Thermosensitive copolymers of N-vinylimidazole as displacers of proteins in immobilised metal affinity chromatography

Synthetic copolymers of N-vinylcaprolactam (VCL) and N-vinylimidazole (VI) were studied as thermosensitive, reusable displacers for immobilised metal affinity chromatography (IMAC) of proteins. The copolymer with weight-average molecular mass of 11700 g/mol prepared by free radical polymerisation at a 9:1 monomer molar ratio was separated into several fractions by IMAC and thermal precipitation. The fraction with an average VI content of 8.5% was most efficient as a reusable displacer for IMAC of ovalbumin, lysozyme and other proteins of egg white on Cu2+-IDA-Sepharose. The displacer exhibited a sharp breakthrough curve and binding capacity of 16-20 mg/ml gel, depending on the flow-rate. The recovery of egg white proteins in the course of displacement chromatography was >95%. The displacer could be removed quantitatively from the protein fractions by thermal precipitation at 48 degrees C. Co-precipitation of lysozyme with the displacer was minimal in the presence of 3% (v/v) acetonitrile, while the lysozyme enzymatic activity in the supernatant was completely retained. Addition of free imidazole to the mobile phase increased the rate of protein desorption and allowed better separation of egg white proteins and the displacer in the course of chromatography. The displacement profile of the egg white extract consisted of three zones with different distributions of individual proteins characterised by SDS-PAGE. Regeneration of the column was easily performed with 0.02 M EDTA in 0.15 M sodium chloride, pH 8.0, followed by washing with distilled water and reloading with Cu2+. The displacer could also be regenerated by thermal precipitation at 48 degrees C and subsequent dialysis against dilute hydrochloric acid (pH 2.5).     

The effect of multi-component adsorption on selectivity in ion exchange displacement systems

This paper examines chemically selective displacement chromatography using affinity ranking plots, batch displacer screening experiments, column displacements, multi-component adsorption isotherms and spectroscopy. The affinity ranking plot indicated that the displacers, sucrose octasulfate (SOS) and tatrazine, should possess sufficient affinity to displace the proteins amyloglucosidase and apoferritin over a wide range of operating conditions. In addition, the plots indicated that the separation of these proteins by displacement chromatography would be extremely difficult. Further, the two proteins were shown to have very similar retention times under shallow linear gradient conditions. When batch displacement experiments were carried out, both tartrazine and SOS exhibited significant selectivity differences with respect to their ability to displace these two proteins, in contrast to the affinity ranking plot results. Column displacement experiments carried out with sucrose octasulfate agreed with the predictions of the affinity ranking plots, with both proteins being displaced but poorly resolved under several column displacement conditions. On the other hand, column displacement with tartrazine as the displacer resulted in the selective displacement and partial purification of apoferritin. Single- and multi-component isotherms of the proteins with or without the presence of displacers were determined and were used to help explain the selectivity reversals observed in the column and batch displacement experiments. In addition, fluorescence and CD spectra suggested that the displacers did not induce any structural changes to either of the proteins. The results in this paper indicate that multi-component adsorption behavior can be exploited for creating chemically selective displacement separations.     

Investigation of particle-based and monolithic columns for cation exchange protein displacement chromatography using poly(diallyl-dimethylammonium chloride) as displacer

The overall topic of the investigation was the separation of basic proteins by cation exchange displacement chromatography. For this purpose two principal column morphologies were compared for the separation of ribonuclease A and alpha-chymotrypsinogen, two proteins found in the bovine pancreas. These were a column packed with porous particles (Macro-Prep S, 10 microm, 1000 A) and a monolithic column (UNO S1). Both columns are strong cation exchangers, carrying -SO3(-)-groups linked to a hydrophilic polymer support. Poly(diallyl-dimethylammonium chloride) (PDADMAC), a linear cationic polyelectrolyte composed of 100-200 quaternary pyrrolidinium rings, was used as displacer. The steric mass action (SMA) model and, in particular, the operating regime and dynamic affinity plots were used to aid method development. To date the SMA model has been applied primarily to simulate non-linear displacement chromatography of proteins using low molar mass displacers. Here, the model is applied to polyelectrolytes with a molar mass below 20000 g mol(-1), which corresponds to a degree of polymerization below 125 and an average contour length of less than 60 nm. The columns were characterized in terms of the adsorption isotherms (affinity, capacity) of the investigated proteins and the displacer.     

Selective displacement chromatography in multimodal cation exchange systems

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.     

Salt effects in anion exchange displacement chromatography: Comparison of pentosan polysulfate and dextran sulfate displacers

Summary Experimental studies were carried out to investigate the utility of pentosan polysulfate as a low molecular weight polyelectrolyte displacer for the purification of proteins in anion-exchange displacement systems. In addition, the influence of mobile phase salt concentration on displacer efficacy, protein-protein resolution, and displacement development were studied for several anionic displacers. It was found that while large polyelectrolytes (50 kd dextran sulfate) were efficient displacers for a wide range of salt concentrations, relatively small polyelectrolytes (3 kd pentosan polysulfate) were seen to act as an efficient displacer only under conditions of high salt micro-environments. In addition, for proteins exhibiting similar affinities, zone mixing at the protein-protein boundary was found to be quite sensitive to the salt concentration. Finally, displacement chromatography was successfully implemented for the separation of proteins from milk whey.     

Displacement chromatography of proteins on hydrophobic charge induction adsorbent column

Displacement chromatography of protein mixtures is proposed on hydrophobic charge induction chromatography (HCIC). We have used an HCIC medium, MEP-Hypercel as the stationary phase and a quaternary ammonium salt, benzethonium chloride, as the displacer. It was found that the multiple interactions between proteins/displacer and the HCIC sorbent, i.e. hydrophobic interaction and charge repulsion, enabled a greater flexibility for the design of displacement processes and ease of column regeneration by adjustment of pH. The capacity factors of proteins and displacers were used to predict their performances in column displacement, and the experimental results agreed well with the prediction. An isotachic displacement train of lysozyme and alpha-chymotrypsinogen A was formed with benzethonium chloride as the displacer at pH 5.0 with good yields and purities of the two proteins. Column regeneration was efficiently achieved by charge repulsion between the displacer and the adsorbent at lower pH values (pH 3 and 4). The results indicate that the displacement chromatography on HCIC is a good alternative to traditional hydrophobic displacement chromatography.     

Purification of complex protein mixtures by ion-exchange displacement chromatography using spacer displacers.

The anion-exchange separation of complex protein mixtures by displacement chromatography using spacer displacers driven by high-affinity final displacers is demonstrated. Guinea pig serum was separated on a medium-resolution adsorbent using a single heterogeneous mixture of carboxymethyldextran displacers to space the protein components. Mouse liver cytosol was separated on a low-resolution adsorbent using six carboxymethyldextran spacer displacers of increasing column affinity. The demonstration of the purification of alkaline phosphatase from E. coli periplasm by displacement chromatography on a high-performance liquid chromatography column is reviewed. The benefits of spacer displacers for separating minor components from complex biological protein mixtures is discussed. A simplified method for preparing carboxymethyldextran displacers is presented.     

Investigation of operating parameters in high-performance displacement chromatography

The effect of operational parameters of displacement chromatography was examined in the separation of various mixtures such as that of the main hydrolysis products of methylfurylbutyrolactone, a potential anticancer drug, the diastereoisomers benzoyl-D- and benzoyl-L-phenylalanyl-L-alanyl-L-proline, as well as polyethylene glycol homologues containing 1-10 ethylene oxide units. The chromatograph was assembled from modules generally used in analytical high-performance liquid chromatography (HPLC) and the column effluent was analyzed by an on-line HPLC unit at 30-sec intervals. Octadecyl-silica was used throughout as the stationary phase. Derivatives of ethylene glycol and propylene glycol as well as tetrabutylammonium bromide and n-butanol were used as displacers. The throughput was used as the measure of efficiency. In the absence of axial dispersion, for a given separation various displacers are expected to yield the same efficiency if the slope of the operating line is kept the same by appropriate adjustment of displacer concentrations. In practice, however, the optimum slope of the operating line has to be determined experimentally as most available chromatographic systems depart from ideal behavior. The dependence of the throughput on the flow-rate and feed load also indicated the presence of non-equilibrium phenomena and the optimum value of these parameters was established experimentally. In most cases water was used as the carrier solvent but the separation of poorly soluble peptides required the use of hydro-organic carriers. Results obtained with octadecyl-silicas of different origin and a given displacer were found to vary significantly suggesting that even for stationary phases of the same type the selection of displacer requires special consideration. Most experiments were carried out with columns having dimensions customary in analytical HPLC. Increasing the inner diameter of the column did not result in the expected increase in throughout probably due to poor distribution of the sample at the column entrance. Therefore scaling-up the process requires careful engineering of inlet conditions. Throughput can be increased by connecting a small inner diameter column to the outlet of a large diameter preparative column. As theoretical predictions for ideal displacement chromatography do not hold in practice when axial dispersion is significant, optimization of the process requires experimental support. The results obtained in the separation of a variety of mixtures shed light on the most important operational aspects of displacement chromatography and suggest approaches to find optimum conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Displacement chromatography of chemotactic peptides

Displacement chromatography was successfully used to separate a binary peptide mixture, n-formyl-Met–Phe and n-formyl-Met–Trp, on a reversed-phase column. Displacement parameters such as choice of displacer, displacer concentration, mobile phase organic level, and flow-rate were critically examined in the context of maximizing productivity. Since the feed composition was limited by solubility, optimal productivity was sought as a function of feed volume. The impurities contained in the commercial displacers used in this study did not seem to affect the overall separation quality. In most cases the final pattern of contiguous rectangular bands was not attained; nevertheless, separations of high productivity were achieved using benzethonium chloride and tris[2-(2-methoxyethoxy)ethyl]amine as displacers. In some cases further increase in productivity was not possible only because of solubility constraints. Loading of feed at low initial organic modulator level coupled with displacements at higher modulator level was found to give efficient separations.     

Mass influences in the performance of oligomeric poly(diallyldimethylammonium chloride) as displacer for cation-exchange displacement chromatography of proteins

A novel type of linear polyelectrolyte, namely poly-DADMAC [poly(diallyldimethylammonium chloride)], was prepared and studied as a displacer for cation-exchange displacement chromatography of proteins. In contrast to the commercially available polymers of that chemistry, the novel type of poly-DADMAC introduced here is characterized by a homogeneous linear structure, a narrow distribution of the (adjustable) molar mass as well as by a defined and homogeneous affinity for the stationary phase. Five poly-DADMACs of different size (17900 to 88000 g/mol) were prepared and compared with regard to their stationary phase affinity and protein separation potential, taking a mixture of basic proteins, namely lysozyme, cytochrome C, and ribonuclease A (from bovine pancreas), as an example. The steric mass action model was employed to aid method development. Under the chosen conditions (low ionic strength of the mobile phase guaranteeing strong binding of both the proteins and the displacer) the poly-DADMAC with the lowest molar masses proved to be the most efficient displacers for the basic proteins with a stationary phase affinity constant of 5.3 x 10(16) and a steric factor of 224. Using this substance as displacer, a sample mixture containing up to three proteins was separated and the proteins recovered at high yields (80-97%) and in high purity and concentration.     

The effect of feed composition on the behavior of chemically selective displacement systems

In this paper we examine whether adding a more retained protein to the feed will mitigate displacer–protein interactions in the column, thus affecting the displacement modality that occurs (chemically selective vs. traditional displacement chromatography). STD-NMR experiments were carried out to probe displacer–protein interactions for the chemically selective displacer chloroquine diphosphate and the results indicated that this displacer only had measurable interactions with the protein α-chymotrypsinogen A. For a two component feed mixture containing ribonuclease A and α-chymotrypsinogen A, the separation resulted in the displacement of ribonuclease A, with the more hydrophobic α-chymotrypsinogen A remaining on the column. On the other hand, when the experiment was repeated with cytochrome c added to the feed, all three feed proteins were displaced. Column simulations indicated that the combination of sample self-displacement occurring during the introduction of the feed, along with the dynamics of the initial displacement process at the column inlet was responsible for this behavior. These results indicate that for this class of hydrophobic-based selective displacers, in order for the protein to be selectively retained, the protein should be the most strongly retained feed component.     

Ion-exchange displacement chromatography of proteins Dextran-based polyelectrolytes as high affinity displacerss

Dextran-based polyelectrolyte displacers were successfully employed for the displacement purification of proteins in ion-exchange displacement systems. The effect of molecular mass was investigated by examining the efficacy of DEAE-dextran and dextran sulfate displacers of various molecular masses in cation- and anion-exchange systems, respectively. Induced salt gradients produced during these displacement experiments were measured in order to study their effect on the protein separations. The unique characteristics of these displacements were well predicted by simulations obtained from a steric mass action (SMA) ion-exchange model. These displacements differ from the traditional vision of displacement chromatography in several important ways: the isotherm of the displacer does not necessarily lie above the feed component isotherms; the concentration of the displaced proteins can sometimes exceed that of the displacer; higher-molecular-mass displacers are not necesarily more efficacious than lower-molecular-mass compounds; and the salt gradients induced by the adsorption of the displacer produce different salt micro-environments for each displaced protein.     

Unique selectivity windows using selective displacers/eluents and mobile phase modifiers on hydroxyapatite

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.     

An affinity-based strategy for the design of selective displacers for the chromatographic separation of proteins

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.     

Displacer concentration effects in displacement chromatography: Implications for trace solute detection

In this paper, the utility of ion-exchange displacement chromatography for the concentration and enrichment of trace proteins is examined. Separations with varying displacer concentrations (1–25 mM neomycin sulfate) indicate that higher concentrations result in elevated protein concentrations, at the price of reduced yields. The results demonstrate that displacement chromatography carried out at relatively low displacer concentrations (2.5 mM) can produce significant concentration (8.5-fold) and enrichment (18-fold) of trace proteins present in the feed. Parametric simulations using the steric mass action model are carried out to investigate the concentration effects and enrichment factors observed over a wide range of feed, displacer and buffer counter-ion concentrations, and solute separation factors. The simulations confirm that trace components can be readily concentrated and enriched by displacement chromatography and that these effects will be more pronounced as the separation factor between trace and abundant components is increased. The results presented in this paper indicate the potential of displacement chromatography for improved separations where trace enrichment is critical such as proteomic applications.     

High performance displacement chromatography of proteins: Separation of β-Lactoglobulins A and B

Summary β-Lactoglobulins A and B were separated by high performance displacement chromatography on an anion-exchanger column with chondroitin sulfate as the displacer. A sample of 100 mg containing a mixture of the two β-lactoglublins was separated on a column of 75×7.5 mm in a single chromatographic run. The separation process followed the rules of the classical displacement development: the two proteins formed contiguous rectangular bands and their concentrations were dependent on the displacer concentration. The results demonstrate that high performance displacement chromatography is a useful technique for the separation of proteins in preparative amounts with columns and instrumentation typically used in analytical HPLC. Furthermore, it has the potential to become the method of choice in large scale separation of proteins.     

Displacement Chromatography of Proteins using Low Molecular Weight Anionic Displacers

A major impediment to the implementation of displacement chromatography has been the lack of suitable displacer compounds. Recently, it has been shown that low molecular weight dendritic polymers, protected amino acids and antibiotics can be successfully employed for displacement purification in cation-exchange systems. In this paper, a variety of low molecular weight anionic displacers are identified for the resolution of a bovine -lactoglobulin mixture into two closely related forms (A and B). A Dynamic Affinity plot is employed to evaluate the affinity of these low molecular weight compounds under various displacement conditions. In contrast to large polyelectrolyte displacers, the efficacy of these low molecular weight displacers are shown to be dependent on displacer concentration. In fact, the Dynamic Affinity Plot qualitatively predicts the transition from a displacement to a desorption regime with these low molecular weight displacers. In addition to the fundamental interest generated by low molecular weight displacers, it is likely that these displacers will have significant operational advantages as compared to large polyelectrolyte displacers. Furthermore, the ability to carry out selective displacement chromatography with these low molecular weight displacers offers significant potential for developing robust large scale displacement processes.     

Liquid chromatography of polymer mixtures applying a combination of exclusion and full adsorption mechanisms. 5. Six-component blends of chemically similar polymers

The separation of six-component blends of chemically similar homopolymers utilising the full adsorption-desorption (FAD) process is presented. The main advantage of the FAD approach over other methods represents the successive and independent size- exclusion chromatography (SEC) characterisation of all blend components. The method is based on the full adsorption and retention of all n or n−1 components of the polymer blend from an adsorption promoting liquid (ADSORLI) in a small FAD column. Nonadsorbed macromolecules are forwarded directly into SEC for molecular characterisation. Next, appropriate displacers are successively applied to the FAD column to selectively release preadsorbed blend constituents into the on-line SEC column. Dynamic integral desorption isotherms for single constituents, as well as for polymer blends to be analysed, allow identification of optimal displacer compositions to release just one kind of macromolecule. Model polymer blends containing polystyrene (PS), poly(lauryl methacrylate), poly(butyl methacrylate), poly(ethyl methacrylate), poly(methyl methacrylate) and poly(ethylene oxide) (PEO) or, alternatively, PS, poly(2-ethylhexyl acrylate), poly(butyl acrylate), poly(ethyl acrylate), poly(methyl acrylate) and PEO of similar molar masses can be separated and characterised in one multistep run using nonporous silica FAD packing, toluene as an ADSORLI and its mixtures with a desorption promoting liquid such as ethyl acetate, tetrahydrofuran or dimetylformamide to form displacers with appropriate desorption strength. Received: 9 September 1998 Accepted in revised form: 16 November 1998     

Tandem use of carboxypeptidase Y reactor and displacement chromatograph for peptide synthesis

A packed-bed enzyme reactor with immobilized carboxypeptidase Y was used in tandem with a displacement chromatograph for the preparation of N-benzoyl-L-arginyl-L-methioninamide, from N-benzoyl-L-arginine and L-methioninamide. The pumps and valves of the coupled enzyme reactor and displacement chromatograph were controlled by a microprocessor. The enzyme was immobilized on microparticulate amino-silica by glutaraldehyde and packed into a 60 X 4.6 mm I.D. column. The packed-bed reactor was used in the recirculating mode and components of the reaction mixture were subsequently separated by displacement chromatography on a 250 X 4.6 mm octadecyl-silica column using butoxyethoxyethanol as the displacer. Unreacted L-methioninamide was returned to the reaction mixture. Both the progress of the reaction and the extent of separation by displacement chromatography were monitored by high-performance liquid chromatographic analysis. The system was designed so that enzymatic peptide synthesis, separation by displacement chromatography, and column regeneration were carried out simultaneously by using two identical columns in parallel. An amount of 460 mg of N-benzoyl-L-arginyl-L-methioninamide having purity greater than 99% could be obtained in 24 h with this system. The tandem operation of the enzyme reactor and liquid chromatograph operated in the displacement mode offers a means for the synthesis and purification of peptides.