Mixed-mode chromatography (MMC), which combines features of ion exchange chromatography (IEC) and hydrophobic interaction chromatography (HIC), is an interesting method for protein separation and purification. The design of MMC processes is challenging as adsorption equilibria are influenced by many parameters, including ionic strength and the presence of different salts in solution. Systematic studies on the influence of those parameters in MMC are rare. Therefore, in the present work, the influence of four salts, namely, sodium chloride, sodium sulfate, ammonium chloride, and ammonium sulfate, on the adsorption of lysozyme on the mixed-mode resin Toyopearl MX-Trp-650M at pH 7.0 and 25°C was studied systematically in equilibrium adsorption experiments for ionic strengths between 0 mM and 3000 mM. For all salts, a noticeable adsorption strength was observed over the entire range of studied ionic strengths. An exponential decay of the loading of the resin with increasing ionic strength was found until approx. 1000 mM. For higher ionic strengths, the loading was found to be practically independent of the ionic strength. At constant ionic strength, the highest lysozyme loadings were observed for ammonium sulfate, the lowest for sodium chloride. A mathematical model was developed that correctly describes the influence of the ionic strength as well as the influence of the studied salts. The model is the first that enables the prediction of adsorption isotherms of proteins on mixed-mode resins in a wide range of technically interesting conditions, accounting for the influence of the ionic strength and four salts of practical relevance.

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The separation and purification of proteins is often carried out by chromatography. Mixed-mode adsorbents are interesting as they may combine favorable features of different chromatographic methods, such as ion-exchange chromatography (IEC) and hydrophobic interaction chromatography (HIC). Systematic experimental studies and models of adsorption isotherms of proteins on mixed-mode resins covering a wide range of parameters are still rare, which hampers both the scientific analysis of the complex processes that occur upon the adsorption of proteins on these resins as well as the practical separation design. Therefore, such studies were carried out in the present work for a model system: the adsorption of bovine serum albumin (BSA) on the mixed-mode resin Toyopearl MX-Trp-650M, which combines features of IEC and HIC resins. Adsorption isotherms were measured using sodium chloride, sodium sulfate, ammonium chloride, and ammonium sulfate at ionic strengths up to 3000 mM and for pH 4.0, 4.7, and 7.0 at 25 $$^\circ$$ ∘ C. In the studied pH ranges, BSA exhibits strongly varying net charges and undergoes a conformational change. At pH 4.0 and 4.7, an exponential decay of the BSA adsorption with increasing ionic strength was found at ionic strengths up to approximately 1000 mM, while a rather linear increase was observed at higher ionic strengths for all studied salts; for all salts and ionic strengths, decreasing adsorption with increasing pH value was found. Moreover, a mathematical model was developed, which enables the prediction of equilibrium adsorption isotherms of BSA on Toyopearl MX-Trp-650M for any ionic strength of the studied salts.

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.

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