A liquid chromatographic method for determination of the modification degree of proteins: PEGylated arginase as an example

Abstract A liquid chromatographic method was developed to determine the modification degree of PEGylated proteins. This method effectively separated free polyethylene glycol (PEG) from other species in conjugation mixtures on a C4 reversed-phase column using water–acetonitrile gradient elution. Then the concentrations of free PEG were determined according to the integrated area under the curve of its evaporative light scattering detector (ELSD) signal, which was normalized by the PEG standard with similar molecular weights. The actual numbers of PEG attached to proteins, not those of lysines modified, were calculated. This method was performed with PEGylated arginase mixtures as an example and showed clear advantages over 2,4,6-trinitrobenzenesulfonic acid (TNBS) assays.