PEGylation is the covalent binding of at least one chain of polyethylene glycol (PEG) to a protein, often resulting in a heterogeneous population of conjugated species and unmodified protein. Despite some recent developments, one of the major challenges in the area of PEGylated proteins is related to the purification. Sequential chromatographic steps have been extensively used for the resolution of the different PEGylated species. This study, presents the use of a chemically modified support as an alternative for the separation of PEGylated ribonuclease (RNase) species from its unmodified counterpart as well as the resolution of the different PEGylated species in a single chromatographic step using hydrophobic interaction chromatography CHIC) media. Sepharose-6B was subjected to chemical modification (covalent addition of PEG 550, PEG 2000 and PEG 5000 g/mol) to increase the selectivity of this media for PEGylated RNase A. The effect of ammonium sulfate concentration as well as the length of the PEG molecule on the chromatographic behavior of purified monoPEGylated and diPEGylated RNase was evaluated. The complete separation of native RNase from its PEGylated conjugates was achieved using a linear gradient elution of 1.5 M ammonium sulfate in 25 mM potassium phosphate pH 7.0. With step gradient optimization, the resolution of PEGylated conjugates (mono and di-PEG RNase) was also achieved injecting an actual PEGylation reaction mixture as sample, using the same elution buffers and Sepharose 6B-PEG5000 as stationary phase. The purity obtained for monoPEGylated RNase A was 85% with a yield of 96% comparable to what has been reported previously for other HIC/SEC media. The use of PEGylated Sepharose 6B was demonstrated as an attractive alternative for the separation and resolution of PEGylated proteins in a single chromatographic step. (C) 2014 Elsevier B.V. All rights reserved.