inhibiting clot formation. In the case of empowerment, the warming is one of those recommended by the US Pharmacopoeia (USP) to eliminate interference, since denatures proteins that cause endotoxin enhancement lossless methods. In this paper the determination of bacterial endotoxins in IgG was standardized by the LAL method, which is quick and easy to implement method, which can be implemented as a routine test in quality control and thus allows us to streamline releases Lots of these products.
Subject(s)
Humans , Male , Female , Immunoglobulins , Endotoxins , Gram-Negative Bacteria , Lipopolysaccharides , Antibody-Producing CellsABSTRACT
Objetivo: valorar las endotoxinas bacterianas por la técnica del lisado del amebocito de Limulus para el producto inyectable ácido zoledrónico, por el método de gelificación. Métodos: el ensayo se realizó mediante dos pruebas: 1) confirmación de la sensibilidad del lisado etiquetado, para lo cual se preparó una curva estándar con diluciones seriadas dobles de endotoxina por cuadruplicado; y 2) el ensayo del producto de inhibición, en el que se prepararon diluciones seriadas dobles de endotoxina con agua apirogénica y con las muestras de los lotes a ensayar sin sobrepasar la máxima dilución válida. Se determinó el punto final y se calculó la media geométrica. Se definió la dilución de trabajo, la cual se validó por cuadruplicado en tres lotes consecutivos. Resultados: la sensibilidad del lisado resultó 0,03125 UE/mL. La máxima dilución válida fue de 112 UE/mL y la dilución de trabajo 1/100. La cantidad de endotoxinas bacterianas presentes en tres lotes del producto inyectable no sobrepasó el límite establecido, por lo que cumplió con las especificaciones de calidad establecidas para el ensayo. Conclusión: la estandarización de las condiciones del método por gelificación, hace que este resulte eficaz, confiable, rápido y de fácil ejecución, por lo que puede emplearse como ensayo de rutina en el control de la calidad del inyectable analizado
Objective: To asses the presence of bacterial endotoxins in Zoledronic Acid injectable drug by using the Limulus amebocyte lysate test, particularly by the gelling procedure. Methods: The assay was performed in two tests: the first was the confirmation of labeled lysate sensitivity by preparing a standard curve with serial double dilutions of endotoxins four times, and the second was the inhibition product test in which serial double dilutions of endotoxins were prepared with apyrogenic water and with samples from the batches to be tested, without exceeding the maximum valid dilution. The end point was determined and the geometric mean was calculated. Working dilution was defined and then validated four times in three consecutive batches. Results: The lysate sensitivity was 0.03125 EU/mL). The maximum valid dilution and the working dilution were 112 EU/mL and 1/100 EU/mL) respectively. The amount of bacterial endotoxins present in three batches of the injectable drug did not exceed the set limit, so it complied with the quality specifications for this test. Conclusions: The standardization of the gelling method conditions makes it possible to state that this method is effective, reliable, quick and easy-to-perform, so it can be used as a regular test in the quality control of the analyzed parenteral drug
Subject(s)
Endotoxins , Gels , Limulus Test/methodsABSTRACT
A comparison of methodologies for detection of pyrogens in pharmaceutical products was performed. The rabbit pyrogen test was optimized and the dose-response curve was obtained for the 2nd International Standard for bacterial endotoxins, establishing 13.81 EU/mL/kg as the concentration of endotoxin necessary to induce a temperature rise of 0.5ºC. The 0.5ºC cut-off was shown to give results that were more compatible with the pyrogenic doses for humans. The Limulus amoebocyte lysate test (LAL) was standardized with gel-clot and chromogenic endpoints, and used for the comparative evaluation of pharmaceutical products showing good agreement. The use of beta-glucan-reactive and non-reactive LAL reagents identified some products with false-positive results. The interference test was carried out and the specifications validated for some new products as the maximum valid dilution. The results emphasized the importance and limitations of the assays recommended for the evaluation of purity and quality control of parenteral medicinal products, improving the existing methodologies in the context of reduction and replacement in the use of animal models.
Realizou-se a comparação de metodologia para avaliação de pirogênios em produtos farmacêuticos. Otimizou-se o teste da hipertermia em coelhos elaborando a curva dose-resposta com o 2º Padrão Internacional de endotoxinas bacterianas, com base na qual determinou-se a concentração de 13,81 UE/mL por kg de peso corporal, necessária para produzir aumento de temperatura de 0,5ºC. Observou-se que o limite de 0,5ºC forneceu resultados comparáveis com as doses pirogênicas para o homem. Padronizou-se o teste do lisado de amebócitos do Limulus (LAL) com determinação do ponto final cromogênico e por geleificação, que foram utilizados para a avaliação de produtos farmacêuticos obtendo-se resultados concordantes. Avaliaram-se as respostas de reagentes LAL reativos e não-reativos a beta-glicanos, observando diferenças que poderiam reprovar amostras com base em resultados falso-positivos. Executou-se o teste de interferências, validou-se o procedimento e estabeleceu-se a máxima diluição válida para produtos farmacêuticos sem especificações farmacopéicas. Os resultados enfatizam a importância e as limitações dos ensaios preconizados para avaliação da pureza e controle da qualidade de produtos farmacêuticos parenterais, contribuindo para aprimorar as metodologias existentes no contexto da redução e substituição dos modelos animais.