Research Articles
Tungsten-induced protein aggregation: Solution behavior

https://doi.org/10.1002/jps.21778Get rights and content

Abstract

Tungsten has been associated with protein aggregation in prefilled syringes (PFSs). This study probed the relationship between PFSs, tungsten, visible particles, and protein aggregates. Experiments were carried out spiking solutions of two different model proteins with tungsten species obtained from the extraction of tungsten pins typically used in syringe manufacturing processes. These results were compared to those obtained with various soluble tungsten species from commercial sources. Although visible protein particles and aggregates were induced by tungsten from both sources, the extract from tungsten pins was more effective at inducing the formation of the soluble protein aggregates than the tungsten from other sources. Furthermore, our studies showed that the effect of tungsten on protein aggregation is dependent on the pH of the buffer used, the tungsten species, and the tungsten concentration present. The lower pH and increased tungsten concentration induced more protein aggregation. The protein molecules in the tungsten-induced aggregates had mostly nativelike structure, and aggregation was at least partly reversible. The aggregation was dependent on tungsten and protein concentration, and the ratio of these two and appears to arise through electrostatic interaction between protein and tungsten molecules. The level of tungsten required from the various sources was different, but in all cases it was at least an order of magnitude greater than the typical soluble tungsten levels measured in commercial PFS. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4695–4710, 2009

Section snippets

Abbreviations

DLS, dynamic light scattering; SEC, size exclusion chromatography; ICP, inductively coupled plasma; UV, ultraviolet; CD, circular dichroism; FTIR, Fourier-transformed infrared; PFS, prefilled syringe.

INTRODUCTION

In recent years, the range of protein biopharmaceuticals in the marketplace has changed dramatically, with an increase in the number of approved products, in the modalities of the proteins (cytokines, growth factors, monoclonal antibodies, etc.) and in the types of delivery devices and/or primary containers used (vial, PFS, etc.).1,2 With the change in complexity of protein products and delivery devices comes increased complexity in the interactions of the protein with components of the device

Materials

The two proteins studied were purified at Amgen (Thousand Oaks, CA) and are >98% pure by SEC. The two proteins were chosen as models to represent protein classes with high helical content and Fc fusion proteins, respectively. The alpha-helical protein contains four aspartic acid, nine glutamic acid, four lysine, five arginine, and five histidine for the charged amino acids. The numbers of each of the charged amino acids in the Fc fusion protein are: 20 aspartic acid, 25 glutamic acid, 26

Spiking of an Alpha-Helical Protein With Soluble Na2WO4 and Tungsten Pin Extract in Buffer A

The alpha-helical protein was spiked with soluble Na2WO4 at 100, 500, and 1000 ppm and with tungsten pin extract at 1, 10, 100, 500, and 1000 ppm of tungsten in plastic microfuge tubes, then filled into luer tip syringes. This provided the typical exposure to silicone oil that the protein–tungsten mixtures saw in the PFS where the aggregation was first observed and kept other factors constant while varying the amount and species of tungsten. Filling into PFS would have risked possible exposure

CONCLUSION

Our studies suggest that the tungsten species produced by high temperatures and contact with glass during PFS manufacturing are more potent in inducing the formation of protein oligomers and particles than any of the other tungstate species tested. Tungsten induces protein aggregation mainly through electrostatic interactions where the conformations of the proteins remain unchanged. The tungsten-induced protein aggregation was at least partially reversible depending on the pH of the solution,

Acknowledgements

The authors would like to thank David Brems for reviewing the manuscript and helpful discussions, Tiansheng Li and Merrill Goldenberg for their work on the tungsten model compounds and for allowing us to include their Raman spectra, Jenny Li for preparing the samples, the Amgen Quality Analytical Lab members for their assay support, Rob Swift for obtaining the tungsten pins from our supplier and helpful input and Amgen, Inc. for financial support. The authors would also like to thank John

REFERENCES (18)

There are more references available in the full text version of this article.

Cited by (0)

View full text