Fast and high-resolution fractionation of positional isomers of a PEGylated protein using membrane chromatography

The fractionation of positional isomers of a PEGylated protein is quite challenging as these have similar molecular weight, and only very slightly different surface charge. In this study, cation exchange z² laterally-fed membrane chromatography (z²LFMC), which has been shown to be suitable for high-speed, high-resolution protein purification, was used to fractionate positional isomers of mono-PEGylated lysozyme. The performance of the z²LFMC device was compared with a commercial preparative cation exchange column having the same volume and ligand. PEGylated lysozyme purification experiments showed that while the positional isomers of mono-PEGylated lysozyme could not be satisfactorily resolved using the preparative commercial cation exchange column, almost baseline resolution of these could be achieved using the z²LFMC device. Moreover, the z²LFMC device-based process was faster by an order of magnitude. The results discussed in this paper demonstrate that z²LFMC is a superior alternative to column-based chromatography for challenging protein separations, such as the one discussed here, both in terms of speed and resolution.