Continuous Protein Modification and Separation Process with Chromatography

Abstract

Modification of proteins with polyethylene glycol (PEGylation) contributes to improve the stability of the proteins. However, homogeneous liquid-phase reactions generally give a mixture of multiple PEGylated isomers with different stability and functions. Ion exchange chromatography (IEC) can potentially be applied to separate these isomers on the basis of the mechanistic model of IEC elaborated with respect to non-modified proteins. To clarify this point, PEGylated proteins on the IEC column with the linear salt gradient elution were analyzed on the basis of the model with mono-PEGylated bovine serum albumin (BSA) and random-PEGylated lysozyme as model samples. The model predicted that the number of binding sites of PEGylated proteins on the resin of IEC was the same as that of the non-modified ones. Furthermore, solid-phase PEGylation reaction and separation with IEC were simultaneously controlled by selecting the salt concentration in the mobile phase on the basis of the model. The number of isomers of PEGylated lysozyme could be reduced by the solid-phase PEGylation and the isoform of interest could be purified on the same IEC column. A recycle-type solid-phase continuous reaction and separation operation was designed, and successfully perfomed at high yields.