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Pharmaceutical Research

, Volume 28, Issue 7, pp 1661–1667 | Cite as

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

  • Michael James Wilson JohnstonEmail author
  • Grant Frahm
  • Xuguang Li
  • Yves Durocher
  • Mary Alice Hefford
Research Paper

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.

KEY WORDS

circular dichroism differential scanning calorimetry glycosylation interferon 

ABBREVIATIONS

CD

circular dichroism

EDQM

European Directorate for the Quality of Medicines

IFN

interferon

Tm

melting temperature

Notes

ACKNOWLEDGMENTS

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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Copyright information

© Her Majesty the Queen in Right of Canada  2011

Authors and Affiliations

  • Michael James Wilson Johnston
    • 1
    Email author
  • Grant Frahm
    • 1
  • Xuguang Li
    • 1
    • 3
  • Yves Durocher
    • 2
  • Mary Alice Hefford
    • 1
    • 3
  1. 1.Centre for Vaccine Evaluation Biologics & Genetic Therapies DirectorateHealth CanadaOttawaCanada
  2. 2.National Research Council Canada, Animal Cell Technology Group Bioprocess SectorBiotechnology Research InstituteMontrealCanada
  3. 3.Department of Biochemistry, Microbiology and ImmunologyUniversity of OttawaOttawaCanada

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