Purpose:  Tungstate polyoxyanions have been implicated in the aggregation of proteins in prefilled syringes.  Assays based on SEC-HPLC and turbidity were used to determine protein sensitivity to tungstate and to investigate the effects of pH and ionic strength on the aggregation of proteins by tungstate polyoxyanions.

Methods: Sensitivity to aggregation by tungstate was evaluated by incubating proteins at  concentrations of 0.1- 1 mg/mL in microfuge tubes in 20 mM buffers pH 4-7 with tungstate at 1-100ppm.  Stock solutions of tungstate and NaCl were prepared in the same buffers and readjusted to the nominal pH.  After incubation overnight at 4°C, turbidity was measured spectrophotometrically at 350 nm.  Protein precipitates were removed by centrifugation and the soluble protein was analyzed by SEC-HPLC.

Results:  Protein aggregation in the presence of tungstate was observed from pH 4-5.5, but not at 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.

Conclusions:  Assays based on turbidity and SEC-HPLC 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 polyoxyanions and aggregation by tungstate polyoxyanions 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 that are free of both silicone oil and tungsten, such as Daikyo Crystal Zenith^® resin, should be considered for use with sensitive biomolecules.

Authors:
Lloyd Waxman, Tadd Steeley and Vinod Vilivalam

For more information, contact Vinod Vilivalam, Ph.D., Director, Strategic Operations, at 610-594-3147 or Vinod.Vilivalam@westpharma.com.