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

ABSTRACT

BACKGROUND PEGylation, defined as the covalent attachment of polyethylene glycol, allows the synthesis of PEGylated therapeutic proteins with enhanced physicochemical properties. Traditional alkylating Nterminal 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.