These tungsten residuals have been reported to have a marked effect on drug products stored in prefilled syringes. Lee et
al. examined the aggregation of proteins resulting from leached tungsten (11). They report that concentrations of tungsten
as low as 1 ppm in the drug product can produce measurable protein aggregation. Analysis of 500 syringes from various manufacturers
indicated that, in general, the level of leached tungsten is well below this critical concentration. These authors suggest
that tungsten speciation in solution is an important factor in protein aggregation, with large tungstate polyanions (the dominant
aqueous species at low pH) having a greater potential impact on aggregation than the smaller tungstate anion that dominates
tungsten speciation at higher pH values.
Similarly, Osterberg reports a situation in which tungsten leached from a syringe caused the oxidation of the contained drug
product, resulting in drug degradation and aggregation (12). This interaction ultimately led to an unacceptable drug product.
Rosenberg and Worobec also report the tungsten-induced aggregation of a protein arising from oxidation mediated by leached
tungsten (13), and Markovic documents a case where leached tungsten oxide triggered protein oxidation followed by aggregation
(14). In the latter case, the aggregation was mitigated by switching from a tungsten to a platinum filament. Finally, Wen
et al. report a case in which protein aggregation results from small particles of tungsten that were present in the drug product
as a result of its storage in a prefilled syringe (15).
oil. Silicon oil is a common lubricant used in many container–closure systems, including prefilled syringes. The potential influence
that silicone oil can have on the viability of protein-drug molecules is well documented.
Considering prefilled syringes specifically, Sharma notes that silicone oil has been implicated in the induction of protein
aggregation, although evidence in large changes in the protein in the presence of the lubricant is limited (16). Jones et
al. report a significant induction of protein aggregation (of four proteins of various molecular weights) occurred in the
presence of silicone oil and suggest that the most likely explanation for aggregation is that the silicon oil directly affects
intermolecular protein interactions or exerts an effect via a solvent (17). Markovic documents an instance in which the drug
product developed thread-like, gelatinous particles when stored in syringes for only a short period of time (less than one
hour) because of product interaction with the silicone oil (14). Alternatively, Overcashier demonstrated that the degradation
profiles two proteins of varying size were similar when the stored in either glass vials or prefilled syringes (18).
Leaching. One of the most widely documented instances of an unanticipated incompatibility between a container–closure system and a
protein-drug product is that of "Eprex" (epoetinum alfa) (Janssen-Ortho, ON, Canada) and its prefilled syringe packaging system
(19–21). At some point in its product lifetime (1998), Eprex, a product containing recombinant human erythropoietin, was reformulated with polysorbate 80, which replaced human serum
albumin as a formulation stabilizer. Shortly after this change, there were increased incidents of antibody-mediated pure red
cell aplasia (PCRA) with Eprex use by chronic renal failure patients. The cause of PCRA was directly linked to the formation
of neutralizing antibodies to both recombinant and endogenous erythropoietin in patients adminstered Eprex.
A considerable, crossfunctional technical effort was undertaken to establish the root cause of this phenomenon. One potential
root cause involved leached substances. The presence of previously unidentified leachables was suggested as new peaks in the
tryptic map of Eprex. Leaching studies determined that the polysorbate 80 extracted low levels of vulcanizing agents (and
related substances) from the uncoated rubber components of the prefilled syringe. This leaching issue was addressed by replacing
the rubber components with components coated with a fluoropolymer. Because the fluoropolymer is an effective barrier to migration,
the leaching of the rubber components was greatly reduced. Since the conversion from the uncoated to the coated components,
the incidence of PRCA has returned to the baseline rate seen for all marketed epoetin products. This is strong circumstantial
evidence that leaching of the vulcanizing agent was, in fact, the root cause of the observed effect. The circumstantial case
against rubber leachables as the root cause was further strengthened when the adjuvant effect of the leached vulcanizing agent
(and related substances) was confirmed in animal models. Nevertheless, it is noted that the link between the extracted vulcanizing
agent and the adverse patient effect has not been conclusively established and there are alternate proposals as to the root
cause of this phenomenon.