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A library of displacer analogues with varying degrees of electrostatic, hydrophobic and hydrogen bonding moieties was evaluated for their ability to enhance the selectivity of multimodal (MM) chromatography under high loading conditions. The library was screened for displacement of model proteins using a robotic liquid handling system and selective batch separations were achieved for proteins that were inseparable with linear gradient chromatography. Trends in protein displacement were identified and displacers with higher hydrophobicity and net charge exhibited improved protein displacements. Proteins that interacted with the resins primarily via electrostatic interactions were more readily displaced than those that possessed a significant hydrophobic contribution to their binding. In addition, multimodal displacers were found to be more selective than single mode electrostatic displacers. Column chromatography studies were also carried out and baseline separations were achieved for model protein pairs using selective displacement. Finally, operation of these columns in the desorption mode resulted in baseline separation of model proteins which were not separable by selective displacement chromatography. This study indicates that the inherent selectivity of MM resins can be augmented by the selectivity of the displacer under non-linear competitive binding conditions, creating new opportunities for protein separations not possible using traditional gradient operations.

We describe an affinity-based strategy for designing selective protein displacers for the chromatographic purification of proteins. To design a displacer that is selective for a target protein, we attached a component with affinity for the target protein to a resin-binding component; we then tested the ability of such displacers to selectively retain the target protein on a resin relative to another protein having a similar retention time. In particular, we synthesized displacers based on biotin, which selectively retained avidin as compared to aprotinin on SP Sepharose high performance resin. In addition, we have extended this approach to develop an affinity-peptide-based displacer that discriminates between lysozyme and cytochrome c. Here, a selective displacer was designed from a lysozyme-binding peptide that had been identified and optimized previously using phage-display technology. Our results suggest a general strategy for designing highly selective affinity-based displacers by identifying molecules (e.g., peptides) that bind to a protein of interest and using an appropriate linker to attach these molecules to a moiety that binds to the stationary phase.

Design of peptide affinity ligands against biological targets is important for a broad range of applications. Here, we report on de novo and combinatorial strategies for the design of high-affinity and high-specificity peptides against S-protein as a target. The peptide libraries employed in this study contain (1) consensus motif (CM) sequences identified from high-throughput phage combinatorial screening, (2) point mutations of CM sequences, and (3) de novo sequences rationally designed based on stereo-chemical information of the complex between S-protein and its natural ligand, S-peptide. In general, point mutations to CM allowed for modulating peptide affinity and specificity over a broad range. This is particularly useful in designing peptides with varying affinities and specificities for the target. De novo sequences, especially those based on the S-protein binding pocket, on average bound with higher affinities within a narrow range (10–100 nM) as compared to point mutations to CM (1 nM–2 $$\mu $$ M). As such, the approaches described here serve as a general guide for optimizing the design of peptide affinity ligands for a wide range of target proteins or applications.

A detailed study was carried out to combine the unique selectivity of ceramic hydroxyapatite (CHA) with the separation power of selective displacement chromatography. A robotic liquid handling system was employed to carry out a parallel batch screen on a displacer library made up of analogous compounds. By incorporating positively charged, metal chelating and/or hydrogen bonding groups into the design of the displacer, specific interaction sites on CHA were targeted, thus augmenting the selectivity of the separation. The effect of different mobile phase modifiers, such as phosphate, sulfate, lactate and borate, were also investigated. Important functional group moieties and trends for the design of CHA displacers were established. Selective batch separations were achieved between multiple protein pairs which were unable to be resolved using linear gradient techniques, demonstrating the applicability of this technique to multiple protein systems. The specific interaction moieties used on the selective displacer were found to dictate which protein was selectively displaced in the separation, a degree of control not possible using a mono-interaction type resin in displacement chromatography. Mobile phase modifiers were also shown to play a crucial role, augmenting the selectivity of a displacer in a synergistic fashion. Column separations were carried out using selective displacers and mobile phase modifiers identified in the batch experiments, and baseline separation of the previously unresolved protein pairs was achieved. Further, the elution order in these systems was able to be reversed while still maintaining baseline separations. This work establishes a new class of separations which combine the selectivities of multi-modal resins, displacers/eluents, and mobile phase modifiers to create unique selectivity windows unattainable using traditional modes of operation.

A library of molecular analogues to the selective displacer, N'1'-(4-methylquinolin-2-yl)ethane-1,2-diamine dinitrate, was employed to study the effects of changes in displacer chemistry on their efficacy for selective separations. High throughput screens were carried out using a robotic liquid handling system to examine the ability of these compounds to selectively displace proteins in batch adsorption systems. Experiments were conducted using the model protein pairs ribonuclease A/alpha-chymotrypsinogen A and cytochrome C/lysozyme on a strong cation exchanger. Selectivity pathway and DC-50 plots were constructed from the analogue screen data, and results indicated that minor changes in the molecular design of the displacer can have a significant impact on the separation behavior. Specifically, charge density and spacing of resin and protein interaction moieties were found to be important. The screen also identified a new displacer, 4-methyl-2-piperazin-1-yl-quinoline, which produced a more selective displacement than previously reported with the original compound. A steric mass action dynamic affinity plot was constructed to validate that this new displacer was acting as a chemically selective, rather than a steric mass action selective displacer. Finally, saturation transfer difference NMR experiments were conducted to examine protein-displacer interactions with these displacers and protein pairs. These results demonstrate how subtle changes in displacer design can be employed to fine-tune the separation performance of chemically selective displacers.