BACKGROUND: The implementation of efficient processes for the development, recovery and purification of biological products is a main challenge for the bioengineering field. PEGylation can enhance the therapeutic properties of the protein, and often results in heterogeneous population of conjugated species and unmodified protein that presents a protein separations challenge. Non-chromatography strategies, such as countercurrent distribution in aqueous two-phase systems (CCD-ATPS) and ultrafiltration (UF) for the fractionation of PEGylated RNase A conjugates represent attractive alternative worth of consideration. RESULTS: In this work the potential application of CCD-ATPS for the separation of closely related proteins (i.e. mono-PEGylated and di-PEGylated RNase A) was performed under selected conditions. Process conditions involved PEG 8000 13% (w/w), phosphate 10.4% (w/w), at pH 7.0 and volume ratio of 1.0 resulted, after 30 transfers in CCD, in distinguishable peaks that can be attributed to the mono-PEGylated protein (61%) and di-PEGylated (58%) form of RNase A. Alternatively, an ultrafiltration-based strategy resulted in the partial separation of mono-PEGylated and di-PEGylated protein, 38% and 60%, respectively, in the concentrate with a 30 kDa membrane. Using a 50 kDa membrane the mono-PEGylated RNase A accumulates in the filtrate (43%) and the di-PEGylated in the concentrate (45%). CONCLUSIONS: The results reported here can be considered for the separation of PEGylated proteins, as a first step in the implementation of a purification process. (C) 2012 Society of Chemical Industry