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

Abstract

Background: Interferon (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.

Results: In 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.

Conclusions: The 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.

Keywords: Circulation half-life; Fusion protein; Glycosylation; IFN-α/Fc; Pichia pastoris.

Fig. 1

Schematic diagram for IFN-α/Fc fusion proteins. The dimer is composed of two molecules of IFN-α joined to dimeric Fc, and the monomer has a single molecule of IFN-α linked to monomeric Fc. Black circles N-glycosylation site in the wild-type IgG1 Fc region; DB disulfide bridges between dimeric Fc; F-hinge full hinge region of IgG1; P-hinge partial hinge with the amino acid sequence of HTCPPCP

Fig. 2

Expression of the IFN-α/Fc-MD fusion protein. The transformed colonies of IFN-α/Fc-MD were screened by dot blot (a); and further confirmed by Western blot (b). a1–c8: the colony number. c The time period for the expression of IFN-α/Fc-MD during induction in fermentation was analyzed by Western blot under reduced and non-reduced conditions

Fig. 3

Characterization of purified IFN-α/Fc fusion proteins. a Comparison of purified IFN-α/Fc fusion proteins using SDS-PAGE under reduced and non-reduced conditions. b Western blot analysis of the purified IFN-α/Fc fusion proteins. The indicated samples were separated via reduced SDS-PAGE and identified through Western blot analysis with goat anti-human IgG-HRP conjugate or mouse anti-human IFN-α monoclonal antibodies, followed by rabbit anti-mouse IgG-HRP conjugate. c PAS staining of purified IFN-α/Fc fusion proteins via SDS-PAGE under reduced conditions. WT: IFN-α/Fc-WT, MD: IFN-α/Fc-MD, SC: IFN-α/Fc-SC, M: prestained protein marker

Fig. 4

Antiviral activity of IFN-α/Fc fusion proteins. The protective effects of IFN-α/Fc fusion proteins and two controls (recombinant human IFN-α and pegylated interferon PEG-IFN-α) at the indicated concentrations were evaluated in antiviral assays using human WISH (a) and bovine MDBK (b) challenged with the VSV virus. All data are presented here as mean ± standard deviation of the OD values

Fig. 5

Anti-proliferative activity of IFN-α/Fc fusion proteins. Daudi cells were incubated for 72 h in the presence of increasing concentrations of the indicated samples with triple parallel wells in 96-well plates. Cell proliferation was assessed using the MTT method. All data are presented here as mean ± standard deviation of the OD values

Fig. 6

Pharmacokinetics of IFN-α/Fc fusion proteins. SD rats (n = 3 per group) were intravenously administered a single dose of 30 µg/kg of the indicated samples via the vena caudalis. Blood samples were drawn prior to treatment and at 0.2, 8, 24, 48, 72, 96, 120 and 144 h after the treatment. The serum human IFN-α level was quantified using the CBA Human IFN-α Flex Set. All data are presented here as mean ± standard deviation of the concentrations