Abstract
Over the last decade, ion-exchange chromatography (IEC) has been extensively explored for protein purification at both small and large scales. Despite several IEC columns are commercialized, the physical phenomena underlying the adsorption of proteins on ion-exchange columns performance has not been thoroughly investigated. In this work, the influence of operating conditions on the adsorption of lactoperoxidase (LP) and lactoferrin (LF) on cation exchange chromatography adsorbent is experimentally studied in order to understand fundamental pertaining to underlying mechanism. Analysis was carried out in columns with different IDs (7.7 and 16 mm), packed for 100 mm with 90 μm particle size polymer-grafted cation exchanger. The flow distribution was measured using acetone as a non-binding tracer. An evaluation of van Deemter plots was done as well as LP breakthrough curves at different flow rates and LP loading concentrations. The results were compared with two columns in terms of efficiency and the LP binding capacity. The dynamic binding capacity at 10% breakthrough was found to be independent of the applied flow rate. Surprisingly for both systems, LP breakthrough takes place later at higher loading concentrations, which is in contrast to IEC. The results propose a major presence of non-ideal effects as steric shielding and charge repulsion of protein in the adsorption. In addition, the accessibility of binding sites for protein at higher concentrations seems more available than sodium counter-ions in buffer.
Highlights
Ion-exchange chromatography was introduced in the mid-1940s as a separation technique based on charge properties of molecular species [1]
The Péclet number (Pe) values are comparatively identical for each column; AIMS Bioengineering the higher Pe numbers are preferred as they correspond to a uniform distribution of flow to the inlet surface of the column as well as uniform distribution of the binding site properties
The LP breakthrough curves were measured at various flow rates and loading concentrations
Summary
Ion-exchange chromatography was introduced in the mid-1940s as a separation technique based on charge properties of molecular species [1]. Within the last few decades, there has been an increasing interest in liquid chromatographic processes because of the developing biotechnology scope and demands from the pharmaceutical and chemical industries for extremely particular and productive separation methods. IEC is being employed for purification of proteins, peptides, nucleic acids and other charged biomolecules. IEC technique is well-suited for capture, intermediate purification or polishing steps in a purification protocol.
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