Fractionation of different PEGylated forms of a protein by chromatography using environment-responsive membranes

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

“…Liquid‐phase PEGylation of HSA was first carried out, and the reaction mixtures thus obtained were fractionated using stacks of PVDF membranes, both in preparative and analytical mode. The overall objective of this study was to validate the working hypothesis for the fractionation of PEGylated proteins by HIMC as described in our previous work . The objective of the analytical HIMC experiments was to see if the above approach could be used to develop membrane chromatographic methods for the rapid and sensitive analysis of PEGylated HSA and PEGylated proteins in general.…”

“…Chromatographic purification methods for PEGylated proteins exploit the differences in physicochemical properties such as molecular size , surface charge , and relative hydrophobicity . Most chromatographic separation processes reported are based on the use of packed columns .…”

“…Most chromatographic separation processes reported are based on the use of packed columns . Some more recent studies have shown the suitability of membrane chromatography for purification of PEGylated proteins . Membrane chromatography is fast due to the convection‐dominated transport of solutes, it operates at low pressure, and is better suited for purification of bulky macromolecules .…”

“…Membrane chromatography is fast due to the convection‐dominated transport of solutes, it operates at low pressure, and is better suited for purification of bulky macromolecules . In our previous paper, we demonstrated the use of hydrophobic interaction membrane chromatography (HIMC) using a stack of hydrophilized microporous polyvinylidene fluoride (PVDF) membranes with environment‐responsive properties for the purification of PEGylated lysozyme . These membranes become hydrophobic in the presence of lyotropic salt and revert back to the original hydrophilic state when the salt is removed .…”

“…Since the hydrophobicity of the membrane can be varied, the interactions between the different PEGylated proteins and the membrane surface can be manipulated. By being able to do so, the difference in the degree of interaction of the different PEGylated forms with the membrane can be maximized, such that this technique not only separated the PEGylated protein from its unmodified form but was able to resolve different PEGylated forms into separate peaks, which is sometimes not feasible with conventional column‐based hydrophobic interaction chromatography, the media of which are always hydrophobic .…”