Dokument

Zusammenfassung:
Im Rahmen dieser Doktorarbeit wurde eine PCR-basierte Methode zum Nachweis der Abwesenheit von Mykoplasmen (Mollicutes) in der mikrobiologischen Qualitätskontrolle der Firma Novartis Vaccines & Diagnostics (NVD) am Standort Marburg etabliert. Ziel war es, die Grundlagen für die anschließende Validierung und Zulassung dieser Methode durch die Behörden zu schaffen, sodass beide, aktuell zur Freigabe der Impfstoffe verwendeten Test-Methoden (Kultivierungstest und Indikatorzelltest gemäß Europäischem Arzneibuch) durch das neue Verfahren ersetzt werden können. Die hierfür am besten geeignete Methode zur PCR-basierten Detektion der Mollicutes war das CytoInspectTM System der Firma Greiner Bio-One, deren Prinzip auf einer touch-down PCR, gefolgt von einer Microarray Analyse zur Detektion und Identifizierung der PCR-Produkte, beruhte. Im direkten Vergleich wurden drei „ready-to-use“-Kits betrachtet. Die Hauptkriterien waren dabei die in praktischen Versuchen gemessene Sensitivität für Proben mit hohen Zell- (>107 Zellen/ml) und Viruskonzentrationen, die Spezifität und die Positivkontrollen zur Überwachung der Funktionalität jedes einzelnen Tests. Für die Validierung des PCR-basierten Verfahrens und den Vergleich mit den bisherigen Methoden auf Kultivierungsbasis wurden Referenzstandards von zehn verschiedenen Mollicutes-Spezies etabliert. Es handelte sich dabei um aliquotierte und tiefgefrorene Zellsuspensionen. Das Hauptkriterium für die Eignung dieser Standards war ein möglichst niedriges und gleichmäßiges Verhältnis von Genkopien (GC) pro koloniebildender Einheit (KBE) in den einzelnen Aliquots der jeweiligen Zellsuspensionen. Alle zehn Referenzstandards wiesen einen stabilen Titer (KBE/ml) und ein Verhältnis von < 20 GC/KBE auf. Die Sensitivität des CytoInspectTM Kits wurde in Form der Nachweisgrenze bestimmt. Für die zehn, in dieser Arbeit getesteten Mollicutes-Spezies lag sie zwischen 22 und 0,5 GC/ml (entsprach 4,5 0,1 KBE/ml), bei einem Probenvolumen von 10 ml. Sie war vergleichbar mit der Nachweisgrenze des Kultivierungstests, die in dieser Arbeit bei 2 bis 0,1 KBE/ml gemessen wurde. Durch die ebenfalls hohe Sensitivität bei der Detektion freier DNA von bereits abgestorbenen Zellen, bietet diese Methode zusätzlich die Möglichkeit zur Prävention lebender Kontaminationen in den Produktionsanlagen. Die Robustheit des Verfahrens und die spezifische (ausschließliche) Detektion von Mollicutes mit dem CytoInspectTM Kit konnten durch praktische Tests gezeigt werden. Im Rahmen der Etablierung der Methode als Routineanwendung zur Chargenfreigabe von Impfstoffen unter den örtlichen Gegebenheiten in den Laboren der mikrobiologischen Qualitätskontrolle von NVD in Marburg wurde die Sicherheit des Testverfahrens bewertet. Dies erfolgte zum einen durch die Evaluierung der kritischen Schritte, die zu einem falsch-negativen Ergebnis führen könnten. Zum anderen wurde das Risiko für falsch-positive Ergebnisse, die durch Kontaminationen der Proben während der Testdurchführung entstehen könnten, bewertet und durch entsprechende Maßnahmen minimiert.

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