Ion-exchange displacement chromatography of proteins Dendritic polymers as novel displacers

Jayaraman, Guhan; Li, Yufei; Moore, James A.; Cramer, Steven M.

doi:10.1016/0021-9673(94)01007-2

Cited by 84 publications

(52 citation statements)

References 19 publications

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“…Pentaerythritol-based dendritic polyelectrolytes having quaternary amine functionalities and ranging in molecular weights from 480-1620 daltons were shown to be effective displacers in cation exchange systems (Jayaraman et al, 1995). Displacers containing a single arginine or lysine moiety have been shown to be effective for protein purification in cation exchange systems (Kundu et al, 1995a).…”

Section: Introductionmentioning

confidence: 99%

Selective displacement chromatography of proteins

Kundu

Barnthouse

Cramer

1997

Biotechnol. Bioeng.

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In contrast to high molecular weight polyelectrolyte displacers, the efficacy of low molecular weight displacers are dependent on both mobile phase salt and displacer concentration. This sensitivity to the operating conditions opens up the possibility of carrying out selective displacement where the product(s) of interest can be selectively displaced while the low affinity impurities can be desorbed in the induced salt gradient ahead of the displacement train, and the high affinity impurities either retained or desorbed in the displacer zone. This type of displacement combines the operational advantages of step gradient and the high resolution inherent in a true displacement process, in a single operation. Theoretical expressions are presented for establishing selective displacement operating conditions (initial salt concentration, displacer concentration) based on the Steric Mass Action parameters of the displacer and the linear Steric Mass Action parameters of the feed proteins. Experimental results are presented to elucidate the concept of selective displacement in both cation and anion exchange systems. A mixture of α‐lactalbumin and β‐lactoglobulin A and B has been used for anion‐exchange systems; a four‐protein mixture consisting of ribonuclease B, bovine and horse heart cytochrome c, and lysozyme has been employed in cation exchange systems. This article also demonstrates that on‐line monitoring can be readily employed for the selective displacement process, thus facilitating the scale‐up and control of the process. This work sets the stage for the development of robust large scale high resolution separations using selective displacement chromatography. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 119–129, 1997.

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Section: Introductionmentioning

confidence: 99%

Selective displacement chromatography of proteins

Kundu

Barnthouse

Cramer

1997

Biotechnol. Bioeng.

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“…The use of ion-exchange displacement chromatography for the purification of proteins has been investigated by a number of researchers (Peterson and Torres, 1983;Liao et al, 1987;Jen and Pinto, 1991;Gerstner and Cramer, 1992a,b;Jayaraman et al, 1993;Gadam et al, 1993). Conventionally, displacement chromatography is carried out with feed loads small enough to ensure isotachic separations (full development).…”

Section: Introductionmentioning

confidence: 99%

“…To describe the equilibrium ion exchange of proteins, the steric mass action (SMA) formalism has been introduced (Brooks and Cramer, 1992;Gadam et al, 1993;Jayaraman et al, 1993). This model accounts for both the effect of salt on the adsorption of biomolecules and the effect of steric shielding, which occurs during the binding of large molecules to ion-exchange surfaces.…”

Section: Introductionmentioning

confidence: 99%

Transient profiles in ion‐exchange displacement chromatography

Gadam¹,

Gallant²,

Cramer³

1995

AIChE Journal

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“…Displacement chromatography has been successfully employed for the purification of proteins using hydroxyapatite (Freitag and Breier, 1995;Vogt and Freitag, 1997), hydrophobic interaction (Shukla et al, 2000b), and ion exchange chromatographic systems (Barnthouse et al, 1998;Gerstner et al, 1995;Jayaraman et al, 1993Jayaraman et al, , 1995Kundu and Cramer, 1997;Peterson and Torres, 1983;Rege et al, 2005;Shukla et al, 1998Shukla et al, , 2000aTugcu et al, 2001). In particular, ion exchange displacement chromatography has attracted significant attention as a powerful technique for the purification of biomolecules (Jayaraman et al, 1993;Kundu and Cramer, 1997;Tugcu et al, 2003a).…”

Section: Introductionmentioning

confidence: 99%

“…In particular, ion exchange displacement chromatography has attracted significant attention as a powerful technique for the purification of biomolecules (Jayaraman et al, 1993;Kundu and Cramer, 1997;Tugcu et al, 2003a). Two classes of selective displacement chromatography have also been developed, namely, mass action and chemically selective displacement.…”

Section: Introductionmentioning

confidence: 99%

Mechanistic studies of displacer–protein binding in chemically selective displacement systems using NMR and MD simulations

Morrison¹,

Godawat²,

McCallum³

et al. 2008

Biotech & Bioengineering

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A parallel batch screening technique was employed to identify chemically selective displacers which exhibited exclusive separation behavior for the protein pair a-chymotrypsin/ribonuclease A on a strong cation exchange resin. Two selective displacers, 1-(4-chlorobenzyl)piperidin-3-aminesulfate and N 0 1 0 -(4-methyl-quinolin-2-yl)-ethane-1,2-diamine dinitrate, and one non-selective displacer, spermidine, were selected as model systems to investigate the mechanism of chemically selective displacement chromatography. Saturation transfer difference (STD) NMR was used to directly evaluate displacer-protein binding. The results indicated that while binding occurred between the two chemically selective displacers and the more hydrophobic protein, a-chymotrypsin, no binding was observed with ribonuclease A. Further, the non-selective displacer, spermidine, was not observed to bind to either protein. Importantly, the binding event was observed to occur primarily on the aromatic portion of the selective displacers. Extensive molecular dynamic simulations of protein-displacer-water solution were also carried out. The MD results corroborated the NMR findings demonstrating that the binding of selective displacers occurred primarily on hydrophobic surface patches of a-chymotrypsin, while no significant long term binding to ribonuclease A was observed. The non-selective displacer did not show significant binding to either of the proteins. MD simulations also indicated that the charged amine group of the selective displacers in the bound state was primarily oriented towards the solvent, potentially facilitating their interaction with a resin surface. These results directly confirm that selective binding between a protein and displacer is the mechanism by which chemically selective displacement occurs. This opens up many possibilities for future molecular design of selective displacers for a range of applications.

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Ion-exchange displacement chromatography of proteins Dendritic polymers as novel displacers

Cited by 84 publications

References 19 publications

Selective displacement chromatography of proteins

Selective displacement chromatography of proteins

Transient profiles in ion‐exchange displacement chromatography

Mechanistic studies of displacer–protein binding in chemically selective displacement systems using NMR and MD simulations

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