Multistep purification of cytochrome c PEGylated forms using polymer-based aqueous biphasic systems


Chemical PEGylation of proteins has been used to improve their physicochemical properties and kinetics. However, the PEGylation reactions lead to a heterogeneous mixture of PEGylated conjugates and unreacted protein, which is a challenge for the design of an efficient downstream process. The purification of PEGylated proteins should address the two main issues: the separation of PEGylated conjugates from the unreacted protein and the fractionation of the PEGylated conjugates on the basis of their degree of PEGylation. The present study aims at the development of liquid-liquid extraction processes for the purification of PEGylated conjugates. An initial study of the partition behavior of cytochrome c and their PEGylated conjugates (Cyt-c-PEG-4 and Cyt-c-PEG-8) on polyethylene-glycol (PEG) + potassium phosphate buffer (pH = 7) aqueous biphasic systems (ABS) shows that PEGs with intermediate molecular weights (PEG MW = 1000-2000) allow the separation of the PEGylated conjugates from the unreacted protein in a single step. It is further shown that the PEGylated conjugates can be efficiently separated using ABS based on PEGs with high molecular weight (PEG MW = 6000-8000) and a study of the protein stability after purification was carried using circular dichroism. A downstream process to separate Cyt-c, Cyt-c-PEG-4 and Cyt-c-PEG-8 with high purities (96.5% Cyt-c, 85.8% Cyt-c-PEG-4, and 99.0% Cyt-cPEG-8) was developed. The process proposed addresses not only the efficient separation of each of the protein forms but also the recycling of the unreacted protein purified and the ABS phases, which was successfully used in a new step of PEGylation.




Chemistry; Science & Technology - Other Topics


Santos, JHPM; Carretero, G; Coutinho, JAP; Rangel-Yagui, CO; Ventura, SPM

nossos autores


This work was developed within the scope of the project CICECO-Aveiro Institute of Materials, POCI-01-0145-FEDER-007679 (FCT Ref. UID/CTM/50011/2013), financed by national funds through the FCT/MEC and when appropriate co-financed by FEDER under the PT2020 Partnership Agreement. The authors are grateful for the financial support of Sao Paulo Research Foundation - FAPESP (grant # 2016/22065-5) and the Portuguese Foundation for Science and Technology (FCT) for the doctoral grant of SFRH/BD/102915/2014 of Joao H. P. M. Santos. S. P. M. Ventura acknowledges the FCT/MEC (Portugal) for a contract under Investigador FCT 2015 contract number IF/00402/2015.

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