caa a22 4500 445312289 CHVBK 20180317142634.0 cr unu---uuuuu 170323e20110701xx s 000 0 eng 10.1007/s11095-011-0402-0 doi (NATIONALLICENCE)springer-10.1007/s11095-011-0402-0 O-Linked Glycosylation Leads to Decreased Thermal Stability of Interferon Alpha 2b as Measured by Two Orthogonal Techniques [Elektronische Daten] [Michael Johnston, Grant Frahm, Xuguang Li, Yves Durocher, Mary Hefford] 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 = 63.8C +/− 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. Her Majesty the Queen in Right of Canada, 2011 circular dichroism nationallicence differential scanning calorimetry nationallicence glycosylation nationallicence interferon nationallicence CD : circular dichroism nationallicence EDQM : European Directorate for the Quality of Medicines nationallicence IFN : interferon nationallicence Tm : melting temperature nationallicence Johnston Michael Centre for Vaccine Evaluation Biologics & Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, K1A 0K9, Ottawa, Ontario, Canada aut Frahm Grant Centre for Vaccine Evaluation Biologics & Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, K1A 0K9, Ottawa, Ontario, Canada aut Li Xuguang Centre for Vaccine Evaluation Biologics & Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, K1A 0K9, Ottawa, Ontario, Canada aut Durocher Yves National Research Council Canada, Animal Cell Technology Group Bioprocess Sector, Biotechnology Research Institute, 6100 Royalmount Ave., H4P 2R2, Montreal, Quebec, Canada aut Hefford Mary Centre for Vaccine Evaluation Biologics & Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, K1A 0K9, Ottawa, Ontario, Canada aut Pharmaceutical Research Springer US; http://www.springer-ny.com 28/7(2011-07-01), 1661-1667 0724-8741 28:7<1661 2011 28 11095 https://doi.org/10.1007/s11095-011-0402-0 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s11095-011-0402-0 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Johnston Michael Centre for Vaccine Evaluation Biologics & Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, K1A 0K9, Ottawa, Ontario, Canada aut NATIONALLICENCE 700 1- Frahm Grant Centre for Vaccine Evaluation Biologics & Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, K1A 0K9, Ottawa, Ontario, Canada aut NATIONALLICENCE 700 1- Li Xuguang Centre for Vaccine Evaluation Biologics & Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, K1A 0K9, Ottawa, Ontario, Canada aut NATIONALLICENCE 700 1- Durocher Yves National Research Council Canada, Animal Cell Technology Group Bioprocess Sector, Biotechnology Research Institute, 6100 Royalmount Ave., H4P 2R2, Montreal, Quebec, Canada aut NATIONALLICENCE 700 1- Hefford Mary Centre for Vaccine Evaluation Biologics & Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, K1A 0K9, Ottawa, Ontario, Canada aut NATIONALLICENCE 773 0- Pharmaceutical Research Springer US; http://www.springer-ny.com 28/7(2011-07-01), 1661-1667 0724-8741 28:7<1661 2011 28 11095 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer