title: Comparison of site-specific PEGylations of the N-terminus of interferon beta-1b: selectivity, efficiency, and in vivo/vitro activity.
abstract: PEGylation, including nonspecific and site-directed approaches, is a well-established and validated strategy to increase the stability, in vivo plasma retention time, and efficacy of protein pharmaceutics together with a reduction in immunogenicity and hydrophobicity. Site-directed conjugation by PEG-aldehyde is the most widely used method for N-terminal modification; however, the generation of multimodified products is inevitable because of lysine chemistry, which always leads to difficulties in purification and quantification. In this study, we developed a specific PEGylation strategy through the periodation of the N-terminus of interferon beta-1b (IFN-β-1b) followed by the coupling of PEG-hydrazide. The prolonged elimination half-life and significantly diminished immunogenicity of the PEG-hydrazide-modified protein indicated the development of an effective process for improving the pharmacology and immunogenicity of IFN-β-1b. We further conducted comparisons on the selectivity, velocity, yield, and pharmacokinetics of the two methods. The results demonstrated that the hydrazide-based conjugation was a highly specific coupling reaction that only produced homogeneous N-terminal mono-PEGylated conjugate but also generated heterogeneous multimodified products in the aldehyde-based process. In addition, a better PEGylation yield was found for the hydrazide conjugation compared with that of the aldehyde strategy. These investigations supply a practical approach for the site-specific modification of proteins with an N-terminal serine or threonine to achieve improved homogeneity of the conjugates as well as enhanced pharmacological properties.
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences , Beijing 100190, China.
authors.forename: Zhan
authors.initials: Z
authors.lastname: Zhou
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Lijing
authors.initials: L
authors.lastname: Sun
authors.forename: Guanghui
authors.initials: G
authors.lastname: Ma
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Su
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D006801:Humans; D006834:Hydrazines / Q000737:chemistry*; D000068576:Interferon beta-1b; D016899:Interferon-beta / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D008958:Models, Molecular; D015394:Molecular Structure; D011092:Polyethylene Glycols / Q000737:chemistry*; D051381:Rats; D014709:Vero Cells
chemical_list: D006834:Hydrazines; D000068576:Interferon beta-1b; D011092:Polyethylene Glycols; D016899:Interferon-beta