Protein Sensitivity to Tungsten

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.

WEST

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.

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.

Table I: Biophysical properties of proteins

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.

Results and discussion

Protein aggregation in the presence of tungstate was observed from pH 4 to 5.5, but not at pH 6 or 7. Aggregation was blocked by increasing the ionic strength with NaCl. The concentration of salt required to prevent precipitation was protein-dependent, but also varied with both pH and tungstate concentration. Once formed, precipitates could be resolubilized by raising the pH to 7. Not all proteins precipitated in the presence of tungstate, including those with low isoelectric points. Unlike glass prefilled syringes, which contained measurable amounts of tungsten and silicone, syringes made of plastic were both tungsten and silicone-free.

Aggregation by tungstate requires acidic pH. Assays were carried out with bovine IgG (bIgG) over the pH range 4–7 at 1, 10, and 100 ppm tungstate. Figure 1 shows that the bIgG monomer is quantitatively precipitated by 10 and 100 ppm tungstate at pH 4.0–5.5, but is not affected at pH 6.0 or 7.0. The data also show that lower tungstate (1 ppm) is not as effective as higher concentrations at precipitating bIgG.

Figure 1: Effect of pH on the precipitation of bIgG by tungstate. Soluble monomer was quanititated by size-exclusion chromatography. (ALL FIGURES ARE COURTESY OF THE AUTHORS)

The effect of ionic strength on protein precipitation by tungstate. The effect of ionic strength on the precipitation of proteins by tungstate was investigated as many drug formulations contain salt. Figure 2 shows the effect of ionic strength on the precipitation of solutions of Chymotrypsinogen A (Chy A) and bIgG by tungstate at pH 5.0. The data indicate that 100 mM NaCl was sufficient to completely block the precipitation of ChyA by tungstate. In contrast, more than 500 mM NaCl was required to completely prevent precipitation of bIgG under the same conditions. In addition, higher concentrations of salt were necessary to block aggregation in the presence of 100 ppm tungstate than 10 ppm tungstate, and higher ionic strength was required to block aggregation by tungstate at pH 5.0 than at pH 5.5 (data not shown). These experiments show that the interaction of tungstate with proteins is primarily electrostatic and that the interaction is stronger at lower pH.

Figure 2: Effect of ionic strength on the precipitation of proteins by tungstate. Soluble protein was quantitated by size-exclusion–high-performance liquid chromatography.

Using turbidity to evaluate protein aggregation by tungstate. Measurement of protein turbidity caused by tungstate aggregation provides a rapid alternative to SEC. Turbidity was monitored spectrophotometrically by measuring the change in absorbance at 350 nm, a wavelength at which protein absorbance is negligible. Figure 3 compares data obtained for four proteins using both methods. The concentrations required to precipitate one-half of the protein (IC50s) for both methods are 10–20 ppm tungstate. An assay based on turbidity could also be performed in a microplate format which would increase throughput and reduce sample handling.

Figure 3: Comparison of protein aggregation by size-exclusion–high-performance liquid chromatography (SEC) with turbidity.

Aggregation by tungstate is correlated with protein net charge. The interaction of tungstate polyoxyanions appears to be primarily electrostatic; therefore, proteins with a net positive charge at a particular pH should be precipitable at that pH. When tested at pH 4, there was little difference between the IC50s for bovine serum albumin (BSA, isoelectric point (pI) 4.2–4.9), ovalbumin (pI 4.5–4.7), and the basic proteins of ChyA (pI 8.8–9.6) and lysozyme (pI 10.5–11) (see Figure 4). These four proteins all differ in mass, isoelectric point, net charge, and total number of positive charges (see Table I). However, pepsin, which has a pI below 3 and a net negative charge at pH 4, was not precipitated by tungstate even at 1000 ppm (data not shown). This result is consistent with the proposal that the net charge on a protein is a key determinant of precipitability.

Figure 4: Effect of pH on the precipitation of ovalbumin and lysozyme. Turbidity was measured spectrophotometrically.

Based on these observations, the authors tested whether the pH range over which a protein would be precipitated by tungstate could be predicted. The pH dependence of ovalbumin (pI 4.5–4.7) was compared with lysozyme (pI 10.5–11.0). Figure 4 shows that lysozyme could be precipitated from pH 4 to 5.5, a range over which it remains positively charged. In contrast, ovalbumin was only precipitable at pH 4 and 4.5, but at pH 5 (slightly above its isoelectric point and close to where it is electroneutral), it was no longer precipitable.

The interaction of proteins with tungstate is reversible. To determine whether protein/tungstate precipitates are reversible, a set of precipitates of BSA and bIgG were prepared using pH 5.0 buffer and 100 ppm tungstate, conditions shown to quantitatively precipitate each protein. The precipitates were re-suspended in pH 5.0 or pH 7.0 buffer with or without 0.5 M NaCl. Table II shows that dilution into incubation buffer in the absence of tungstate did not solubilize the precipitates. The addition of 0.5 M NaCl at low pH solubilized about 11% of bIgG. Re-suspension in pH 7.0 buffer solubilized greater than 90% of BSA and ~60% of bIgG. However, inclusion of 0.5 M NaCl in pH 7.0 buffer completely solubilized bIgG.

Table II: Reversibility of protein aggregates

Tungsten- and silicone-free prefilled syringes for sensitive proteins. Unlike staked needle syringes made of glass, manufacture of the specialty plastic syringe system (CZ, Daikyo) does not utilize a tungsten pin to create an opening for inserting the needle. Consequently, this syringe system is expected to be tungsten-free. Analysis showed that the amount of extractable tungsten in the plastic syringes is at the level of the solvent blank whereas the amount of tungsten in syringes made of glass is significant (see Table III).

Table III: Tungsten and silicone content of prefilled syringes

Silicone is present in glass syringes as a lubricant. However, silicone has been observed to cause changes in native protein structure as well as aggregation (7, 8). Analysis showed that specialty plastic syringes, which do not contain silicone oil as a lubricant, are silicon free (see Table III).

Conclusion

Assays based on turbidity and SEC can be used to evaluate protein sensitivity to tungsten in order to optimize formulations for the packaging of biologics in glass prefilled syringes. Ionic strength and pH are important elements in the aggregation of proteins by tungstate polyoxyanions. An acidic pH is necessary for the generation of tungstate polyoxyanions and aggregation may be blocked by increasing the ionic strength of the formulation. This suggests that the interaction of polyoxometalates with proteins is primarily electrostatic. Some proteins precipitate with tungstate more readily than others, but a net positive charge is essential. Although protein size does not appear to be important, other factors related to a protein's structure may play a role in determining binding and aggregation by tungstate polyoxyanions. Syringes made of plastic, which are free of both silicone oil and tungsten, should be considered for use with sensitive biomolecules.

Vinod Vilivalam*, PhD, is director of strategic market and technical development, Lloyd Waxman is a principal scientist, West Analytical Labs, Tadd Steeley is a senior chemist, all at West Pharmaceutical Services, 101 Gordon Drive, Lionville, PA 1934, tel: 610.594.3147, vinod.vilivalam@westpharma.com.

*To whom all correspondence should be addressed.

References

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