The Effect of Length and Structure of Attached Polyethylene Glycol Chain on Hydrodynamic Radius, and Separation of PEGylated Human Serum Albumin by Chromatography.

Parvin Akbarzadehlaleh,Mona Mirzaei,Mahdiyeh Mashahdi-Keshtiban,Hamid Reza Heidari

doi:10.34172/apb.2021.082

Parvin Akbarzadehlaleh, Mona Mirzaei + Show 2 more

Open Access

https://doi.org/10.34172/apb.2021.082

Copy DOI

Abstract

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.

Highlights

PEGylation is the covalent and non-covalent attachment of activated polyethylene glycol (PEG) chain to various biomolecules
As PEGylated human serum albumin (HSA) and dimer HSA have almost the same size and in size exclusion (SEC) they celute at very close retention times, so in this case IExC is more effective than SEC in separation of PEGylated HSA
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

Summary

Introduction

PEGylation is the covalent and non-covalent attachment of activated polyethylene glycol (PEG) chain to various biomolecules. PEG is not toxic and nor immunogenic polymer. This technique is introduced as a strategy to eliminate some clinical disadvantages of biopharmaceuticals. T After PEGylation the hydrodynamic radius of the molecule increases which causes reduced renal excretion and increased maintenance time of the conjugates in the body, and thereby ip allows reducing the dosing frequency of biomolecule in clinic and the patient’s comfort[2,3]. To understand the importance c of size (molecular weight) and structure (linear versus branched) of PEGs on physico-chemical properties of the PEGylated biomolecule, at this study site selective polymer U HSA has a single polypeptide chain and 17 disulfide bridges, and one free cystein residue (Cys. 34) in its structure, and has 585 amino acids (66.5 kDa). This protein is the main protein of n plasma with concentration of about 3.5–5.0 g/dL, and supplies 80% of the colloid osmotic pressure of plasma (25-33 mmHg)[9,10]

Methods

Results

Conclusion