An integrated process combining the reaction and purification of PEGylated proteins†
A downstream process combining PEGylation reaction and the use of enzyme conjugates acting as phase-forming components of aqueous biphasic systems (ABS) is proposed here. In this approach, citrate buffer (pH = 7.0) was used simultaneously to stop the reaction (avoiding the use of hydroxylamine) and as a phase forming agent inducing the phase separation of the PEGylated proteins. The partition of the bioconjugates was assessed using two model enzymes of small size [cytochrome c (Cyt-c) and lysozyme (LYS)], and two of large size [L-asparaginase (ASNase) and catalase (CAT)] as well as reactive PEG of 5, 10, 20 and 40 kDa. The effect of the reaction time on the PEGylation and recovery steps was also evaluated. All reactive PEGs allowed high selectivity in the separation of PEGylated proteins from native proteins (S > 100). A positive effect in terms of selectivity was found for longer reaction times. It allowed greater amounts of PEGylated proteins, with an increase of the PEG-protein rich-phase volume (top phase), allowing 100% of recovery of PEGylated proteins. More selective systems were obtained for Cyt-c and LYS (S > 100) compared to those for ASNase and CAT (40 < S < 60); nevertheless for all, the native and PEGylated proteins had their biological activity preserved. Envisioning the industrial potential evaluation, an integrated process diagram was defined combining the PEGylation reaction with the purification of the protein conjugates. Two different scenarios were investigated considering the PEGylation reaction performance. For both approaches (complete and incomplete PEGylation reaction), high recovery yields and purities were achieved for the PEGylated conjugates (92.1 ± 0.4% < %RecTCyt-c-PEG < 98.1 ± 0.1%; 84.6% < purity < 100%) and for the unreacted enzyme (%RecBCyt-c = 81 ± 1%; purity = 97.7%), while maintaining their structural integrity.