Purification and analysis of mono-PEGylated HSA by hydrophobic interaction membrane chromatography

We discuss the purification of mono-PEGylated HSA by hydrophobic interaction membrane chromatography. The hydrophobicity difference between the different fractionated species was induced by the addition of a lyotropic salt that caused phase transition of PEG (hydrophilic under normal condition) to a mildly hydrophobic form. The HSA PEGylation reaction mixture was mixed with lyotropic salt and passed through a stack of hydrophilized polyvinylidene fluoride membrane discs. Unmodified HSA was obtained in the flow through, while the PEGylated forms of the protein bound to the membrane and could be eluted by reducing the salt concentration. Among the three major PEGylated forms of HSA present in the feed (i.e. mono-, di-, and tri-), mono-PEGylated HSA was eluted first and could be resolved from the others. The purified material was analyzed by SDS-PAGE, dynamic light scattering, and SEC combined with multi-angle light scattering. All these analytical techniques indicated the presence of species that has a molar mass consistent with mono-PEGylated HSA. A scaled-down version of the membrane chromatographic methods could be used for the rapid and sensitive analysis of PEGylated proteins.