The effect of feed composition on the behavior of chemically selective displacement systems

• Published in: Journal of Chromatography A 2010-02, Vol.1217 (8), p.1249-1254
• Main Authors: Evans, Steven T., Morrison, Christopher J., Freed, Alexander, Cramer, Steven M.
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Feed composition effect; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Ion exchange; Protein purification; Proteins; Selective displacement chromatography
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2009.12.020

In this paper we examine whether adding a more retained protein to the feed will mitigate displacer–protein interactions in the column, thus affecting the displacement modality that occurs (chemically selective vs. traditional displacement chromatography). STD-NMR experiments were carried out to probe displacer–protein interactions for the chemically selective displacer chloroquine diphosphate and the results indicated that this displacer only had measurable interactions with the protein α-chymotrypsinogen A. For a two component feed mixture containing ribonuclease A and α-chymotrypsinogen A, the separation resulted in the displacement of ribonuclease A, with the more hydrophobic α-chymotrypsinogen A remaining on the column. On the other hand, when the experiment was repeated with cytochrome c added to the feed, all three feed proteins were displaced. Column simulations indicated that the combination of sample self-displacement occurring during the introduction of the feed, along with the dynamics of the initial displacement process at the column inlet was responsible for this behavior. These results indicate that for this class of hydrophobic-based selective displacers, in order for the protein to be selectively retained, the protein should be the most strongly retained feed component.