Skip to main content
Log in

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

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

CD:

circular dichroism

EDQM:

European Directorate for the Quality of Medicines

IFN:

interferon

Tm:

melting temperature

REFERENCES

  1. Almeida AJ, Souto E. Solid lipid nanoparticles as a drug delivery system for peptides and proteins. Adv Drug Deliv Rev. 2007;59:478–90.

    Article  PubMed  CAS  Google Scholar 

  2. Graddis TJ, Remmele Jr RL, McGrew JT. Designing proteins that work using recombinant technologies. Curr Pharm Biotechnol. 2002;3:285–97.

    Article  PubMed  CAS  Google Scholar 

  3. Veronese FM, Mero A. The impact of PEGylation on biological therapies. BioDrugs. 2008;22:315–29.

    Article  PubMed  CAS  Google Scholar 

  4. Sola RJ, Griebenow K. Effects of glycosylation on the stability of protein pharmaceuticals. J Pharm Sci. 2009;98:1223–45.

    Article  PubMed  CAS  Google Scholar 

  5. Jelkmann W. Developments in the therapeutic use of erythropoiesis stimulating agents. Br J Haematol. 2008;141:287–97.

    Article  PubMed  CAS  Google Scholar 

  6. Dummer R, Mangana J. Long-term pegylated interferon-alpha and its potential in the treatment of melanoma. Biologics. 2009;3:169–82.

    PubMed  CAS  Google Scholar 

  7. Sola RJ, Griebenow K. Glycosylation of therapeutic proteins: an effective strategy to optimize efficacy. BioDrugs. 2010;24:9–21.

    Article  PubMed  CAS  Google Scholar 

  8. Loignon M, Perret S, Kelly J, Boulais D, Cass B, Bisson L, et al. Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells. BMC Biotechnol. 2008;8:65.

    Article  PubMed  Google Scholar 

  9. Adolf GR, Kalsner I, Ahorn H, Maurer-Fogy I, Cantell K. Natural human interferon-alpha 2 is O-glycosylated. Biochem J. 1991;276(Pt 2):511–8.

    PubMed  CAS  Google Scholar 

  10. Johnson WC. Analyzing protein circular dichroism spectra for accurate secondary structures. Proteins. 1999;35:307–12.

    Article  PubMed  CAS  Google Scholar 

  11. Greenfield NJ. Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions. Nat Protoc. 2006;1:2527–35.

    Article  PubMed  CAS  Google Scholar 

  12. Larocque L, Blui A, Xu R, Diress A, Wang J, Lin R, et al. Bioactivity determination of native and variant forms of therapeutic interferons. J Biomed Biotech. 2011;in press.

  13. Johnston MJ, Nemr K, Hefford MA. Influence of bovine serum albumin on the secondary structure of interferon alpha 2b as determined by far UV circular dichroism spectropolarimetry. Biologicals. 2010;38:314–20.

    Article  PubMed  CAS  Google Scholar 

  14. Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS. Stability of protein pharmaceuticals: an update. Pharm Res. 2010;27:544–75.

    Article  PubMed  Google Scholar 

  15. Philo JS, Arakawa T. Mechanisms of protein aggregation. Curr Pharm Biotechnol. 2009;10:348–51.

    Article  PubMed  CAS  Google Scholar 

  16. Marshall SA, Lazar GA, Chirino AJ, Desjarlais JR. Rational design and engineering of therapeutic proteins. Drug Discov Today. 2003;8:212–21.

    Article  PubMed  CAS  Google Scholar 

  17. Worn A, Auf der Maur A, Escher D, Honegger A, Barberis A, Pluckthun A. Correlation between in vitro stability and in vivo performance of anti-GCN4 intrabodies as cytoplasmic inhibitors. J Biol Chem. 2000;275:2795–803.

    Article  PubMed  CAS  Google Scholar 

  18. Di NL, Whitson LJ, Cao X, Hart PJ, Levine RL. Proteasomal degradation of mutant superoxide dismutases linked to amyotrophic lateral sclerosis. J Biol Chem. 2005;280:39907–13.

    Article  Google Scholar 

  19. Willuda J, Honegger A, Waibel R, Schubiger PA, Stahel R, Zangemeister-Wittke U, et al. High thermal stability is essential for tumor targeting of antibody fragments: engineering of a humanized anti-epithelial glycoprotein-2 (epithelial cell adhesion molecule) single-chain Fv fragment. Cancer Res. 1999;59:5758–67.

    PubMed  CAS  Google Scholar 

  20. Cha SS, Kim JS, Cho HS, Shin NK, Jeong W, Shin HC, et al. High resolution crystal structure of a human tumor necrosis factor-alpha mutant with low systemic toxicity. J Biol Chem. 1998;273:2153–60.

    Article  PubMed  CAS  Google Scholar 

  21. Durocher Y, Butler M. Expression systems for therapeutic glycoprotein production. Curr Opin Biotechnol. 2009;20:700–7.

    Article  PubMed  CAS  Google Scholar 

  22. Narhi LO, Arakawa T, Aoki KH, Elmore R, Rohde MF, Boone T, et al. The effect of carbohydrate on the structure and stability of erythropoietin. J Biol Chem. 1991;266:23022–6.

    PubMed  CAS  Google Scholar 

  23. Runkel L, Meier W, Pepinsky RB, Karpusas M, Whitty A, Kimball K, et al. Structural and functional differences between glycosylated and non-glycosylated forms of human interferon-beta (IFN-beta). Pharm Res. 1998;15:641–9.

    Article  PubMed  CAS  Google Scholar 

  24. Yesilyurt BT, Gielens C, Meersman F. Thermal stability of homologous functional units of Helix pomatia hemocyanin does not correlate with carbohydrate content. FEBS J. 2008;275:3625–32.

    Article  PubMed  CAS  Google Scholar 

  25. Spiriti J, Bogani F, van der Vaart A, Ghirlanda G. Modulation of protein stability by O-glycosylation in a designed Gc-MAF analog. Biophys Chem. 2008;134:157–67.

    Article  PubMed  CAS  Google Scholar 

  26. Ceaglio N, Etcheverrigaray M, Kratje R, Oggero M. Novel long-lasting interferon alpha derivatives designed by glycoengineering. Biochimie. 2008;90:437–49.

    Article  PubMed  CAS  Google Scholar 

  27. Ceaglio N, Etcheverrigaray M, Conradt HS, Grammel N, Kratje R, Oggero M. Highly glycosylated human alpha interferon: an insight into a new therapeutic candidate. J Biotechnol. 2010;146:74–83.

    Article  PubMed  CAS  Google Scholar 

  28. Silva MM, Gaines-Das RE, Jones C, Robinson CJ. Biological activity of EDQM CRS for Interferon alfa-2a and Interferon alfa-2b—assessment in two in vitro bioassays. Pharmeur Bio. 2007;2007:1–6.

    CAS  Google Scholar 

  29. Klaus W, Gsell B, Labhardt AM, Wipf B, Senn H. The three-dimensional high resolution structure of human interferon alpha-2a determined by heteronuclear NMR spectroscopy in solution. J Mol Biol. 1997;274:661–75.

    Article  PubMed  CAS  Google Scholar 

  30. Simons B, Scholl D, Cyr T, Hefford MA. Effects of increased loop flexibility on the structure and stability of a de novo designed helical protein. Protein Pept Lett. 2001;8:89–96.

    Article  CAS  Google Scholar 

  31. Kumaran J, Wei L, Kotra LP, Fish EN. A structural basis for interferon-alpha-receptor interactions. FASEB J. 2007;21:3288–96.

    Article  PubMed  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Michael James Wilson Johnston.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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). https://doi.org/10.1007/s11095-011-0402-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-011-0402-0

KEY WORDS

Navigation