Abstract
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.
Highlights
Chromatography is an important separation and purification technique in biotechnological processes
Very high ionic strengths lead to a complete occupation of the cation exchange sites of the resin with the cations in solution, such that ionic interactions between lysozyme and the resin are less pronounced in this regime
This behavior can be attributed to the superposition of the two discussed opposing effects: the increasing ionic strength leads to a weakening of the cation exchange mechanism of the adsorption whereas it favors the hydrophobic interactions by a salting-out effect, which leads to a better adsorption
Summary
Chromatography is an important separation and purification technique in biotechnological processes Besides longestablished techniques, such as ion exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), and affinity chromatography (AC), hybrid variants known as mixed-mode chromatography (MMC) exist and have attracted much interest in recent years [1, 2]. Higher ionic strengths weaken the ionic interactions and lead to an increased attraction between the target molecules and the hydrophobic part of the resin [4], i.e., it becomes more similar to a HIC resin. Such mixed-mode resins provide wider operating ranges than IEC resins in terms of the salt concentration [5]. MMC is thereby applied for challenging separation processes, such as the purification of recombinant proteins, including monoclonal antibodies [15]
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