Impact of operating conditions on chromatographic column performance: experimental studies on adsorption of high-value minor whey proteins
Ajay K. Ray
1 Department of Chemical and Biochemical Engineering, University of Western Ontario, London, Ontario, Canada
2 Faculty of Engineering and Applied Science, Memorial University of Newfoundland, St John's, Newfoundland and Labrador, Canada
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.
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