Reactive aqueous two-phase systems for the production and purification of PEGylated proteins

Background: PEGylation, defined as the covalent attachment of polyethylene glycol, allows the synthesis of PEGylated therapeutic proteins with enhanced physicochemical properties. Traditional alkylating N terminal PEGylation reactions on amine groups involve the use of modified linear mono-methoxy polyethylene glycol (mPEG) molecules looking for the synthesis of mono-PEGylated products. However, this approach requires different purification steps since inevitably undesired cross-linked products are synthesized. Herein, we propose the use of reactive aqueous two-phase systems (ATPS) to produce and purify PEGylated therapeutic conjugates using Ribonuclease A (RNase A) as a model protein. Results: Selected linear 5 kDa and 20 kDa mPEG - potassium phosphate systems were produced according to equilibrium data obtained from constructed binodal curves. All reactive systems were able to generate biphasic systems and to PEGylate RNase A. Two 5 kDa and two 20 kDa systems were selected based on the reaction yield percentage and the feasibility of purifying the mono-PEGylated RNase A from the diPEGylated and native RNase A by contrasting the differences in their partition behaviors. The remnant biological activity was of 94% and of 100% for the mono-PEGylated RNase A purified from the 5 kDa and 20 kDa mPEG systems when compared to the mono-PEGylated conjugate obtained by standard procurement methods. Conclusions: This novel approach using reactive ATPS shows that it is feasible to simultaneously produce and purify PEGylated therapeutic proteins with conserved biological activity and presents another exam-ple where reactive ATPS can be successfully implemented. How to cite: Campos-Garcia VR, Benavides J, Gonzalez-Valdez J. Reactive aqueous two-phase systems for the production and purification of PEGylated proteins. Electron J Biotechnol 2021;54. https://doi.org/10. 1016/j.ejbt.2021.09.003 (c) 2021 Pontificia Universidad Catolica de Valparaiso. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).