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 °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.

Influence of pH value and salts on the adsorption of lysozyme in mixed-mode chromatography.

Mixed-mode chromatography (MMC) is an interesting technique for challenging protein separation processes which typically combines adsorption mechanisms of ion exchange (IEC) and hydrophobic interaction chromatography (HIC). Adsorption equilibria in MMC depend on multiple parameters but systematic studies on their influence are scarce. In the present work, the influence of the pH value and ionic strengths up to 3000 mM of four technically relevant salts (sodium chloride, sodium sulfate, ammonium chloride, and ammonium sulfate) on the lysozyme adsorption on the mixed-mode resin Toyopearl MX-Trp-650M was studied systematically at 25℃. Equilibrium adsorption isotherms at pH 5.0 and 6.0 were measured and compared to experimental data at pH 7.0 from previous work. For all pH values, an exponential decay of the lysozyme loading with increasing ionic strength was observed. The influence of the pH value was found to depend significantly on the ionic strength with the strongest influence at low ionic strengths where increasing pH values lead to decreasing lysozyme loadings. Furthermore, a mathematical model that describes the influence of salts and the pH value on the adsorption of lysozyme in MMC is presented. The model enables predicting adsorption isotherms of lysozyme on Toyopearl MX-Trp-650M for a broad range of technically relevant conditions.