title: Solid-phase polyethylene glycol conjugation using hydrophobic interaction chromatography.
abstract: PEGylation is a widely applied approach to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. The current solution-phase PEGylation protocols often suffer from poor yield of homogeneously PEGylated bioactive products and hence fall short of being commercially attractive. To improve upon these techniques, here we developed a novel, solid-phase PEGylation methodology using a hydrophobic interaction chromatography (HIC) resin. Two variations of the HIC-based PEGylation are described that are tailored towards conjugation of proteins with hydrophobicity index above (lysozyme) and below (fibroblast growth factor 1, FGF-1) that of the mPEG-butyraldehyde (mPEG) chain used. In the case of lysozyme, the protein was first immobilized on the HIC, and the HIC-bound protein was then conjugated by passing over the column. In the case of FGF-1, the mPEG solution was first immobilized on the HIC, and the FGF-1 solution was then passed through the column. Circular dichroism (CD) spectroscopy demonstrated HIC-based PEGylation almost retained the secondary structures of proteins. Bioactivity assay showed that the recovery of activity of HIC-based PEGylated rhFGF-1 (i.e. 92%) was higher than that of liquid-phase PEGylated rhFGF-1 (i.e. 61%), while HIC-based PEGylated lysozyme showed the same activity recovery (i.e. 7%) as the liquid-phase PEGylated form. For specific proteins, the HIC-based solid-phase PEGylation maybe offer a more promising alternative than the current PEGylation methods and is expected to have a major impact in the area of protein-based therapeutics.
authors.affiliation: School of Pharmacy, Wenzhou Medical University, Wenzhou 325035, Zhejiang, China; Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041, Sichuan, China.
authors.forename: Jianlou
authors.initials: J
authors.lastname: Niu
authors.affiliation: School of Pharmacy, Wenzhou Medical University, Wenzhou 325035, Zhejiang, China.
authors.forename: Yanlin
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: School of Pharmacy, Wenzhou Medical University, Wenzhou 325035, Zhejiang, China.
authors.forename: Yaoyao
authors.initials: Y
authors.lastname: Xie
authors.affiliation: School of Pharmacy, Wenzhou Medical University, Wenzhou 325035, Zhejiang, China.
authors.forename: Lintao
authors.initials: L
authors.lastname: Song
authors.affiliation: School of Pharmacy, Wenzhou Medical University, Wenzhou 325035, Zhejiang, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Shi
authors.affiliation: School of Pharmacy, Wenzhou Medical University, Wenzhou 325035, Zhejiang, China.
authors.forename: Junjie
authors.initials: J
authors.lastname: Lan
authors.affiliation: Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041, Sichuan, China.
authors.forename: Bailin
authors.initials: B
authors.lastname: Liu
authors.affiliation: School of Pharmacy, Wenzhou Medical University, Wenzhou 325035, Zhejiang, China.
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Li
authors.affiliation: School of Pharmacy, Wenzhou Medical University, Wenzhou 325035, Zhejiang, China. Electronic address: nyuhuang@gmail.com.
authors.forename: Zhifeng
authors.initials: Z
authors.lastname: Huang
mesh_terms: D002845:Chromatography / Q000379:methods; D002942:Circular Dichroism; D016220:Fibroblast Growth Factor 1 / Q000032:analysis; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D009113:Muramidase / Q000032:analysis; D011092:Polyethylene Glycols / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000032:analysis; D012996:Solutions
chemical_list: D011994:Recombinant Proteins; D012996:Solutions; D016220:Fibroblast Growth Factor 1; D011092:Polyethylene Glycols; C028210:monomethoxypolyethylene glycol; D009113:Muramidase
keywords: FGF-1; Hydrophobic interaction chromatography; Lysozyme; Solid-phase PEGylation; mPEG-butyraldehyde