{"id":22686,"date":"2019-05-17T11:14:16","date_gmt":"2019-05-17T09:14:16","guid":{"rendered":"https:\/\/kymos.com\/news\/analisi-de-proteines-de-cellules-hostes\/"},"modified":"2019-05-17T11:14:16","modified_gmt":"2019-05-17T09:14:16","slug":"analisi-de-proteines-de-cellules-hostes","status":"publish","type":"post","link":"https:\/\/kymos.com\/ca\/news\/analisi-de-proteines-de-cellules-hostes\/","title":{"rendered":"An\u00e0lisi de prote\u00efnes de c\u00e8l\u00b7lules hostes"},"content":{"rendered":"

El desenvolupament i la validaci\u00f3 d’un assaig espec\u00edfic del proc\u00e9s per a la determinaci\u00f3 de prote\u00efnes de la c\u00e8l\u00b7lula hoste (HCP) en prote\u00efnes recombinants terap\u00e8utiques ha de seguir les especificacions que figuren a la monografia 2.6.34 de la Farmacopea Europea amb un enfocament pas a pas:<\/span><\/p>\n

\n
\n
\n
\n

1. Producci\u00f3 dels ant\u00edgens HCP<\/b><\/span><\/p>\n

Aquesta fase la duu a terme el client en una simulaci\u00f3 del proc\u00e9s de fabricaci\u00f3 de la subst\u00e0ncia activa utilitzant una l\u00ednia cel\u00b7lular nul\u00b7la. Aquesta simulaci\u00f3 de producci\u00f3 ha d’imitar el pitjor dels casos pel que fa a la generaci\u00f3 d’HCP. S’han d’utilitzar els m\u00e8todes anal\u00edtics actuals per a la subst\u00e0ncia activa per assegurar que no hi hagi traces de la subst\u00e0ncia activa. S’ha de dur a terme una purificaci\u00f3 m\u00ednima (filtraci\u00f3, concentraci\u00f3) per assegurar que no es perdi cap component de l’HCP durant aquest proc\u00e9s. <\/span><\/p>\n

2. Caracteritzaci\u00f3 dels ant\u00edgens HCP<\/b><\/span><\/p>\n

L’antigen contra les prote\u00efnes HCP obtingut en la producci\u00f3 simulada s’analitza mitjan\u00e7ant SDS-PAGE i\/o electroforesi 2D utilitzant un m\u00e8tode de tinci\u00f3 sensible (blau de Coomassie o plata) i els resultats obtinguts s’han de comparar amb els resultats obtinguts a partir d’una producci\u00f3 est\u00e0ndard de la subst\u00e0ncia activa. Es recomana tenir mostres amb la m\u00ednima purificaci\u00f3 possible i altres que hagin seguit el proc\u00e9s est\u00e0ndard posterior. <\/span><\/p>\n

3. Producci\u00f3 de reactiu d’anticossos policlonals<\/b><\/span><\/p>\n

Les prote\u00efnes antig\u00e8niques HCP obtingudes en la producci\u00f3 simulada s’administren a animals (normalment rabins) per induir la resposta immunit\u00e0ria en aquests animals amb l’objectiu d’obtenir s\u00e8rums amb un t\u00edtol elevat d’anticossos contra els ant\u00edgens HCP. Un cop el t\u00edtol dels anticossos s\u00e8rics mesurats amb una prova ELISA sigui prou alt, cada s\u00e8rum s’ha de caracteritzar mitjan\u00e7ant Western Blot i tots els s\u00e8rums s’han d’agrupar per obtenir la m\u00e0xima cobertura de l’HCP en l’assaig ELISA. <\/span><\/p>\n

4. Purificaci\u00f3 i caracteritzaci\u00f3 final del reactiu d’anticossos policlonals<\/b><\/span><\/p>\n

Els s\u00e8rums agrupats s’han de purificar addicionalment mitjan\u00e7ant cromatografia de prote\u00efna A o G i caracteritzar-se mitjan\u00e7ant SDS-PAGE, titraci\u00f3 i Western Blot.<\/span><\/p>\n

5. Desenvolupament d’un assaig ELISA quantitatiu per a la determinaci\u00f3 d’HCP<\/b><\/span><\/p>\n

S’hauria de desenvolupar un ELISA quantitatiu tenint en compte els seg\u00fcents aspectes:<\/span><\/p>\n

    \n
  • Triar el format ELISA m\u00e9s adequat: directe, sandvitx, competitiu, etc.<\/span><\/li>\n
  • Etiquetatge de reactius.<\/span><\/li>\n
  • Optimitzaci\u00f3 de les condicions d’assaig.<\/span><\/li>\n
  • Caracteritzaci\u00f3 de l’assaig: sensibilitat, especificitat, LLOQ, rang, diluci\u00f3 m\u00ednima, etc.<\/span><\/li>\n
  • Definici\u00f3 de la prova d’idone\u00eftat.<\/span><\/li>\n<\/ul>\n

    6. Validaci\u00f3 d’un assaig ELISA quantitatiu per a la determinaci\u00f3 d’HCP<\/b><\/span><\/p>\n

    Un cop desenvolupat el m\u00e8tode ELISA, s’ha de dur a terme una validaci\u00f3 del m\u00e8tode avaluant els par\u00e0metres seg\u00fcents:<\/span><\/p>\n

      \n
    • Especificitat<\/span><\/li>\n
    • Rang\/corba de calibratge<\/span><\/li>\n
    • Precisi\u00f3 i precisi\u00f3<\/span><\/li>\n
    • L\u00edmit de detecci\u00f3 i l\u00edmit de quantificaci\u00f3<\/span><\/li>\n
    • Estabilitat<\/span><\/li>\n
    • Robustesa<\/span><\/li>\n
    • Linealitat de diluci\u00f3<\/span><\/li>\n<\/ul>\n

      7. An\u00e0lisi de mostres<\/b><\/span><\/p>\n

      Arribat a aquest punt, el m\u00e8tode anal\u00edtic es pot utilitzar per a cada lot de fabricaci\u00f3 com a m\u00e8tode de control de qualitat per a l’alliberament. Cada an\u00e0lisi ha d’incloure la prova d’idone\u00eftat establerta en la fase de desenvolupament d’ELISA i realitzada en un laboratori GMP per al prop\u00f2sit esmentat anteriorment. <\/span><\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"

      El desenvolupament i la validaci\u00f3 d’un assaig espec\u00edfic del proc\u00e9s […]<\/p>\n","protected":false},"author":6,"featured_media":15014,"comment_status":"","ping_status":"","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[97],"tags":[],"class_list":["post-22686","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-cmc-ca"],"acf":[],"yoast_head":"\nAn\u00e0lisi de prote\u00efnes de c\u00e8l\u00b7lules hostes | Kymos<\/title>\n<meta name=\"description\" content=\"M\u00e9s informaci\u00f3 sobre els passos i les especificacions que ha de seguir la determinaci\u00f3 de HCP en assaigs de prote\u00efnes recombinants terap\u00e8utiques.\" \/>\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\/ca\/news\/analisi-de-proteines-de-cellules-hostes\/\" \/>\n<meta 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