BACKGROUNDPurification of PEGylated proteins from reactions is still a challenge due to the formation of isomers. In this work, the recovery of PEGylated lysozyme was studied in a novel integrative approach called in situ aqueous two-phase systems (ATPS). The excess of PEG in the lysozyme PEGylation reaction (LPR) was used as part of the phase forming chemicals with different polymer (UCON, ficoll, dextran) and salt (sodium phosphate, potassium sulphate, sodium sulphate, ammonium sulphate, sodium carbonate) solutions. RESULTSThe best option for the in situ ATPS formation was the addition of a 4 mol L-1 ammonium sulphate in 20 mmol L-1 Tris-HCl (pH 7.0) solution to the PEGylation reaction. PEGylated conjugates of lysozyme exhibited a preferential partition to the top (PEG-rich) phase while native protein was partitioned to the bottom (salt-rich) phase. Di-PEGylated lysozyme partitioned entirely to the top phase (recovery of 100%), while the mono-PEGylated protein presented a recovery yield of 56% in the top phase. On its part, 97.7% of the native lysozyme was concentrated in the bottom phase. CONCLUSIONThe proof-of-concept presented in this work allows the recovery of PEGylated proteins directly from a PEGylation reaction. It is the first time that PEGylated proteins have been fractionated using a PEG-ammonium sulphate system taking advantage of the unreacted PEG left in a PEGylation reaction. Based on reaction engineering, in situ ATPS are a potential mechanism for PEGylation control, a good alternative for packed-bed' or on-column PEGylation processes and a pioneer strategy in the development of phase transfer catalysis in PEGylation. (c) 2017 Society of Chemical Industry