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.
Similar content being viewed by others
Abbreviations
- CD:
-
circular dichroism
- EDQM:
-
European Directorate for the Quality of Medicines
- IFN:
-
interferon
- Tm:
-
melting temperature
REFERENCES
Almeida AJ, Souto E. Solid lipid nanoparticles as a drug delivery system for peptides and proteins. Adv Drug Deliv Rev. 2007;59:478–90.
Graddis TJ, Remmele Jr RL, McGrew JT. Designing proteins that work using recombinant technologies. Curr Pharm Biotechnol. 2002;3:285–97.
Veronese FM, Mero A. The impact of PEGylation on biological therapies. BioDrugs. 2008;22:315–29.
Sola RJ, Griebenow K. Effects of glycosylation on the stability of protein pharmaceuticals. J Pharm Sci. 2009;98:1223–45.
Jelkmann W. Developments in the therapeutic use of erythropoiesis stimulating agents. Br J Haematol. 2008;141:287–97.
Dummer R, Mangana J. Long-term pegylated interferon-alpha and its potential in the treatment of melanoma. Biologics. 2009;3:169–82.
Sola RJ, Griebenow K. Glycosylation of therapeutic proteins: an effective strategy to optimize efficacy. BioDrugs. 2010;24:9–21.
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.
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.
Johnson WC. Analyzing protein circular dichroism spectra for accurate secondary structures. Proteins. 1999;35:307–12.
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.
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.
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.
Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS. Stability of protein pharmaceuticals: an update. Pharm Res. 2010;27:544–75.
Philo JS, Arakawa T. Mechanisms of protein aggregation. Curr Pharm Biotechnol. 2009;10:348–51.
Marshall SA, Lazar GA, Chirino AJ, Desjarlais JR. Rational design and engineering of therapeutic proteins. Drug Discov Today. 2003;8:212–21.
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.
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.
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.
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.
Durocher Y, Butler M. Expression systems for therapeutic glycoprotein production. Curr Opin Biotechnol. 2009;20:700–7.
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.
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.
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.
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.
Ceaglio N, Etcheverrigaray M, Kratje R, Oggero M. Novel long-lasting interferon alpha derivatives designed by glycoengineering. Biochimie. 2008;90:437–49.
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.
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.
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.
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.
Kumaran J, Wei L, Kotra LP, Fish EN. A structural basis for interferon-alpha-receptor interactions. FASEB J. 2007;21:3288–96.
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
Corresponding author
Rights 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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11095-011-0402-0