Recently work in our lab has lead to the discovery of a new mode of displacement chromatography where a selected solute or solutes are pulled back into the displacement zone, separating them from the displacement train.  This technique has been termed Selective Displacement and utilizes the design of the displacer to add an orthogonal dimension of selectivity to the normal displacement mode.  Current work in our lab has been directed toward the understanding and expansion of this technique for bioseparations in ion exchange chromatography.

     We have adapted robotic liquid handling systems to carry out high throughput displacer screens of commercially availble chemical libraries in order to search for new chemically selective displacer leads.  At the same time novel chemically selective displacers are being rationally designed to target specific biomolecules for custom separations.  Multiple outside techniques, such as saturation transfer difference NMR, SPR and MD simulations have been brought in to further the understanding and design of these displacers.  This has led to multiple ongoing collaborations including the Kane, Garde and Dordick groups of Chemical and Biological Engineering, the Moore and Breneman groups of Chemistry and Chemical Biology and Dr. McCallum of the Center for Biotechnology and Interdisciplinary Studies at RPI.  Proof of concept studies have already been carried out on this technique with future work being directed towards analytical applications for targeted classes of proteins and preparative applications for desired products or targeted impurity clearance.
 

Figures:  

Diagram of the column output from a selective displacement train separation (left).  Demonstrates how a normal displacement separation  is carried out while a selected solute is heald back in the displacer zone, adding an orthogonal dimension of selectivity.  

An example DC-50 plot of a Chemically Selective Displacer batch separation on two different protein pairs, AChyA/RNase A and CytC/Lys (right).  Demonstrates how almost 100% separations may be achieved using chemically selective displacers on protein pairs that have identical IEX gradient retention. 

An example saturation transfer difference NMR result on a selective displacer and non-selective displacer (below).  Demonstrates how the chemically selective displacer binds to the selected solute while not binding to the displaced solute.  The non-selective displacer also shows no binding.  Binding has been found to play an important role in the mechanism of chemically selective displacement.    
 

Relevant Papers:

Tugcu, N.; Ladiwala, A.; Breneman, C.M.; Cramer, S.M.  Identification of Chemcially Selective Displacers Using Parallel Batch Screening Experiments and Quantitative Structure Efficacy Relationship Models.  Anal. Chem. 2003, 75, 5806-16.

Liu, J.; Hilton, Z.A.; Cramer, S.M.  Chemically Selective Displacers for High Resolution Protein Separations in Ion-exchange Systems: Effect of displacer-protein interactions.  in press.

Morrison, C.J.; Cramer, S.M.  Characterization and Design of Chemically Selective Cationic Displacers Utilizing a Robotic High Throughput Screen. in press.
 

Representative Abstract:

Morrison, Christopher J.; McCallum, Scott A.; Godawat, Rahul; Moore, J.A.; Garde, Shekhar; Cramer, Steven M.  Investigation of chemically selective displacers using robotic high throughput screening, SPR, NMR and MD simulations.  Abstracts of Papers, 234th ACS National Meeting, Bosoton, MA, US, Aug 19-23, 2007.

High throughput screening was employed in concert with several analytical techniques to identify and evaluate the behavior of chemically selective displacers for protein purification in ion exchange systems.  A robotic liquid handling system was adapted to efficiently carry out this parallel batch screen of selective displacers on multiple protein pairs.  The results identified potential selective displacers and important functional group chemistries.  The screen also indicated that this selectivity was due primarily to the specific binding between the displacer and targeted proteins.  NMR was then conducted on several protein/displacer mixtures verifying the binding of the selective displacers to the targeted proteins and the location of the binding event.  Surface plasmon resonance experiments and molecular dynamic simulations were also carried out to corroborate the NMR results.  This proof of concept study shows that more specific selectivities may also be possible by utilizing affinity based selective displacers for explicit protein systems.