- 1:34 PM

Capillary Electrophoresis of Pegylated Proteins

Jessica R. Molek, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406 and Andrew L. Zydney, Department of Chemical Engineering, The Pennsylvania State University, 220 Fenske Laboratory, University Park, PA 16802.

There is growing clinical interest in the use of PEGylated recombinant proteins, formed by covalent attachment of polyethylene glycol, due to their increased stability and bioavailablility. Although capillary electrophoresis has been used to analyze the composition of PEGylated protein mixtures, there is little understanding of the physical characteristics of these biomolecules. The objective of this study was to obtain quantitative data for the electrophoretic mobility and resolution of PEGylated alpha-lactalbumin using capillary electrophoresis. Experiments were performed using several buffers, with best resolution and peak symmetry obtained in the presence of 0.1 M glycine. Data were also obtained with a series of acetylated alpha-lactalbumins having equal number of modifications as the PEGylated species, providing a direct measure of the additional hydrodynamic drag associated with the PEG. Experimental data for the drag ratio were in good agreement with model calculations using the effective size of the PEGylated proteins determined by size exclusion chromatography assuming that the protein charge was uniformly distributed over the outer surface of the sphere. These results provide important insights into the use of capillary electrophoresis for analysis of PEGylated proteins and provide additional insights into the underlying structure of the PEGylated species.