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Updated: Jun 3, 2026

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α
Published on: June 14, 2018
O-linked glycosylation leads to decreased thermal stability of interferon alpha 2b as measured by two orthogonal
Michael James Wilson Johnston1, Grant Frahm, Xuguang Li
1Centre for Vaccine Evaluation Biologics & Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, Ottawa, Ontario, K1A 0K9, Canada. michael.johnston@hc-sc.gc.ca
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.
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