RESUMEN
Sterile pharmaceuticals require they be developed and manufactured using suitable container closure systems to maintain sterility until product opening. Characterizing container closure integrity (CCI) in relation to rubber stopper displacement was controversially discussed during the Annex 1 revision process. An automated inspection system can reject units with displaced rubber stoppers, and the related acceptance criteria for such in-process testing can be established by adequate studies. In this manuscript, we describe a novel helium leak CCI testing method to study the relation of rubber stopper displacement and CCI. Ten different commonly used vial-rubber stopper combinations were characterized, which led to robust test results. Pronounced differences between the different vial-rubber stopper combinations were observed, clearly showing that the combination of different stoppers, vials, and caps led to significant differences in allowable stopper displacement for routine manufacture.
Asunto(s)
Embalaje de Medicamentos/normas , Automatización , Industria Farmacéutica , Diseño de Equipo , Helio , Ensayo de Materiales , Control de Calidad , Reproducibilidad de los Resultados , Goma , Esterilización , Tecnología FarmacéuticaRESUMEN
To examine the dynamics of tight junctions (TJs) in living cells, chimera between the TJ-associated protein ZO-1 and green fluorescent protein (GFP) were constructed. If ZO-1 fused to the C-terminus of GFP (ZO1-CGFP) was stably expressed in MDCK cells, it was fully incorporated into TJs and colocalized with endogenous ZO-1. The GFP tag did not influence cell growth, transepithelial electrical resistance, and paracellular mannitol transport. The morphology of the transfected cells was unchanged. The ZO1-CGFP MDCK cell line thus represents an excellent tool to study TJ dynamics. The influence of the external calcium ion concentration on the formation and dynamics of TJs in living cells was thus explored. Upon opening of the TJs under short-term treatment with EGTA (up to 20 min), the localization of ZO1-CGFP at the membrane persisted. The rim-like pattern around the individual cells appeared fuzzier than in non-treated cells. Long-term calcium depletion resulted in the localization of ZO1-CGFP in the cytoplasm and the nucleus. After restoration of normal Ca(2+) concentrations, cell-cell contacts were restored and the localization of ZO1-CGFP was indistinguishable from the one in control cells kept at normal Ca(2+) concentrations. It remains open how the different localizations of ZO-1 correspond to changes in the signal transduction activity of the molecule.