The aggregation of protein in therapeutic products has the potential to cause severe immune responses in patients. Silicone oil, which is used to lubricate glass prefillable syringes, has been reported to induce protein aggregation, and in several cases, the aggregation of therapeutic proteins has been attributed to the presence of tungsten particles in prefilled syringes (1, 2). The source of tungsten in glass prefilled syringes appears to be tungsten oxide vapor deposits in the syringe-funnel area from the heated tungsten pins used to produce the channel through which the needle is mounted (3). Two recent in vitro studies have identified conditions required to induce protein aggregation by tungsten and have shown that soluble tungsten polyanions generated at acidic pH are responsible for tungsten induced protein aggregation (4, 5, 6). In addition to being primarily electrostatic in nature and dependent on the concentration of both protein and tungsten, the interaction is partially reversible, and calculations have shown that the protein coprecipitates with polytungstate as a charge-neutral complex (5). Both reports used soluble tungsten species from commercial sources and concluded that acidified sodium tungstate (Na2WO4) was the most potent. Extracts prepared from used tungsten pins were also effective at inducing formation of protein aggregates (4).
Materials and methodsPreparation of reagents. Sensitivity to aggregation by tungstate was evaluated by incubating proteins at concentrations of 0.1- 1 mg/mL at pH 4–7 with tungstate at 1–100 ppm. Buffers used in these studies had a concentration of 20 mM: sodium acetate was used at pH 4.0, 4.5, 5.0 and 5.5, sodium citrate was used at pH 6.0 and sodium phosphate at pH 7.0. Stock solutions of tungstate were prepared at a concentration of 10,000 ppm by dissolving sodium tungstate powder in each buffer and readjusting the pH. Other concentrations were prepared by serial dilution of the 10,000 ppm solutions. Stock solutions of 1 M NaCl were prepared at each pH to study the effect of ionic strength on protein precipitation by tungstate.
Protein aggregation assays. Assays were carried out in microfuge tubes in a volume of 1200 μL. The reaction mixture consisted of 120 μL protein solution, 960 μL buffer, and 120 μL tungstate stock solution at the same pH. The final concentration of protein was typically 0.1–0.5 mg/mL. The mixtures were stored overnight at 4 °C prior to analysis. Controls contained buffer in place of tungstate to determine whether pH alone had an effect on protein aggregation. To examine the effect of ionic strength, aliquots of stock solutions of NaCl prepared at various pHs were added to the incubation mixtures instead of the buffer.
Two methods were used to measure protein aggregation:
Tungsten and silicone content of prefilled syringes. Tungsten was analyzed using inductively coupled plasma–mass spectroscopy (ICP-MS, SCIEX Elan DRC II, Perkin Elmer). Tungsten was extracted from glass or plastic syringes by drawing up 1 mL 2% HNO3 or 1 mL 5% NH4OH and sonicating the filled syringes for 1 h at 50 °C. The extracts were dispensed and the syringes were flushed with an additional 1 mL of solvent. The extract and the rinsate were pooled and brought to 3 mL with water. The lower limit of quantification is ~2.5 ng/mL, corresponding to ~1 μg/syringe.
Silicone was determined by atomic absorption spectroscopy (Analyst 100, Perkin Elmer). The needles were cut off four 1 mL long plastic syringes (Crystal Zenith (CZ), Daikyo) or four glass syringes (~100 cm2 barrel surface area, Becton Dickson). The syringes were extracted with 50 mL methyl isobutyl ketone to solubilize the silicone. Blanks were prepared similarly. The lower limit of quantification was approximately 8 μg/mL, corresponding to 100 μg/syringe.