Adsorption of conjugates of lysozyme and fluorescein isothiocyanate in hydrophobic interaction chromatography

Bioconjugates, such as antibody-drug conjugates or fluorescent-labeled proteins, are highly interesting for various applications in medicine and biology. In their production, not only the synthesis is challenging but also the downstream processing, for which hydrophobic interaction chromatography (HIC) is often used. However, in-depth studies of the adsorption of bioconjugates in HIC are still rare. Therefore, in the present work, three different conjugates of lysozyme and fluorescein isothiocyanate (FITC) were synthesized and isolated, and their adsorption on the hydrophobic resin Toyopearl PPG-600 M was systematically studied in batch experiments. The influence of sodium chloride and ammonium sulfate with ionic strengths up to 2000 mM on the adsorption isotherms was investigated at pH 7.0 and 25 degrees C, and the results were compared to those for pure lysozyme. The conjugation leads to an increase of the adsorption in all studied cases. All studied conjugates contain only a single FITC and differ only in the position of the conjugation on the lysozyme. Despite this, strong differences in the adsorption behavior were observed. Moreover, a mathematical model was developed, which enables the prediction of the adsorption isotherms in the studied systems for varying ionic strengths.