Solid-phase protein PEGylation: Achieving mono-PEGylation through molecular tethering
Thesis DisciplineChemical Engineering
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Protein PEGylation (covalent attachment of poly(ethylene glycol) or PEG to proteins) is an excellent example of a drug delivery system that improves pharmacokinetics and pharmacodynamic properties of therapeutics. However, although PEGylation is clinically proven and attracts both scientific and commercial interest, the technique is associated with many process constraints, in particular related to controlling the number of conjugated PEG chains. A novel, solid-phase PEGylation methodology was attempted to overcome the drawbacks of the commonly used solution-phase methods for preparing PEGylated products. The solid-phase PEGylation methodology involved conjugating protein onto a tethered PEG derivative attached onto a solid matrix, followed by hydrolytic cleavage of the PEG chain from the solid matrix under mild conditions to yield PEGylated protein in free solution.
PEGs with molecular weights (MWs) 2000 and 4000 Da were used and a heterobifunctional PEG derivative, α-(β-alanine)-ω-carboxy PEG, with a cleavable β-alanine ester terminal was prepared for surface grafting and protein conjugation. The amine terminal of this PEG derivative was used for grafting PEG onto carboxy functionalized hydrophilic Sephadex and hydrophobic polystyrene derivatives. The free carboxyl terminal was used for protein conjugation via amine coupling. A kinetic study of PEG-surface grafting was performed to understand the influence of a number of parameters on the PEG surface concentration and its conformation, including temperature, reaction time, nature of the matrix, solvent and base, and MW of PEG. PEG grafted matrices were characterized using various surface characterization tools including Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS).
Higher PEG grafting was observed with polystyrene matrices (up to 0.3 mmol/g) than either of the Sephadex derivatives (less than 0.15 mmol/g) using both molecular weights. Detailed surface characterization using XPS studies showed a layer thickness of 11.87 nm was achieved with polystyrene matrices using 4000 Da PEG derivatives after a grafting period of 72 hours at 40°C, indicating the presence of brush conformations for the grafted PEGs. In contrast, mushroom conformations were observed for PEG molecules grafted on both carboxymethyl and carboxypentyl Sephadex derivatives after the same reaction period, with a layer thickness of 2.62 nm and 4.14 nm respectively.
Optimized PEG grafting and hydrolysis conditions were developed for solid-phase protein PEGylation using Cytochrome c as a model protein. The presence of PEGylated species were detected by size exclusion chromatography (SEC) from Sephadex derivatives but were absent when using polystyrene matrices. Both Sephadex derivatives gave mainly multi-PEGylated species with poor yields, in place of the expected mono-PEGylated products. A solution-phase PEGylation using the same PEG derivatives was performed successfully and various PEGylated species were identified and characterized using SEC and gel electrophoresis, based on their viscosity radius.
An examination of the surface characteristics of the PEG-grafted was carried out by XPS, showing that protein conjugation was greatly influenced by surface force interactions, which depended on the PEG grafting densities and the nature of the solid matrices. Finally, fluorescent images obtained using confocal microscope with fluorescein isothiocyanate labelled Cytochrome c provided supporting evidence regarding the factors that constrained the solid-phase PEGylation process.