PEGylation, the covalent attachment of a polyethylene glycol (PEG) chain to a small protein, has been successfully used to improve the efficacy of therapeutic proteins. One of the challenges in producing PEGylated proteins is the purification of the desired conjugate, typically the singly PEGylated protein from both the residual unreacted PEG and native protein and from multiply PEGylated conjugates produced during the reaction. Considerable work has focused on size exclusion and ion exchange chromatography; however, the resolution is often poor and the overall capacity/throughput is low due to the steric hindrance provided by the attached PEG. The objective of this talk is to summarize recent work on the use of ultra?ltration with electrically-charged membranes for the purification of a model PEGylated protein.Experimental studies were performed using a-lactalbumin PEGylated with a 20 kDa PEG, with ultrafiltration performed using negatively-charged composite regenerated cellulose membranes. Data were analyzed using a recently developed theoretical model accounting for the increase in effective size associated with PEGylation and the ion exclusion from the PEG layer. The PEGylated a-lactalbumin was effectively separated from the unreacted protein and PEG using a negatively charged membrane with relatively large pore size at low ionic strength, conditions that maximize the electrostatic exclusion of the PEGylated protein while allowing the unreacted (neutral) PEG and small a-lactalbumin to pass into the filtrate. The singly PEGylated a-lactalbumin was separated from the more highly PEGylated conjugates by exploiting the difference in both size and net electrical charge of the different PEGylated species; the more heavily PEGylated proteins have a greater negative charge due to the further substitution via (positive) lysine groups. Opportunities for the simultaneous reaction and separation of a desired PEGylated protein are also discussed. These results clearly demonstrate the potential of using ultra?ltration for the production and purification of desired PEGylated proteins.

Recent advances in purification of PEGylated proteins using charge-modified ultrafiltration membranes

243rd National Meeting (2012)

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