Evaluating Separations of PEGylated Proteins using Gel Filtration Chromatography

The use of polyethylene glycols (PEG) is widespread in the pharmaceutical industry as a method to improve the pharmacokinetic qualities of protein and peptide therapeutics. However, such modifications also increase the heterogeneity of protein structures and generate difficulties in purifying such modified species away from the native protein. As PEGylation exhibits the majority of its effect on the size of protein and peptides, gel filtration chromatography is an excellent method for separating proteins by their degree of PEGylation. Efforts were undertaken to evaluate using gel filtration as a method for purifying PEGylated proteins from their unmodified precursors. Several different proteins and PEGylation chemistries were evaluated on BioSep SEC 2000 HPLC columns. Results show that retention of PEGylated species is directly related to the size of the PEG reagent used; proteins modified with larger PEG moieties generally demonstrate greater resolution from unmodified proteins than small modifications. Such results show the utility of GFC using BioSep 2000 as a means of characterizing and purifying PEGylated proteins.