A comprehensive evaluation of mixed mode interactions of HEA and PPA HyperCel™ chromatographic media.

Mixed mode or multimodal chromatography has been developed for rational use of multiple interactions in a controlled manner, in contrast to non-specific interactions. Indeed, as the term "mixed mode" suggests, these resins allow different types of interactions within a single chromatographic medium. In this paper, HEA HyperCel™, PPA HyperCel™ mixed-mode chromatographic media have been studied. These mixed-mode sorbents typically involve hydrophobic pseudo-affinity interactions for binding and essentially ionic interactions (charge repulsion) for elution. We identified and characterized these different interactions in chromatographic experiments by exploiting specific properties of proteins using protein standards and complex mixtures. We highlighted the major intervention of at least two types of interactions in these media: hydrophobic and electrostatic interactions. We observed the behaviour of these resins at different pH, ionic strength, with different salts and buffers types and in the presence of different organic compounds.

A study on the nature of interactions of mixed-mode ligands HEA and PPA HyperCel using phenylglyoxal modified lysozyme.

Mixed mode chromatography, or multimodal chromatography, involves the exploitation of combinations of several interactions in a controlled manner, to facilitate the rapid capture of proteins. Mixed-mode ligands like HEA and PPA HyperCel™ facilitate different kinds of interactions (hydrophobic, ionic, etc.) under different conditions. In order to better characterize the nature of this multi-modal interaction, we sought to study a protein, lysozyme, which is normally not retained by these mixed mode resins under normal binding conditions. Lysozyme was modified specifically at Arginine residues by the action of phenylglyoxal, and was extensively studied in this work to better characterize the mixed-mode interactions of HEA HyperCel™ and PPA HyperCel™ chromatographic supports. We show here that the adsorption behaviour of lysozyme on HEA and PPA HyperCel™ mixed mode sorbents varies depending on the degree of charge modification at the surface of the protein. Experiments using conventional cation exchange and hydrophobic interaction chromatography confirm that both charge and hydrophobicity modification occurs at the surface of the protein after lysozyme reaction with phenylglyoxal. The results emanating from this work using HEA and PPA HyperCel sorbents strongly suggest that mixed mode chromatography can efficiently separate closely related proteins of only minor surface charge and/or hydrophobicity differences.

Rapid screening of purification strategies for the capture of a human recombinant F(ab')2 expressed in baculovirus-infected cells using a micro-plate approach and SELDI-MS.

The development of a capture step of a human recombinant F(ab')(2) produced and expressed in baculovirus-infected cells was investigated by screening three mixed-mode chromatography sorbents (HEA HyperCel, PPA HyperCel and MEP HyperCel) and two ion exchangers (Q Ceramic HyperD F, S Ceramic HyperD F) sorbents using a 96-well plate format and SELDI-MS. HEA HyperCel gave the best separation performance therefore the conditions tested in micro-plate were transferred to laboratory scale chromatographic experiments, confirming that the recombinant F(ab')(2) was effectively captured on the mixed-mode sorbent without any pre-treatment of the crude extract with a 82% recovery and a 39-fold purification.