257125 Influence of Ammonium Sulfate and Sodium Chloride On Adsorption of Pegylated Lysozyme On a Hydrophobic Resin
PEGylation of pharmaceutical proteins, i.e. the covalent binding of poly(ethylene glycol) (PEG) to the protein, can lead to improved biocompatibility, stability and residence time of the drug in the body. Usually, the protein is already purified before PEGylation. Therefore, the purification task after PEGylation is reduced to a separation of the PEGylated isoforms or a fraction with a certain PEGylation degree from the unPEGylated protein. The PEGylation changes the hydrophobicity of the proteins which leads to different interactions in hydrophobic chromatography systems. Therefore, hydrophobic interaction chromatography (HIC) is a promising technique for the separation of PEGylated protein isoforms. Lysozyme is a protein which is often used as a model protein due to its well-known structure and surface properties. Lysozyme can be PEGylated at three lysine residues, resulting in mono-, di- and tri- PEGylated isoforms.
In the present work, the adsorption of different forms of PEGylated lysozyme as well as native lysozyme and pure PEGs on a hydrophobic resin was studied by measuring both the adsorption equilibrium isotherms and the specific enthalpies of adsorption along the isotherms. This gives a complete thermodynamic picture of the adsorption as both Δgads and Δhads are determined experimentally so that T Δsadscan be calculated. The measurements of the adsorption equilibrium isotherms were carried out using a fully automated robotic liquid handling station. For the calorimetric measurements, a high precision isothermal titration micro-calorimeter was used.
The different forms of PEGylated lysozyme studied in the present work are: mono-, di- and tri-PEGylated lysozyme for 5 and 10 kDa PEG each. The adsorption material is Toyopearl PPG-600M, a mildly hydrophobic resin. The studied salts are sodium chloride and ammonium sulfate; a sodium phosphate buffer was used to adjust the pH value. It was found, that adsorption mechanism changes completely upon variation of the salt. A molecular picture is discussed which describes the complex adsorption mechanisms.