Research ArticlesTungsten-induced protein aggregation: Solution behavior
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)
- et al.
Challenges in the development of high protein concentration formulations
J Pharm Sci
(2004) - et al.
Highy concentrated monoclonal antibody solutions: Direct analysis of physical structure and thermal stability
J Pharm Sci
(2007) - et al.
Some sodium tungstate-organic acid protein precipitants
Clin Chim Acta
(1973) - et al.
Isopolymolybdates and isopolytungstates
Inorg Chem Radiochem
(1976) - et al.
Root cause analysis of tungsten induced protein aggregation in pre-filled syringes
PDA Journal
(2009) - Narhi LO,Wen Z,Jiang Y.Tungsten and protein aggregation at 2007 Protein Stability Conference, Breckenridge, CO,...
- Faulkner E.Container closure interactions with biopharmaceutical parenteral products at Formulation Strategies for...
- et al.
Tungsten: Properties, chemistry, technology of the element, alloys, and chemical compounds
(1999) - et al.
Polyoxometalate binding to human serum albumin: A thermodynamic and spectroscopic approach
Phys Chem
(2007)