{"id":23118,"date":"2019-05-17T11:14:16","date_gmt":"2019-05-17T09:14:16","guid":{"rendered":"https:\/\/kymos.com\/news\/analisis-de-proteinas-de-la-celula-huesped\/"},"modified":"2019-05-17T11:14:16","modified_gmt":"2019-05-17T09:14:16","slug":"analisis-de-proteinas-de-la-celula-huesped","status":"publish","type":"post","link":"https:\/\/kymos.com\/es\/news\/analisis-de-proteinas-de-la-celula-huesped\/","title":{"rendered":"An\u00e1lisis de prote\u00ednas de la c\u00e9lula hu\u00e9sped"},"content":{"rendered":"<p><span style=\"font-size: 14pt;\">El desarrollo y validaci\u00f3n de un ensayo espec\u00edfico de proceso para la determinaci\u00f3n de prote\u00ednas de c\u00e9lulas hu\u00e9sped (HCP) en prote\u00ednas recombinantes terap\u00e9uticas debe seguir las especificaciones contenidas en la monograf\u00eda 2.6.34 de la Farmacopea Europea en un enfoque paso a paso:<\/span><\/p>\n<div class=\"blog-content\">\n<div class=\"field field-name-body field-type-text-with-summary field-label-hidden\">\n<div class=\"field-items\">\n<div class=\"field-item even\">\n<p><span style=\"font-size: 14pt;\"><b>1. Producci\u00f3n de los ant\u00edgenos HCP<\/b><\/span><\/p>\n<p><span style=\"font-size: 14pt;\">Esta fase la lleva a cabo el cliente en una simulaci\u00f3n del proceso de fabricaci\u00f3n de la sustancia activa utilizando una l\u00ednea celular nula. Esta simulaci\u00f3n de producci\u00f3n debe simular el peor escenario posible en cuanto a la generaci\u00f3n de HCP. Se deben emplear los m\u00e9todos anal\u00edticos actuales para la sustancia activa a fin de garantizar la ausencia de trazas de la misma. Se debe realizar una purificaci\u00f3n m\u00ednima (filtraci\u00f3n, concentraci\u00f3n) para garantizar que no se pierda ning\u00fan componente del HCP durante este proceso.   <\/span><\/p>\n<p><span style=\"font-size: 14pt;\"><b>2. Caracterizaci\u00f3n de los ant\u00edgenos HCP<\/b><\/span><\/p>\n<p><span style=\"font-size: 14pt;\">El ant\u00edgeno contra las prote\u00ednas HCP obtenido en la producci\u00f3n simulada se analiza mediante SDS-PAGE y\/o electroforesis 2D utilizando un m\u00e9todo de tinci\u00f3n sensible (azul de Coomassie o plata). Los resultados obtenidos deben compararse con los obtenidos en una producci\u00f3n est\u00e1ndar de la sustancia activa. Se recomienda contar con muestras con la m\u00ednima purificaci\u00f3n posible y otras que hayan seguido el proceso est\u00e1ndar posterior. <\/span><\/p>\n<p><span style=\"font-size: 14pt;\"><b>3. Producci\u00f3n de reactivo de anticuerpo policlonal<\/b><\/span><\/p>\n<p><span style=\"font-size: 14pt;\">Las prote\u00ednas antig\u00e9nicas HCP obtenidas en la producci\u00f3n simulada se administran a animales (generalmente rabinos) para inducir la respuesta inmunitaria en ellos con el objetivo de obtener sueros con un alto t\u00edtulo de anticuerpos contra los ant\u00edgenos HCP. Una vez que el t\u00edtulo de anticuerpos s\u00e9ricos medido mediante una prueba ELISA es suficientemente alto, cada suero debe caracterizarse mediante Western Blot y todos los sueros deben agruparse para obtener la m\u00e1xima cobertura del HCP en el ensayo ELISA. <\/span><\/p>\n<p><span style=\"font-size: 14pt;\"><b>4. Purificaci\u00f3n y caracterizaci\u00f3n final del reactivo de anticuerpo policlonal<\/b><\/span><\/p>\n<p><span style=\"font-size: 14pt;\">Los sueros agrupados deben purificarse a\u00fan m\u00e1s mediante cromatograf\u00eda de prote\u00edna A o G y caracterizarse mediante SDS-PAGE, titulaci\u00f3n y Western Blot.<\/span><\/p>\n<p><span style=\"font-size: 14pt;\"><b>5. Desarrollo de un ensayo ELISA cuantitativo para la determinaci\u00f3n de HCP<\/b><\/span><\/p>\n<p><span style=\"font-size: 14pt;\">Se debe desarrollar una prueba ELISA cuantitativa considerando los siguientes temas:<\/span><\/p>\n<ul>\n<li><span style=\"font-size: 14pt;\">Elecci\u00f3n del formato ELISA m\u00e1s adecuado: Directo, s\u00e1ndwich, competitivo, etc.<\/span><\/li>\n<li><span style=\"font-size: 14pt;\">Etiquetado de reactivos.<\/span><\/li>\n<li><span style=\"font-size: 14pt;\">Optimizaci\u00f3n de las condiciones de ensayo.<\/span><\/li>\n<li><span style=\"font-size: 14pt;\">Caracterizaci\u00f3n del ensayo: Sensibilidad, especificidad, LLOQ, rango, diluci\u00f3n m\u00ednima, etc.<\/span><\/li>\n<li><span style=\"font-size: 14pt;\">Definici\u00f3n de la prueba de idoneidad.<\/span><\/li>\n<\/ul>\n<p><span style=\"font-size: 14pt;\"><b>6. Validaci\u00f3n de un ensayo ELISA cuantitativo para la determinaci\u00f3n de HCP<\/b><\/span><\/p>\n<p><span style=\"font-size: 14pt;\">Una vez desarrollado el m\u00e9todo ELISA, se debe realizar una validaci\u00f3n del m\u00e9todo evaluando los siguientes par\u00e1metros:<\/span><\/p>\n<ul>\n<li><span style=\"font-size: 14pt;\">Especificidad<\/span><\/li>\n<li><span style=\"font-size: 14pt;\">Rango\/curva de calibraci\u00f3n<\/span><\/li>\n<li><span style=\"font-size: 14pt;\">Exactitud y precisi\u00f3n<\/span><\/li>\n<li><span style=\"font-size: 14pt;\">L\u00edmite de detecci\u00f3n y l\u00edmite de cuantificaci\u00f3n<\/span><\/li>\n<li><span style=\"font-size: 14pt;\">Estabilidad<\/span><\/li>\n<li><span style=\"font-size: 14pt;\">Robustez<\/span><\/li>\n<li><span style=\"font-size: 14pt;\">Linealidad de diluci\u00f3n<\/span><\/li>\n<\/ul>\n<p><span style=\"font-size: 14pt;\"><b>7. An\u00e1lisis de muestras<\/b><\/span><\/p>\n<p><span style=\"font-size: 14pt;\">Una vez alcanzado este punto, el m\u00e9todo anal\u00edtico puede utilizarse para cada lote de fabricaci\u00f3n como m\u00e9todo de control de calidad para la liberaci\u00f3n. Cada an\u00e1lisis debe incluir la prueba de idoneidad establecida en la etapa de desarrollo de ELISA, realizada en un laboratorio con certificaci\u00f3n GMP para dicho fin. <\/span><\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"<p>El desarrollo y validaci\u00f3n de un ensayo espec\u00edfico de proceso  [&#8230;]<\/p>\n","protected":false},"author":6,"featured_media":21712,"comment_status":"","ping_status":"","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[183],"tags":[],"class_list":["post-23118","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-cmc-es"],"acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v27.3 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>An\u00e1lisis de prote\u00ednas de la c\u00e9lula hu\u00e9sped | Kymos<\/title>\n<meta name=\"description\" content=\"Conozca m\u00e1s sobre los 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Dou","image":{"@type":"ImageObject","inLanguage":"es","@id":"https:\/\/secure.gravatar.com\/avatar\/263d7ff1c7891f5821a5075a1a362a1e513db5931dc882455e887fe1c1ccf924?s=96&d=mm&r=g","url":"https:\/\/secure.gravatar.com\/avatar\/263d7ff1c7891f5821a5075a1a362a1e513db5931dc882455e887fe1c1ccf924?s=96&d=mm&r=g","contentUrl":"https:\/\/secure.gravatar.com\/avatar\/263d7ff1c7891f5821a5075a1a362a1e513db5931dc882455e887fe1c1ccf924?s=96&d=mm&r=g","caption":"Joan Puig de Dou"},"description":"Active in the pharmaceutical marketing for over 30 years President of CataloniaBIO&amp;HealthTech CEO and Qualified Person at Kymos Leader of Kymos team, guaranteeing a trustable, ethical and scientific correct editorial publications. Experience Joan Puig de Dou started his career at Laboratorios Menarini first as scientist later as Qualified Person and finally as Operations General Manager. 2010 he entered Kymos where he became CEO in 2015. 2022 Joan Puig de Dou was elected as president of CataloniaBio&amp;HealthTech to represent the Catalonian biotech industry nationally and internationally. Expertise Joan Puig de Dou has extensive experience in writing scientific content adopting a didactic approach that fits any reader\u2019s level of expertise. He publishes regularely on Kymos\u2019 webpage, social media and in the specialized press. Education He graduated from Universitat de Barcelona as Pharmacist and from IESE Business School\u2019s Programa Diecci\u00f3n General (PDG) Want to know him better? Read his interview in PharmaBoardrooom in January 2019 and more about his presidency of CataloniaBio at Parc Sc\u00edentific de Barcelona.","url":"https:\/\/kymos.com\/es\/news\/author\/joan-puig-de-dou\/"}]}},"_links":{"self":[{"href":"https:\/\/kymos.com\/es\/wp-json\/wp\/v2\/posts\/23118","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/kymos.com\/es\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/kymos.com\/es\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/kymos.com\/es\/wp-json\/wp\/v2\/users\/6"}],"replies":[{"embeddable":true,"href":"https:\/\/kymos.com\/es\/wp-json\/wp\/v2\/comments?post=23118"}],"version-history":[{"count":0,"href":"https:\/\/kymos.com\/es\/wp-json\/wp\/v2\/posts\/23118\/revisions"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/kymos.com\/es\/wp-json\/wp\/v2\/media\/21712"}],"wp:attachment":[{"href":"https:\/\/kymos.com\/es\/wp-json\/wp\/v2\/media?parent=23118"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/kymos.com\/es\/wp-json\/wp\/v2\/categories?post=23118"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/kymos.com\/es\/wp-json\/wp\/v2\/tags?post=23118"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}