PEGylation of human serum albumin: The effect of linear mPEG-Maleimide with different molecular weights on hydrodynamic radius of PEGylated HSA and on its retention profile in ion exchange chromatography

Abstract

Introduction: Increasing the hydrodynamic volume of proteins is one of the most important advantages of PEGylation which is the covalent attachment of activated Poly Ethylene Glycol (PEG) to protein. At this study PEGylation of Human Serum Albumin (HSA) is studied. HSA has a molecular weight of about 66.5kDa and possesses one free thiol at Cys34 which is targeted as PEGylation site. Aims: The aims are to site-specifically mono-PEGylate HSA with 5kDa, 10kDa and 20kDa PEG, to develop and optimize an ion exchange chromatography method to separate PEGylated HSA, and to study the effect of various molecular weights of linear mPEG-Maleimide on hydrodynamic radius of PEGylated HSA. Methods: Site-specific PEGylation of HSA was done on Cys34 by 5kDa, 10kDa and 20kDa linear mPEG-maleimide. PEGylated HSA was separated and purified by fractionating the corresponding peak from size exclusion chromatography (SEC) and ion exchange chromatography (IEX). Various IEX methods were examined to achieve a high-resolution separation. Fractions were analyzed by SEC, IEX and discontinuous sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) to estimate purity and weight. Hydrodynamic radius was calculated using a calibration curve of standard proteins and by the previously reported equations. Results: HSA was successfully PEGylated at Cys-34. Chromatography methods were optimized and PEGylated HSA was effectively purified. The hydrodynamic radius of native HSA and PEGylated HSA with 5kDa, 10kDa and 20kDa PEG was found to be 33Å, 39Å, 48Å and 57Å, respectively. Conclusion: In addition to developing an IEX chromatography method to achieve the highly purified PEGylated HSA, Based on obtained results it can be concluded that IEX is a better method for purification of PEGylated HSA than SEC method. Also, results are indicating the linear relation between the molecular weight of linear PEG and hydrodynamic radius of PEGylated HSA.