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Purpose: This study focuses on the effect of length and structure of attached polyethylene glycol (PEG) chain on hydrodynamic radius (Rh) and chromatographic retention of PEGylated protein. To this aim human serum albumin (HSA) as a standard protein was PEGylated site specifically with mPEG-maleimide. Methods: Separated PEG_HSA fractions were analyzed by size exclusion and anion exchange chromatography (AExC). The purity of fractions and the relative mobility of PEGylated and native proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Hydrodynamic radius was determined based on the retention time of fractions on size exclusion chromatography (SEC), and also according to the previously reported equations. Results: A linear relation was shown between the molecular weight of attached PEG and Rh of PEGylated HSA. No significant difference between Rh of proteins modified with linear and branched PEG was shown. In SDS-PAGE, the delaying effect of branched PEG on movement of PEGylated protein was higher than that of linear PEG. Conclusion: As PEGylated HSA and dimer HSA have almost the same size and in SEC they elute at very close retention times, so in this case ion exchange chromatography (IExC) is more effective than SEC in separation of PEGylated HSA. Branched PEG- HSA showed earlier elution on anion exchange chromatography compared to linear PEG-HSA, that this can explain the different shielding effect of various structures of attached PEGs. The smaller size of PEGylated HSA in compare to the sum of the hydrodynamic radiuses of native HSA and attached PEG could be as a result of shielded attachment of polymer around protein. [ABSTRACT FROM AUTHOR]