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Commercially available, uncoated elastomeric closures were examined in regard to a potential contribution of extracted compounds from the rubber stoppers to protein aggregation under worst-case conditions. All rubber stoppers were confirmed to comply with Ph. Eur. quality requirements. Extraction with 2-propanol under reflux-conditions for 3h led to closure-specific extraction profiles of the tested samples. One type of rubber stopper exhibited a considerably greater number and higher content of extractables. Four extracted compounds were identified as trialkyl benzene-1, 2, 4–tricarboxylates (trivial name: trimellitates), a substance class which is increasingly established as an alternative to phthalates. A highly concentrated aqueous solution of total extractables from this rubber stopper facilitated the formation of soluble and non-soluble high-molecular aggregates when incubated with model biopharmaceuticals (recombinant human immunoglobulin G (IgG) and recombinant erythropoietin (EPO)) under stress conditions (IgG: 60–64°C for 130min, EPO: 55°C for 8days). Furthermore, it was shown that the surfactant concentration (polysorbate 20, 0.1m/v% vs. 1.0m/v%) decisively influenced the formation of high-molecular aggregates. In case of EPO, the 10 fold increased concentration of surfactant was sufficient to prevent the aggregate formation completely.This study suggests the necessity of revisiting the current test system of Ph. Eur. monograph 3.2.9 for appropriate rubber stopper quality evaluation.