Isoform separation and binding site determination of mono-PEGylated lysozyme with pH gradient chromatography

Covalent attachment of PEG to proteins, known as PEGylation, is currently one of the main approaches for improving the pharmacokinetics of biopharmaceuticals. However, the separation and characterization especially of positional isoforms of PEGylated proteins are still challenging tasks. A common purification strategy uses ion exchange chromatography with increasing ionic strength by shallow salt gradients. This paper presents a method which applies a linear pH gradient chromatography to separate five of six possible isoforms of mono-PEGylated lysozyme, modified with 5 kDa and 10 kDa mPEG-aldehyde. To identify the corresponding PEGylation sites a comparison of elution pH values and calculated isoelectric points of each isoform, was used. The resulting correlation showed an R2 > 0.99. Fractionation, tryptic digestion and subsequent MALDI-MS analysis of each peak, verified the predicted elution order. Based on UV areas the N-terminal amine at lysine 1 exhibited the highest reactivity, followed by the lysine 33 residue.

► High resolution separation of PEG-lysozyme isoforms using pH gradients. ► Significant increase in resolution compared to salt gradient runs demonstrated. ► In silico prediction and MALDI-MS validation of isoform identification.