Protected amino acids as novel low-molecular-weight displacers in cation-exchange displacement chromatography

Although the ability to carry out simultaneous concentration and purification in a single displacement step has significant advantages for downstream processing of pharmaceuticals, a major impediment to the implementation of displacement chromatography has been the lack of suitable displacer compounds. An important recent advance in the state of the art of displacement chromatography has been the discovery that low-molecular-weight dendritic polymers can be successfully employed as displacers for protein purification in ion-exchange systems. In this article, protected amino acid esters (based on arginine and lysine) are shown to be useful displacers for protein purification in cation-exchange systems. A dynamic affinity plot is employed to evaluate the affinity of these low-molecular-weight compounds under dis-placement conditions. In contrast to large polyelectroyte displacers, the efficacy of these low-molecular-weight displacers was shown to be dependent on both the initial carrier salt concentration and the displacer concentration. In addition to the funcamental interest generated by low-molecular-weight displacers, it is likely that these displacers will have significant operatioal advantages as compared with large polyelectrolyte displacers. (c) 1995 John Wiley & Sons, Inc.     

Purification of oligonucleotides by high affinity, low molecular weight displacers

High affinity, low molecular weight anionic displacers were successfully employed for the purification of antisense oligonucleotides. Several important structural characteristics were identified that contribute to the affinity of low molecular weight displacers to a hydrophilized polystyrene divinyl benzene anion exchanger. Sulfonic acid groups were found to possess higher affinity than carboxylic acid and phosphate functionalities, and nonspecific interactions (particularly hydrophobic interactions) were shown to play a major role in the retention process on this stationary phase material. Using this information, two high affinity, low molecular weight displacers were identified. These molecules are relatively inexpensive organic dyes that possess multiple sulfonic acid moieties, as well as aromatic functionalities, which increase nonspecific interactions with the stationary phase. These high affinity displacers, which can be readily detected, were then employed to displace several strongly retained antisense oligonucleotides that could not be displaced by previously established low molecular weight displacers. The displacement process resulted in very high purities of the antisense oligonucleotides. The results presented in this paper are significant in that they demonstrate that low molecular weight displacers for ion-exchange chromatography can possess equal to or greater affinities than their higher molecular weight counterparts, when nonspecific interactions with the stationary phase are exploited. In addition, the results illustrate the high resolutions possible with displacement chromatography and demonstrate an attractive technology for the process scale purification of oligonucleotides.     

Characterization and design of chemically selective cationic displacers using a robotic high‐throughput screen

A robotic high‐throughput displacer screen was developed and employed to identify chemically selective displacers for several protein pairs in cation exchange chromatography. This automated screen enabled the evaluation of a wide range of experimental conditions in a relatively short period of time. Displacers were evaluated at multiple concentrations for these protein pairs, and DC‐50 plots were constructed. Selectivity pathway plots were also constructed and different regimes were established for selective and exclusive separations. Importantly, selective displacement was found to be conserved for multiple protein pairs, demonstrating the technique to be applicable for a range of protein systems. Although chemically selective displacers were able to separate protein pairs that had similar retention in ion exchange but different surface hydrophobicities, they were not able to distinguish protein pairs with similar surface hydrophobicities. This corroborates that displacer‐protein hydrophobic interactions play an important role for this class of selective displacers. Important functional group moieties were established and efficient displacers were identified. These results demonstrate that the design of chemically selective displacers requires a delicate balance between the abilities to displace proteins from the resin and to bind to a selected protein. The use of robotic screening of displacers will enable the extension of chemically selective displacement chromatography beyond hydrophobic displacer‐protein interactions to other secondary interactions and more selective displacement systems. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Titanium dioxide as a chemo-affinity solid phase in offline phosphopeptide chromatography prior to HPLC-MS/MS analysis

We have developed a new offline chromatographic approach for the selective enrichment of phosphorylated peptides that is directly compatible with subsequent analysis by online nano electrospray ionization tandem mass spectrometry. In this technique, a titanium dioxide (TiO2)-packed pipette tip is used as a phosphopeptide trap that acts as an offline first-dimension separation step in a two-dimensional chromatography system. This is followed by online nano reversed-phase high-performance liquid chromatography. Here, we present suitable methods for enrichment, optimized separately for each step: sample loading, washing and elution from the TiO2-filled tips. To increase the trapping selectivity of the TiO2 column, we used the sodium salt of 1-octanesulfonic acid combined with 2,5-dihydroxybenzoic acid as ion-pairing agents and displacers for acidic peptides. These agents also improve the binding of phosphorylated peptides and block the binding of non-phosphorylated ones. This enrichment procedure takes 30 min, followed by a 100-min HPLC program, including washing and an elution gradient.     

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

A parallel batch screening technique was employed to identify chemically selective displacers which exhibited exclusive separation behavior for the protein pair α‐chymotrypsin/ribonuclease A on a strong cation exchange resin. Two selective displacers, 1‐(4‐chlorobenzyl)piperidin‐3‐aminesulfate and N′1′‐(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, α‐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 α‐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. Biotechnol. Bioeng. 2009;102: 1428–1437. © 2008 Wiley Periodicals, Inc.     

Application of hydrophobic interaction displacement chromatography for an industrial protein purification

Recently it has been established that low molecular weight displacers can be successfully employed for the purification of proteins in hydrophobic interaction chromatography (HIC) systems. This work investigates the utility of this technique for the purification of an industrial protein mixture. The study involved the separation of a mixture of three protein forms, that differed in the C-terminus, from their aggregate impurities while maintaining the same relative ratio of the three protein forms as in the feed. A batch high-throughput screening (HTS) technique was employed in concert with fluorescence spectroscopy for displacer screening in these HIC systems. This methodology was demonstrated to be an effective tool for identifying lead displacer candidates for a particular protein/stationary-phase system. In addition, these results indicate that surfactants can be employed at concentrations above their CMCs as effective displacers. Displacement of the recombinant proteins with PEG-3400 and the surfactant Big Chap was shown to increase the productivity as compared to the existing step-gradient elution process.     

Displacement of DL-[3H]-2-amino-4-phosphonobutanoic acid ( [3H]APB) binding with methyl-substituted APB analogues and glutamate agonists

The binding of the excitatory amino acid antagonist DL-2-amino-4-phosphonobutanoic acid (DL-APB) to rat brain synaptic plasma membranes was characterized. As determined by Scatchard analysis, the binding was saturable and homogeneous with a Kd = 6.0 microM and Bmax = 380 pmol/mg of protein. The binding was dependent on the presence of Ca2+ and Cl- ions and was diminished upon freezing. The association rate constant was 6.8 X 10(-3) microM-1 min-1, and the dissociation rate constant was 2.0 X 10(-2) min-1. The L isomers of APB, glutamate, and aspartate were more potent as displacers of APB binding than the D isomers. Previously determined inhibition data obtained for APB-sensitive inputs to hippocampal granule cells are compared to the present displacement data in an attempt to identify this binding protein as the recognition site of the receptor mediating the APB-induced inhibition of synaptic transmission. With the exception of kynurenic acid, all compounds examined in both systems were more potent as displacers of APB binding than as inhibitors of synaptic transmission. This difference in potency was most pronounced for agonists at dentate granule cells. L-Glutamate, D-glutamate, and L-glutamate tetrazole were between 140- and 7500-fold more potent as displacers of DL-APB binding than as inhibitors of synaptic transmission. D-2-Amino-5-phosphonopentanoic acid and alpha-methyl-APB were between 10- and 20-fold more potent as displacers of binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Displacement analysis of binding inhomogeneities in crude extracts of receptors

Guidelines are given to distinguish different kinds of binding inhomogeneities of non-radiolabeled ligands in crude extracts of receptors if an appropriate 'binding analogue' of the displacers is available in radiolabeled form. Three minimal models for the simplest types of binding inhomogeneities are analysed theoretically. These models include a cooperative system (with two interacting sites on the same receptor molecule) and two non-cooperative systems (one of them with a single-site receptor having two conformational states in equilibrium and the other with two single-site receptors independent of each other). In certain cases one can distinguish these systems experimentally. Furthermore, if a group of displacers is already classified according to the above models, then dissociation constants can be determined. The quantitative comparison of these displacers on the basis of their dissociation constants is more appropriate (e.g. in Quantitative Structure Activity Relationship studies) than on the basis of their ID50 and Ki values or Hill coefficients, which is often done.     

Immobilized serum albumin: rapid HPLC probe of stereoselective protein-binding interactions

A human serum albumin-based HPLC chiral stationary phase (HSA-CSP) has been examined as a tool to investigate binding of chiral drugs to HSA and drug-drug protein-binding interactions. Rac-oxazepam hemisuccinate (OXH) was used as a model compound and the chromatographic retention (k') of its enantiomers was determined after addition of displacers to the mobile phase. Compounds known to bind at the same site as OXH and at different sites were tested for their displacing capacities. Competitive binding interactions between the OXH enantiomers and displacers in the mobile phase were reflected by decreases in the k's of (R)- and (S)-OXH. The results indicate that retention on the HSA-CSP accurately reflects binding to native HSA and the technique can determine enantioselective and competitive binding interactions at specific sites on HSA. The HSA-CSP was also able to recognize separate binding areas for (S)- and (R)-OXH.     

Selective displacement chromatography of proteins

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 alpha-lactalbumin and beta-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. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 119-129, 1997.     

Evaluation of selectivity changes in HIC systems using a preferential interaction based analysis

It is well established that salt enhances the interaction between solutes (e.g., proteins, displacers) and the weak hydrophobic ligands in hydrophobic interaction chromatography (HIC) and that various salts (e.g., kosmotropes, chaotropes, and neutral) have different effects on protein retention. In this article, the solute affinity in kosmotropic, chaotropic, and neutral mobile phases are compared and the selectivity of solutes in the presence of these salts is examined. Since solute binding in HIC systems is driven by the release of water molecules, the total number of released water molecules in the presence of various types of salts was calculated using the preferential interaction theory. Chromatographic retention times and selectivity reversals of both proteins and displacers were found to be consistent with the total number of released water molecules. Finally, the solute surface hydrophobicity was also found to have a significant effect on its retention in HIC systems.     

Hydrophobic displacement chromatography of proteins

Displacement chromatography of proteins was successfully carried out in both hydrophobic interaction and reversed-phase chromatographic systems using low-molecular weight displacers. The displacers employed for hydrophobic displacement chromatography were water soluble, charged molecules containing several short alkyl and/or aryl groups. Spectroscopy was employed to verify the absence of structural changes to the proteins displaced on these hydrophobic supports. Displacement chromatography on a reversed-phase material was employed to purify a growth factor protein from its closely related variants, demonstrating the high resolutions that can be achieved by hydrophobic displacement chromatography. This process combines the high-resolution/high-throughput characteristics of displacement chromatography with the unique selectivity of these hydrophobic supports and offers the chromatographic engineer a powerful tool for the preparative purification of proteins.     

High affinity binding site for (+) amphetamine in rat hypothalamus: Fact or artefact?

We studied the binding of (+)³H-amphetamine to membrane sites from rat hypothalamus. Our experiments demonstrate the possible existence of artefactual results in previous studies due to the inadequate use of a filtration technique. In addition, it seems that monoamine oxidase A might be a component of the complex mixture of binding sites which are recognized by (+)³H-amphetamine since harmaline and monoamine oxidase A selective inhibitors are good displacers of this ligand.     

Ion-exchange displacement chromatography of serum proteins, using carboxymethyldextrans as displacers

A system of displacers comprising carboxymethyldextrans with progressively higher content of carboxyl groups forms a displacement train in which absorbed proteins find positions according to their affinities for the adsorbent, an anion exchanger. Because little or no salt need be used, effluent fractions can be evaluated directly by gel electrophoresis. Application to the fractionation of serum proteins is demonstrated.     

The GABAA receptor complex in the developing chick optic tectum: characterization of [H]muscimol binding sites

As part of an ongoing study on the GABA_A receptor complex in the developing chick optic tectum we describe some properties of the agonist site, as labeled by [³H]muscimol, including methodological, kinetic and pharmacological aspects. 16-day embryos and 10-day chicks have been selected as representative age points for the initial characterization of the receptor, prior to more detailed developmental studies. Our data indicate the existence, in both embryos and young birds, of a single class of statistically equivalent, high-affinity, saturable binding sites, with a dissociation constant (K_d) of 80–90 nM in freeze-thawed/washed membranes, and about 8 nM in membranes additionally extracted with low concentrations of Triton X-100. Maximal densities of binding sites are nearly identical in both membrane preparations, ranging from 2 to 3 pmol/mg for the two age points considered.

The pharmacological profiles suggest that avian receptors for [³H]muscimol are generally similar to the corresponding mammalian sites, behaving as typical bicuculline-sensitive, baclofen-insensitive type A GABA receptor sites. However, bicuculline and its derivatives are less efficient displacers of [³H]muscimol in detergent-extracted membrane preparations, being in all cases, as usually, much less effective displacers than GABA agonists.

The effect of Triton X-100 on the muscimol site in the GABA_A receptor, increasing the affinity for the radioligand by a factor of 10, and diminishing the efficiency of antagonists, is considered here in terms of structural changes in the receptor, induced by the action of the detergent on the membrane microenvironment.     

α2-Adrenergic Receptors in Neuronal and Glial Cultures: Characterization and Comparison

Membranes prepared from either neuronal or glial cultures contain alpha 2-adrenergic receptors as determined by the characteristics of [3H]yohimbine [( 3H]YOH) binding. The binding was rapid, reversible, saturable, dependent on the protein concentration used, and reached equilibrium by 5 min in membranes from both neuronal and glial cultures. Scatchard analyses of saturation isotherms revealed similar KD values of 13.7 +/- 1.35 nM (n = 10) for neuronal cultures and 18.42 +/- 2.34 nM (n = 10) for glial cultures. Glial cultures contained many more binding sites for [3H]YOH than neuronal cultures, having a Bmax of 1.6 +/- 0.33 pmol/mg protein (n = 10) compared with 0.143 +/- 0.018 pmol/mg protein (n = 10) in neurons. Drugs selective for alpha 2-adrenergic receptors were the most effective displacers of [3H]YOH binding in both neuronal and glial cultures, i.e., the alpha 2-adrenergic antagonists rauwolscine and yohimbine were better displacers than the other catecholamine antagonists prazosin, corynanthine, or propranolol. The agonists showed the same pattern with the alpha 2-selective drugs clonidine and naphazoline being the most effective competitors for the [3H]YOH site. GTP and its nonhydrolyzable analog. 5'-guanylyl-imidodiphosphate, were able to lower the affinity of the alpha 2-receptors for agonists but not antagonists in membranes from both neuronal and glial cultures, suggesting that the receptors are linked to a G protein in both cell types. The presence of alpha 2-adrenergic receptors in neuronal cultures was also substantiated by light microscopic autoradiography of [3H]YOH binding. In summary, we have demonstrated that both neuronal and glial cultures contain alpha 2-adrenoceptors.     

NAD-sensitive hydrogel for the release of macromolecules

A hydrogel membrane containing immobilized ligands and receptors was synthesized and investigated for the controlled diffusion of test proteins (cytochrome C and hemoglobin). Both Cibacron blue (ligand) and lysozyme (receptor) were covalently linked to dextran molecules that were subsequently crosslinked to form a gel. The resulting stable hydrogels contained both covalent and affinity crosslinks such that their intrinsic porosities were sensitive to competitive displacers of the affinity interaction between lysozyme and Cibacron blue. Transport experiments in a twin chamber diffusion cell showed that as NAD was added to the donor side, the dissociation of the binding sites between the Cibacron blue and the lysozyme led to an increase in protein diffusion through the hydrogel. The results showed that addition of NAD caused a saturable concentration-dependent increase in the transport of both cytochrome C and hemoglobin. This effect was shown to be both specific and reversible.     

Effects of Guanyl Nucleotides on [3H]Flunitrazepam Binding to Rat Hippocampal Synaptic Membranes: Equilibrium Binding and Dissociation Kinetics

The effects of guanyl nucleotides on the binding of [3H]flunitrazepam to rat hippocampal synaptic membranes were studied. In equilibrium binding studies, gamma-amino-n-butyric acid (GABA) increased and GTP decreased the binding affinity of [3H]flunitrazepam; GTP also caused a decrease in binding capacity. The effect, however, is variable. In studies of the dissociation kinetics of [3H]flunitrazepam using diazepam and the antagonist Ro 15-1788 as the displacers, there was evidence of two dissociation rate constants. GTP increased both the fast- and slow-dissociation rate constants and increased the ratio of the slow-dissociation binding state. The effect of GTP was mimicked by its nonhydrolyzable analogue 5'-guanylylimidodiphosphate but not by ATP and occurred when diazepam, but not when Ro 15-1788, was used as the displacer. GABA antagonized the effect of GTP on the dissociation of [3H]flunitrazepam. The nature of the benzodiazepine receptor, its actions, and the possible role of cyclic AMP as a second messenger are discussed.     

Quantitative Receptor Autoradiography: Application to the Characterization of Multiple Receptor Subtypes

Abstract

In vitro autoradiographic techniques combined with computer assisted microdensitometry were used to analyze the characteristics and distribution of multiple recognition sites for the neurotransmitters acetylcholine (M₁ and M₂) and serotonin (5-HT_1A and 5-HT_1B). For this purpose, binding competition experiments were performed using non-subtype selective ³H-labeled ligands and selective unlabeled compounds. Consecutive tissue sections were incubated in the presence of increasing concentrations of displacers. By using this approach, maximal densities of binding sites, as well as competition profiles of several drugs could be analyzed and quantified in microscopic brain areas. Our results reveal the presence of brain structures enriched in one class of muscarinic or serotonergic-1 recognition sites. This provides a tool for better characterization of the proposed “subtype-selective” ligands and suggests physiological functions for these receptor subtypes. It is concluded that quantitative autoradiographic techniques provide a level of anatomical and pharmacological information on neurotransmitter receptor subtypes, which is difficult to attain using membrane binding studies.