{"id":27254,"date":"2019-05-17T11:14:16","date_gmt":"2019-05-17T09:14:16","guid":{"rendered":"https:\/\/kymos.com\/news\/analyse-des-proteines-de-la-cellule-hote\/"},"modified":"2019-05-17T11:14:16","modified_gmt":"2019-05-17T09:14:16","slug":"analyse-des-proteines-de-la-cellule-hote","status":"publish","type":"post","link":"https:\/\/kymos.com\/fr\/news\/analyse-des-proteines-de-la-cellule-hote\/","title":{"rendered":"Analyse des prot\u00e9ines de la cellule h\u00f4te"},"content":{"rendered":"

Le d\u00e9veloppement et la validation d’un dosage sp\u00e9cifique au processus pour la d\u00e9termination des prot\u00e9ines de cellules h\u00f4tes (HCP) dans les prot\u00e9ines recombinantes th\u00e9rapeutiques doivent suivre les sp\u00e9cifications contenues dans la monographie 2.6.34 de la Pharmacop\u00e9e europ\u00e9enne selon une approche \u00e9tape par \u00e9tape :<\/span><\/p>\n

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\n
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\n

1. Production des antig\u00e8nes HCP<\/b><\/span><\/p>\n

Cette phase est r\u00e9alis\u00e9e par le client lors d’une simulation du proc\u00e9d\u00e9 de fabrication de la substance active, en utilisant une lign\u00e9e cellulaire nulle. Cette simulation doit reproduire le sc\u00e9nario le plus d\u00e9favorable concernant la g\u00e9n\u00e9ration de prot\u00e9ines de la cellule h\u00f4te (HCP). Les m\u00e9thodes analytiques actuelles de la substance active doivent \u00eatre utilis\u00e9es afin de garantir l’absence de traces de cette derni\u00e8re. Une purification minimale (filtration, concentration) doit \u00eatre effectu\u00e9e pour \u00e9viter toute perte de composants des HCP au cours de ce proc\u00e9d\u00e9. <\/span><\/p>\n

2. Caract\u00e9risation des antig\u00e8nes HCP<\/b><\/span><\/p>\n

L’antig\u00e8ne dirig\u00e9 contre les prot\u00e9ines HCP obtenu lors de la production t\u00e9moin est analys\u00e9 par \u00e9lectrophor\u00e8se sur gel de polyacrylamide en pr\u00e9sence de SDS (SDS-PAGE) et\/ou par \u00e9lectrophor\u00e8se bidimensionnelle (2D) avec une m\u00e9thode de coloration sensible (bleu de Coomassie ou argent). Les r\u00e9sultats obtenus sont compar\u00e9s \u00e0 ceux obtenus lors d’une production standard de la substance active. Il est recommand\u00e9 de disposer d’\u00e9chantillons ayant subi une purification minimale et d’autres ayant suivi le protocole de purification standard. <\/span><\/p>\n

3. Production de r\u00e9actif d’anticorps polyclonaux<\/b><\/span><\/p>\n

Les prot\u00e9ines antig\u00e9niques HCP obtenues lors de la production simul\u00e9e sont administr\u00e9es \u00e0 des animaux (g\u00e9n\u00e9ralement des lapins) afin d’induire une r\u00e9ponse immunitaire et d’obtenir des s\u00e9rums pr\u00e9sentant un titre \u00e9lev\u00e9 d’anticorps dirig\u00e9s contre ces antig\u00e8nes. Lorsque le titre d’anticorps s\u00e9riques, mesur\u00e9 par un test ELISA, est suffisamment \u00e9lev\u00e9, chaque s\u00e9rum est caract\u00e9ris\u00e9 par Western Blot et tous les s\u00e9rums sont regroup\u00e9s afin d’obtenir une couverture maximale des HCP dans le test ELISA. <\/span><\/p>\n

4. Purification et caract\u00e9risation finale du r\u00e9actif d’anticorps polyclonal<\/b><\/span><\/p>\n

Les s\u00e9rums regroup\u00e9s doivent \u00eatre purifi\u00e9s davantage par chromatographie sur prot\u00e9ine A ou G et caract\u00e9ris\u00e9s par SDS-PAGE, titrage et Western Blot.<\/span><\/p>\n

5. Mise au point d’un test ELISA quantitatif pour la d\u00e9termination des HCP<\/b><\/span><\/p>\n

Un test ELISA quantitatif doit \u00eatre d\u00e9velopp\u00e9 en tenant compte des points suivants :<\/span><\/p>\n

    \n
  • Choisir le format ELISA le plus adapt\u00e9 : direct, sandwich, comp\u00e9titif, etc.<\/span><\/li>\n
  • \u00c9tiquetage des r\u00e9actifs.<\/span><\/li>\n
  • Optimisation des conditions d’analyse.<\/span><\/li>\n
  • Caract\u00e9risation du test : sensibilit\u00e9, sp\u00e9cificit\u00e9, LLOQ, plage de mesure, dilution minimale, etc.<\/span><\/li>\n
  • D\u00e9finition du test d’aptitude.<\/span><\/li>\n<\/ul>\n

    6. Validation d’un dosage ELISA quantitatif pour la d\u00e9termination des HCP<\/b><\/span><\/p>\n

    Une fois la m\u00e9thode ELISA mise au point, une validation de la m\u00e9thode doit \u00eatre effectu\u00e9e en \u00e9valuant les param\u00e8tres suivants :<\/span><\/p>\n

      \n
    • Sp\u00e9cificit\u00e9<\/span><\/li>\n
    • Plage\/courbe d’\u00e9talonnage<\/span><\/li>\n
    • Exactitude et pr\u00e9cision<\/span><\/li>\n
    • Limite de d\u00e9tection et limite de quantification<\/span><\/li>\n
    • Stabilit\u00e9<\/span><\/li>\n
    • Robustesse<\/span><\/li>\n
    • lin\u00e9arit\u00e9 de la dilution<\/span><\/li>\n<\/ul>\n

      7. Analyse des \u00e9chantillons<\/b><\/span><\/p>\n

      Une fois ce stade atteint, la m\u00e9thode analytique peut \u00eatre utilis\u00e9e pour chaque lot de fabrication comme m\u00e9thode de contr\u00f4le qualit\u00e9 en vue de sa lib\u00e9ration. Chaque analyse doit inclure le test d’aptitude \u00e9tabli lors de la mise au point du test ELISA et r\u00e9alis\u00e9 dans un laboratoire conforme aux BPF (Bonnes Pratiques de Fabrication) conform\u00e9ment \u00e0 la finalit\u00e9 susmentionn\u00e9e. <\/span><\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"

      Le d\u00e9veloppement et la validation d’un dosage sp\u00e9cifique au processus […]<\/p>\n","protected":false},"author":6,"featured_media":27256,"comment_status":"","ping_status":"","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[267],"tags":[],"class_list":["post-27254","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-cmc"],"acf":[],"yoast_head":"\nAnalyse des prot\u00e9ines de la cellule h\u00f4te | Kymos<\/title>\n<meta name=\"description\" content=\"Apprenez-en davantage sur les \u00e9tapes et les sp\u00e9cifications que doit suivre la d\u00e9termination des HCP dans les essais de prot\u00e9ines recombinantes th\u00e9rapeutiques.\" \/>\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\/fr\/news\/analyse-des-proteines-de-la-cellule-hote\/\" \/>\n<meta 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