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

ABSTRACT 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.