O-linked glycosylation leads to decreased thermal stability of interferon alpha 2b as measured by two orthogonal techniques.

PURPOSE: 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).
METHODS: 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.
RESULTS: 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.
CONCLUSION: We demonstrate that O-linked glycosylation decreases the thermal stability of IFN α-2b compared to a non-glycosylated variant of the protein.