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O-Linked Glycosylation Leads to Decreased Thermal Stability of Interferon Alpha 2b as Measured by Two Orthogonal Techniques



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.

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circular dichroism


European Directorate for the Quality of Medicines




melting temperature


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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.

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Correspondence to Michael James Wilson Johnston.

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Johnston, M.J.W., Frahm, G., Li, X. et al. O-Linked Glycosylation Leads to Decreased Thermal Stability of Interferon Alpha 2b as Measured by Two Orthogonal Techniques. Pharm Res 28, 1661–1667 (2011).

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  • circular dichroism
  • differential scanning calorimetry
  • glycosylation
  • interferon