Elimination of N-glycosylation by site mutation further prolongs the half-life of IFN-α/Fc fusion proteins expressed in Pichia pastoris

Microbial Cell Factories

Research

Changsheng Shao¹  Xiaoping Song²  Hao Jia³  Mingyang Shi³  Yuhui Miao³  Dong Wang³  Rui Li³  Yugang Guo⁴  Jing Wu⁴  Jiajia Ma⁴  Weihua Xiao⁴  Zhigang Tian⁴ 

[1] Hefei National Laboratory for Physical Sciences at the Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China;Anhui Engineering Research Center of Recombinant Protein Pharmaceutical Biotechnology, Institute of Advanced Technology, University of Science and Technology of China, Hefei, China;The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Signaling Network, School of Life Sciences, University of Science and Technology of China, Hefei, China;Department of Pharmacy, Anhui Medical College, Hefei, China;The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Signaling Network, School of Life Sciences, University of Science and Technology of China, Hefei, China;Hefei National Laboratory for Physical Sciences at the Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China;The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Signaling Network, School of Life Sciences, University of Science and Technology of China, Hefei, China;Hefei National Laboratory for Physical Sciences at the Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China;Anhui Engineering Research Center of Recombinant Protein Pharmaceutical Biotechnology, Institute of Advanced Technology, University of Science and Technology of China, Hefei, China;

关键词: IFN-α/Fc;    Fusion protein;    Glycosylation;    Circulation half-life;    Pichia pastoris;   

DOI  :  10.1186/s12934-016-0601-9

 received in 2016-07-30, accepted in 2016-11-23,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundInterferon (IFN)-α has been commonly used as an antiviral drug worldwide; however, its short half-life in circulation due to its low molecular weight and sensitivity to proteases impacts its efficacy and patient compliance.ResultsIn this study, we present an IgG1 Fc fusion strategy to improve the circulation half-life of IFN-α. Three different forms of IgG1 Fc fragments, including the wild type, aglycosylated homodimer and aglycosylated single chain, were each fused with IFN-α and designated as IFN-α/Fc-WT, IFN-α/Fc-MD, and IFN-α/Fc-SC, respectively. The recombinant proteins were expressed in Pichia pastoris and tested using antiviral and pharmacokinetic assays in comparison with the commercial pegylated-IFN-α (PEG-IFN-α). The in vitro study demonstrated that IFN-α/Fc-SC has the highest antiviral activity, while IFN-α/Fc-WT and IFN-α/Fc-MD exhibited antiviral activities comparable to that of PEG-IFN-α. The in vivo pharmacokinetic assay showed that both IFN-α/Fc-WT and IFN-α/Fc-MD have a longer half-life than PEG-IFN-α in SD rats, but IFN-α/Fc-SC has the shortest half-life among them. Importantly, the circulating half-life of 68.3 h for IFN-α/Fc-MD was significantly longer than those of 38.2 h for IFN-α/Fc-WT and 22.2 h for PEG-IFN-α.ConclusionsThe results demonstrate that the elimination of N-glycosylation by mutation of putative N-glycosylation site further prolongs the half-life of the IFN-α/Fc fusion protein and could present an alternative strategy for extending the half-life of low-molecular-weight proteins expressed by P. pastoris for in vivo studies as well as for future clinical applications.

【 授权许可】

CC BY   
© The Author(s) 2016