“Determination of interaction properties between PEGylated proteins and a modified resin by Isothermal Titration Calorimetry (ITC) and FTIR”

Abstract

PEGylated proteins are an increasing important class of therapeutic drugs due to their improved pharmacokinetic characteristics and solubility over their corresponding native forms. PEGylation is the covalent attachment of one or more polyethylene glycol (PEG) molecules to a protein. Despite the many advantages of PEGylated drugs, one of the major challenges is the purification step after the chemical reaction. The main purpose of this project is to determine the nature of chemical interactions between a modified resin with PEG 5000 g/mol and PEGylated proteins that results in a previously demonstrated ability of such resins for the resolution of PEGylated proteins. A chromatographical separation of PEGylated proteins was additionally demonstrated for lysozyme using the modified resin Sepharose 6B-PEG5000 previously reported for PEGylated RNase A. Fourier Transform Infrared (FTIR) spectroscopy provided insight of the resin modification. The interaction thermodynamics associated with PEGylated proteins in hydrophobic interaction chromatography (HIC) with modified resin was carried out in with an ITC (Isothermal Titration Calorimetry) analysis. The specific enthalpy (∆G) was found to be exothermic for both proteins in potassium phosphate buffer with 1.5 M ammonium sulfate at 25ºC. MonoPEGylated proteins showed large negative entropy (-T∆S) values, related to the enhanced hydrophobic interaction between PEG5000 molecules from the resin and PEGylated protein forms. In addition, binding constants (K) of PEGylated proteins to modified resin were slightly higher compared to unmodified proteins.