Progress and Challenges in PEGylated Proteins Downstream Processing: A Review of the Last 8 Years

PEGylation is a well-known process in the bio-pharmaceutical industry mainly due to its great performance in increasing blood circulation half-life of peptides and proteins for health purposes. More than 40 years have been devoted to the study of derived products from PEGylation reaction [native, mono-, di-, multi- PEGylated proteins and unreacted poly(ethylene glycol) (PEG)]. Despite the lack of selectivity and randomness of PEG chains attachment have been overcome by site-specific PEGylation, several drugs that are currently in the market are obtained by means of random PEGylation. In the context of downstream processing, separation of mono-PEGylated forms and their isomers is still a challenge. The growing demand of PEGylated drugs in the pharmaceutical industry requires simple, robust, cost effective and efficient methods for the purification process. The aim of this review is to provide an updated revision along the last 8 years of the methods for the fractionation of PEGylated proteins relative to other contaminant proteins, n-PEGylated forms and their isomers. Chromatographic methods including size exclusion, ion exchange, reversed phase HPLC and affinity have been enlisted. Non-chromatographic complementary techniques such as aqueous two-phase systems and electrophoresis also have been included in this compilation.