Protein Sensitivity to Tungsten

The authors investigate the effect of low pH and ionic strength on aggregation using turbidity measurements and size-exclusion–high-performance liquid chromatography.
Nov 01, 2011
Volume 2011 Supplement, Issue 6

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).

Turbidity measurements and size-exclusion–high-performance liquid chromatography (SEC) can be used to monitor aggregation by Na2WO4. Under the extreme conditions of low pH and ionic strength used to investigate this phenomenon in this study, most proteins are susceptible to precipitation by soluble tungsten. Data suggest that the net charge on a protein is a predictor of precipitation by tungsten. The study also shows the absence of silicon oil and tungsten in prefilled syringes made of plastic. For proteins sensitive to aggregation by tungsten or silicone oil, plastic syringes are an attractive alternative to syringes made of glass.

Materials and methods

Preparation 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.

Table I: Biophysical properties of proteins
Protein solutions were prepared in deionized water or in buffer at a concentration of 1 or 5 mg/mL. The protein concentrations were verified spectrophotometrically using published extinction coefficients. The properties of some of the proteins used in this study are listed in Table I.

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:

  • Protein turbidity was determined spectrophotometrically by measuring the change in absorbance at 350 nm after storage compared to a control containing no tungstate.
  • Aggregation was quantitated by SEC analysis of the same samples following centrifugation (15,000 × g/10 min) to remove insoluble aggregates. Soluble protein was injected onto a GE Amersham Superdex 200 column (1 × 30 cm) controlled by a Waters 2695 liquid chromatography system. Proteins were detected by their absorbance at 280 nm. Recovery was determined by comparing the area under the peak relative to the control. The elution buffer was 20 mM sodium phosphate at pH 7.0 containing either 150 or 500 mM NaCl.

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.

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