{"id":27250,"date":"2019-05-17T11:14:16","date_gmt":"2019-05-17T09:14:16","guid":{"rendered":"https:\/\/kymos.com\/news\/analyse-der-wirtszellproteine\/"},"modified":"2019-05-17T11:14:16","modified_gmt":"2019-05-17T09:14:16","slug":"analyse-der-wirtszellproteine","status":"publish","type":"post","link":"https:\/\/kymos.com\/de\/news\/analyse-der-wirtszellproteine\/","title":{"rendered":"Analyse der Wirtszellproteine"},"content":{"rendered":"

Die Entwicklung und Validierung eines prozessspezifischen Assays zur Bestimmung von Wirtszellproteinen (HCP) in therapeutischen rekombinanten Proteinen sollte schrittweise gem\u00e4\u00df den Spezifikationen der Monographie 2.6.34 des Europ\u00e4ischen Arzneibuchs erfolgen:<\/span><\/p>\n

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1. Herstellung der HCP-Antigene<\/b><\/span><\/p>\n

Diese Phase wird vom Kunden in einem simulierten Herstellungslauf des Wirkstoffs unter Verwendung einer Nullzelllinie durchgef\u00fchrt. Diese simulierte Produktion muss den ung\u00fcnstigsten Fall hinsichtlich der HCP-Generierung abbilden. Es m\u00fcssen g\u00e4ngige Analysemethoden f\u00fcr den Wirkstoff angewendet werden, um sicherzustellen, dass keine Wirkstoffspuren vorhanden sind. Eine minimale Reinigung (Filtration, Konzentrierung) sollte durchgef\u00fchrt werden, um sicherzustellen, dass w\u00e4hrend dieses Prozesses keine Komponente des HCP verloren geht. <\/span><\/p>\n

2. Charakterisierung der HCP-Antigene<\/b><\/span><\/p>\n

Die in der Scheinproduktion gewonnenen Antigene gegen HCP-Proteine werden mittels SDS-PAGE und\/oder 2D-Gelelektrophorese unter Verwendung einer sensitiven F\u00e4rbemethode (Coomassie-Blau oder Silber) analysiert. Die Ergebnisse sollten mit denen einer Standardproduktion des Wirkstoffs verglichen werden. Es wird empfohlen, Proben mit minimaler Aufreinigung sowie solche, die dem Standard-Downstream-Prozess unterzogen wurden, zu verwenden. <\/span><\/p>\n

3. Herstellung von polyklonalem Antik\u00f6rperreagenz<\/b><\/span><\/p>\n

Die in der Scheinproduktion gewonnenen Antigen-HCP-Proteine werden Tieren (\u00fcblicherweise Raben) verabreicht, um eine Immunantwort auszul\u00f6sen und Seren mit einem hohen Antik\u00f6rpertiter gegen die HCP-Antigene zu gewinnen. Sobald der mittels ELISA gemessene Antik\u00f6rpertiter im Serum hoch genug ist, wird jedes Serum mittels Western Blot charakterisiert und alle Seren werden gepoolt, um eine maximale Abdeckung der HCP im ELISA-Test zu gew\u00e4hrleisten. <\/span><\/p>\n

4. Reinigung und abschlie\u00dfende Charakterisierung des polyklonalen Antik\u00f6rperreagenz<\/b><\/span><\/p>\n

Die gepoolten Seren sollten anschlie\u00dfend durch Protein-A- oder Protein-G-Chromatographie weiter aufgereinigt und mittels SDS-PAGE, Titration und Western Blot charakterisiert werden.<\/span><\/p>\n

5. Entwicklung eines quantitativen ELISA-Tests zur Bestimmung von HCP<\/b><\/span><\/p>\n

Ein quantitativer ELISA sollte unter Ber\u00fccksichtigung der folgenden Aspekte entwickelt werden:<\/span><\/p>\n

    \n
  • Auswahl des am besten geeigneten ELISA-Formats: Direkt, Sandwich, kompetitiv usw.<\/span><\/li>\n
  • Reagenzienkennzeichnung.<\/span><\/li>\n
  • Optimierung der Testbedingungen.<\/span><\/li>\n
  • Charakterisierung des Assays: Sensitivit\u00e4t, Spezifit\u00e4t, untere Bestimmungsgrenze (LLOQ), Messbereich, minimale Verd\u00fcnnung usw.<\/span><\/li>\n
  • Definition des Eignungstests.<\/span><\/li>\n<\/ul>\n

    6. Validierung eines quantitativen ELISA-Tests zur Bestimmung von HCP<\/b><\/span><\/p>\n

    Sobald die ELISA-Methode entwickelt ist, sollte eine Validierung der Methode durch Bewertung der folgenden Parameter durchgef\u00fchrt werden:<\/span><\/p>\n

      \n
    • Spezifit\u00e4t<\/span><\/li>\n
    • Kalibrierbereich\/Kalibrierkurve<\/span><\/li>\n
    • Genauigkeit und Pr\u00e4zision<\/span><\/li>\n
    • Nachweisgrenze und Bestimmungsgrenze<\/span><\/li>\n
    • Stabilit\u00e4t<\/span><\/li>\n
    • Robustheit<\/span><\/li>\n
    • Verd\u00fcnnungslinearit\u00e4t<\/span><\/li>\n<\/ul>\n

      7. Probenanalyse<\/b><\/span><\/p>\n

      Sobald dieser Punkt erreicht ist, kann die Analysemethode f\u00fcr jede Produktionscharge als Qualit\u00e4tskontrollverfahren zur Freigabe eingesetzt werden. Jede Analyse sollte den im Rahmen der ELISA-Entwicklung festgelegten Eignungstest umfassen, der in einem GMP-Labor f\u00fcr den zuvor genannten Zweck durchgef\u00fchrt wurde. <\/span><\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"

      Die Entwicklung und Validierung eines prozessspezifischen Assays zur Bestimmung von […]<\/p>\n","protected":false},"author":6,"featured_media":27252,"comment_status":"","ping_status":"","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[266],"tags":[],"class_list":["post-27250","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-cmc"],"acf":[],"yoast_head":"\nAnalyse der Wirtszellproteine | Kymos<\/title>\n<meta name=\"description\" content=\"Erfahren Sie mehr \u00fcber die Schritte und Spezifikationen, die bei der Bestimmung von HCP in therapeutischen rekombinanten Proteinen befolgt werden sollten.\" \/>\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\/de\/news\/analyse-der-wirtszellproteine\/\" \/>\n<meta property=\"og:locale\" content=\"de_DE\" \/>\n<meta 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