On-line characterization of polyethylene glycol-modified proteins

Abstract An high-performance liquid chromatographic method has been developed which simultaneously determines three critical physical properties of polyethylene glycol (PEG)-modified proteins: molecular size, polymer distribution and weight composition. With both UV and refractive index (RI) detectors in series, size-exclusion chromatography (SEC) is used to separate the PEG-protein species according to size. The size analysis of these PEG-proteins is predicted to be accurately calibrated with the viscosity radius (universal calibration), which compensates for the shape differences between PEG and protein structures. The heterogeneity of the PEG-protein grafted copolymer is represented by the polymeric term “polydispersity”, which describes the size distribution. Separate SEC calibrations of the PEG and the protein used for conjugation allow a determination of the weight composition of the PEG-protein (weight PEG/weight protein) by combining UV and RI chromatograms of a PEG-protein sample. This compositional analysis is validated through independent and direct measurement of the PEG on a PEG-protein via acid hydrolysis and quantitative SEC. Comparisons of compositional analysis of PEG-protein with sodium dodecyl sulfate polyacrylamide gel electrophoresis densitometry demonstrate that gel analysis of some proteins is misleading.