O-Linked Glycosylation Leads to Decreased Thermal Stability of Interferon Alpha 2b as Measured by Two Orthogonal Techniques
- 273 Downloads
Thermal stability is considered an indication of protein fold and conformational stability. We investigate the influence of glycosylation on the thermal stability of interferon alpha 2b (IFN α-2b).
Far ultraviolet light circular dichroism spectroscopy (UV CD) and differential scanning calorimetry (DSC) were used to assess the thermal stability of the European Directorate for the Quality of Medicines IFN α-2b reference standards as well as an O-linked glycosylated IFN α-2b produced in human embryonic kidney cells.
Assessment of thermal stability of IFN α-2b and glycosylated IFN α-2b by DSC revealed that non-glycosylated interferon (Tm = 65.7 +/− 0.2°C, n = 3) was more thermally stable than the glycosylated variant (Tm = 63.8 C +/− 0.4°C, n = 3). These observations were confirmed with far UV CD (Tm IFN α-2b = 65.3 +/− 0.4°C, Tm glycosylated IFN α-2b = 63.6 +/− 0.2°C, n = 3). Enzymatic deglycosylation of IFN α-2b resulted in improved thermally stability when assessed with far UV CD and DSC.
We demonstrate that O-linked glycosylation decreases the thermal stability of IFN α-2b compared to a non-glycosylated variant of the protein.
KEY WORDScircular dichroism differential scanning calorimetry glycosylation interferon
European Directorate for the Quality of Medicines
This research is supported by the Government of Canada. We thank Louise Larocque for her assistance in performing the potency assays and Dr. John K. Mark for his assistance with HPLC analysis. We also thank Dr. Jeremy Kunkel and Dr. Richard Isbrucker for their critical reading of the manuscript.
- 12.Larocque L, Blui A, Xu R, Diress A, Wang J, Lin R, et al. Bioactivity determination of native and variant forms of therapeutic interferons. J Biomed Biotech. 2011;in press.Google Scholar
- 19.Willuda J, Honegger A, Waibel R, Schubiger PA, Stahel R, Zangemeister-Wittke U, et al. High thermal stability is essential for tumor targeting of antibody fragments: engineering of a humanized anti-epithelial glycoprotein-2 (epithelial cell adhesion molecule) single-chain Fv fragment. Cancer Res. 1999;59:5758–67.PubMedGoogle Scholar
- 28.Silva MM, Gaines-Das RE, Jones C, Robinson CJ. Biological activity of EDQM CRS for Interferon alfa-2a and Interferon alfa-2b—assessment in two in vitro bioassays. Pharmeur Bio. 2007;2007:1–6.Google Scholar