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

摘要: Abstract Covalent attachment of PEG to proteins, known as PEGylation, is currently one the main approaches for improving pharmacokinetics biopharmaceuticals. However, separation and characterization especially positional isoforms 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 linear pH gradient separate five six possible mono-PEGylated lysozyme, modified 5 kDa 10 kDa mPEG-aldehyde. To identify corresponding PEGylation sites comparison elution values calculated isoelectric points each isoform, was used. The resulting correlation showed an R 2  > 0.99. Fractionation, tryptic digestion subsequent MALDI-MS analysis peak, verified predicted order. Based on UV areas N-terminal amine at lysine 1 exhibited highest reactivity, followed 33 residue.