{"id":27228,"date":"2019-05-17T11:14:16","date_gmt":"2019-05-17T09:14:16","guid":{"rendered":"https:\/\/kymos.com\/news\/analisi-delle-proteine-delle-cellule-ospiti\/"},"modified":"2019-05-17T11:14:16","modified_gmt":"2019-05-17T09:14:16","slug":"analisi-delle-proteine-delle-cellule-ospiti","status":"publish","type":"post","link":"https:\/\/kymos.com\/it\/news\/analisi-delle-proteine-delle-cellule-ospiti\/","title":{"rendered":"Analisi delle proteine delle cellule ospiti"},"content":{"rendered":"

Lo sviluppo e la convalida di un test specifico per il processo di determinazione delle proteine delle cellule ospiti (HCP) nelle proteine ricombinanti terapeutiche devono seguire le specifiche contenute nella monografia 2.6.34 della Farmacopea europea con un approccio graduale:<\/span><\/p>\n

\n
\n
\n
\n

1. Produzione degli antigeni HCP<\/b><\/span><\/p>\n

Questa fase viene eseguita dal cliente in una simulazione del processo di produzione del principio attivo utilizzando una linea cellulare nulla. Questa simulazione di produzione deve simulare il caso peggiore in riferimento alla generazione di HCP. Devono essere impiegati i metodi analitici pi\u00f9 recenti per garantire che non siano presenti tracce di principio attivo. \u00c8 necessario effettuare una purificazione minima (filtrazione, concentrazione) per garantire che nessun componente dell’HCP venga perso durante questo processo. <\/span><\/p>\n

2. Caratterizzazione degli antigeni HCP<\/b><\/span><\/p>\n

L’antigene contro le proteine HCP ottenuto nella produzione simulata viene analizzato mediante SDS-PAGE e\/o elettroforesi 2D utilizzando un metodo di colorazione sensibile (blu di Coomassie o argento) e i risultati ottenuti devono essere confrontati con i risultati ottenuti da una produzione standard del principio attivo. Si raccomanda di disporre di campioni con la minima purificazione possibile e di altri che abbiano seguito il processo standard a valle. <\/span><\/p>\n

3. Produzione del reagente anticorpale policlonale<\/b><\/span><\/p>\n

Le proteine antigeniche HCP ottenute nella produzione simulata vengono somministrate ad animali (solitamente rabbini) per indurre la risposta immunitaria in questi animali, con l’obiettivo di ottenere sieri con un titolo anticorpale elevato contro gli antigeni HCP. Una volta che il titolo anticorpale del siero misurato con un test ELISA \u00e8 sufficientemente elevato, ciascun siero deve essere caratterizzato mediante Western Blot e tutti i sieri devono essere aggregati per ottenere la massima copertura dell’HCP nel test ELISA. <\/span><\/p>\n

4. Purificazione e caratterizzazione finale del reagente anticorpale policlonale<\/b><\/span><\/p>\n

I sieri raccolti devono essere ulteriormente purificati mediante cromatografia proteica A o G e caratterizzati mediante SDS-PAGE, titolazione e Western Blot.<\/span><\/p>\n

5. Sviluppo di un test ELISA quantitativo per la determinazione dell’HCP<\/b><\/span><\/p>\n

Un test ELISA quantitativo dovrebbe essere sviluppato tenendo conto dei seguenti argomenti:<\/span><\/p>\n

    \n
  • Scelta del formato ELISA pi\u00f9 adatto: diretto, sandwich, competitivo, ecc.<\/span><\/li>\n
  • Etichettatura dei reagenti.<\/span><\/li>\n
  • Ottimizzazione delle condizioni di analisi.<\/span><\/li>\n
  • Caratterizzazione del test: sensibilit\u00e0, specificit\u00e0, LLOQ, intervallo, diluizione minima, ecc.<\/span><\/li>\n
  • Definizione del test di idoneit\u00e0.<\/span><\/li>\n<\/ul>\n

    6. Validazione di un test ELISA quantitativo per la determinazione dell’HCP<\/b><\/span><\/p>\n

    Una volta sviluppato il metodo ELISA, \u00e8 necessario effettuare una validazione del metodo valutando i seguenti parametri:<\/span><\/p>\n

      \n
    • Specificit\u00e0<\/span><\/li>\n
    • Intervallo\/curva di calibrazione<\/span><\/li>\n
    • Accuratezza e precisione<\/span><\/li>\n
    • Limite di rilevabilit\u00e0 e limite di quantificazione<\/span><\/li>\n
    • Stabilit\u00e0<\/span><\/li>\n
    • Robustezza<\/span><\/li>\n
    • Linearit\u00e0 della diluizione<\/span><\/li>\n<\/ul>\n

      7. Analisi del campione<\/b><\/span><\/p>\n

      Una volta raggiunto questo punto, il metodo analitico pu\u00f2 essere utilizzato per ogni lotto di produzione come metodo di controllo qualit\u00e0 per il rilascio. Ogni analisi dovrebbe includere il test di idoneit\u00e0 stabilito nella fase di sviluppo del test ELISA e condotto in un laboratorio GMP per lo scopo sopra menzionato. <\/span><\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"

      Lo sviluppo e la convalida di un test specifico per […]<\/p>\n","protected":false},"author":6,"featured_media":27230,"comment_status":"","ping_status":"","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[264],"tags":[],"class_list":["post-27228","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-cmc"],"acf":[],"yoast_head":"\nAnalisi delle proteine delle cellule ospiti | Kymos<\/title>\n<meta name=\"description\" content=\"Scopri di pi\u00f9 sui passaggi e le specifiche che la determinazione dell'HCP nei test delle proteine ricombinanti terapeutiche deve seguire.\" \/>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/kymos.com\/it\/news\/analisi-delle-proteine-delle-cellule-ospiti\/\" \/>\n<meta property=\"og:locale\" content=\"it_IT\" \/>\n<meta property=\"og:type\" 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