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Adsorption of native as well as mono-, di-, and tri-PEGylated lysozyme on Toyopearl PPG-600M, a mildly hydrophobic resin is studied by isothermal titration calorimetry and by independent adsorption equilibrium measurements in sodium phosphate buffer at pH 7.0 and 25 °C. For PEGylation two different PEG sizes are used (5 and 10 kDa) which leads to six different forms of PEGylated lysozyme all of which are systematically studied. Additionally, the adsorption of five pure PEGs is explored. The ammonium sulfate concentration is varied from 600 to 1200 mM. The molar enthalpy of adsorption Δh(p)(ads) is determined from the calorimetric and the adsorption equilibrium data. It is found to be endothermic in all experiments. The comparison of the adsorption of different PEGylated forms shows that the adsorption of PEGylated lysozyme is driven by the adsorption of the PEG chain. The results provide insight into the adsorption mechanisms of polymer-modified proteins on hydrophobic chromatographic resins.

In a recent work (Werner A and Hasse H in J Chromatogr A 2013;1315:135) the influence of mixed electrolytes on the adsorption of the macromolecules lysozyme, PEG and di-PEGylated lysozyme on a hydrophobic resin has been studied, but only at one overall ionic strength (3000 mM). The present work, therefore, extends these studies to other ionic strengths (2400 and 2700 mM), and explores the application of a model to predict the entire data set. The adsorbent is Toyopearl PPG-600M. The solvent is a 25 mM aqueous sodium phosphate buffer at pH 7.0. The studied salts are sodium chloride, sodium sulfate, ammonium chloride and ammonium sulfate. Pure salts as well as binary and ternary mixtures of these salts with varying ratios of the amounts of the salts are studied at 25 °C. The loading of the adsorbent increases with increasing salt concentration for all macromolecules. Synergetic effects of the mixed electrolytes are observed. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1104-1115, 2017.

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