Use of ion-exchange chromatography and hydrophobic interaction chromatography in the preparation and recovery of polyethylene glycol-linked proteins

Cation- and anion-exchange chromatography can be used to purify a polyethylene glycol-linked protein dimer (PEG dimer) made with Mr 20 000 PEG bis-vinylsulfone, even when there are no net charge differences between the components that are being separated. The retention time on ion-exchange generally is inversely proportional to the PEG:protein ratio (on a mass basis). One of the biggest challenges in developing the process for making this PEG dimer was the quality of the PEG linker. Reversed-phase HPLC can be used to determine both size heterogeneity and the degree of end-group activation of Mr 20 000 PEG bis-vinylsulfone. In addition, we have found that hydrophobic interaction chromatography can be used make more size homogeneous preparations of Mr 20000 PEG bis-vinylsulfone, which significantly increased the recovery of the PEG dimer.