Simultaneous detection of free polyethylene glycol and PEGylated proteins by western blot

This study presents detection of free polyethylene glycol (PEG) and conjugated PEG in western blot assay using anti-PEG rabbit monoclonals specific to methoxy group of PEG. In a chromogenic western blot assay, detection limit (DL) for free and PEGylated protein was 2.5 μg and 0.5 μg, respectively. Using chemiluminescence substrate, DL of PEGylated granulocyte colony stimulating factor (GCSF) was 100 pg. Assay applied to PEGylation reaction mixture detected both free and PEGylated protein. Commercially available PEGylated products using western blot assay revealed pharmacologically pure preparations.