The selectivity of carbohydrate-imprinted polymers for several disaccharides, namely cellobiose, maltose, lactose and gentiobiose, is investigated. An ternary ligand–Cu(II)–carbohydrate complex was formed in alkaline solution and captured afterwards in the polymer. The accessibility of the polymer matrix for disaccharides was investigated by HPLC analysis, refractometry and 1H NMR spectroscopy applying excess of the original template during rebinding experiments under saturation conditions in unbuffered, aqueous solution at neutral pH and 20 °C. The selective discrimination of the α- and β-glycosidic linkage of cellobiose and maltose is demonstrated. It is further shown, that the disaccharide-imprinted polymers slightly distinguish between the 1,4-β- and the 1,6-β-glycosidic linkage of cellobiose and gentiobiose, while cellobiose and lactose are not selectively recognized. Due to the weak apparent binding constant of the functional Cu(II) monomers with the targeted disaccharides at physiological pH, the recognition process is dominated by the shape of the created imprinted cavity under the applied conditions.
HPLC-based separations of amino acids and peptides, nucleotide bases, drugs, sugars and steroids using molecularly imprinted polymers (MIPs) have been reviewed in this article. The molecular recognition mechanisms of the template molecules on the MIPs in organic and aqueous eluents were discussed. Furthermore, new polymerization methods suitable for preparations of HPLC columns and packing materials using molecular imprinting techniques, and their applications to HPLC-based separations are also dealt with.
The recovery of cutinase of Fusarium solani pisi produced by the yeast Saccharomyces cerevisiae was studied in a fluidised bed adsorption system directly integrated with a productive fermenter (so-called direct product sequestration; DPS). The relative efficiency of this system was compared with the one of a conventional purification process by discrete sequences of fermentation, broth clarification, ultrafiltration and fixed bed anion exchange chromatography. By direct product sequestration of the extracellular heterologous cutinase it was possible, through only one unit operation: (i) to perform broth clarification, (ii) to obtain a high cutinase concentration factor, and (iii) to recover cutinase with a specific activity that equalled that obtained with the conventional purification process. It was also possible (iv) to substantially reduce the total process time, (v) to improve the overall yield, and (vi) to increase cutinase productivity. Furthermore, the procedure outlined is suitable for large scale bioprocess exploitation.
The physical behavior of the binary phase systems of the non-ionic polyoxyethylene detergent Agrimul NRE 1205 and water was investigated. This technical detergent can be used for the large-scale recovery of biomolecules in detergent based aqueous two-phase systems. The phase diagram was determined. It shows significant and unexpected differences to highly purified detergents. Very similar to neat detergents the phase diagram can be influenced by auxiliary chemicals thus shifting the entire phase diagram in general to lower temperatures. This was demonstrated by lowering the cloud-point by various additions. The concentration factor, as an important parameter of a first capture step in purification was investigated and modeled. Auxiliary chemicals, temperature change and change in detergent concentration also influence the viscosity and density of the phases. These experimental data are shown. They can help to explain the separation behavior of proteins. In large-scale separations aqueous two-phase systems are separated using disc-stack centrifuges. It is demonstrated that this is not a feasible method for detergent-based aqueous two-phase extraction and the physical reason is presented.
Of the many applications of molecular imprinting in analytical separation science, the one with highest potential of soon being used in routine analysis is that of solid-phase extraction. Already several examples of selective pre-concentration of biological and environmental samples have been reported. The interest in imprinted extraction sorbents originates from the high selectivities and affinities obtainable, properties which can be qualitatively and quantitatively pre-determined for a particular analyte and separation by the imprinting process. This review summarises work published on molecular imprinted solid-phase extraction and discusses some imprinted-sorbent specific method development issues.
The parameters important for an optimisation of cloud point extraction in technical scale were investigated using a genetically engineered fusion protein derived from endoglucanase I expressed in Trichoderma reesei and the nonionic polyoxyethylene Agrimul NRE 1205. The key parameters are temperature, detergent concentration, and additional salts. These parameters are interdependent, thus there is an optimum in the partition coefficient with respect to detergent concentration and a maximum for the partition coefficient and the yield with respect to temperature. These results were confirmed for the detergent C12E5 to demonstrate that these optima are due to the nature of polyoxyethylenes. Cloud point extraction was found to be only slightly affected by pH. In the case studied extraction of whole broth is favourable for a high yield and partition coefficient, since fusion protein adhering to the cells can be solubilized. However some loss of detergent which remains in the fungal biomass was observed.
Improved specificity and binding affinity by molecularly imprinted polymers is possible by development of novel functional materials. Furthermore, increasing the cross-link density of imprinted polymers by using cross-linking functional groups was anticipated to improve polymer molecular recognition. A novel cross-linking monomer derived from an L-aspartic acid precursor was synthesized and employed in molecularly imprinted polymers to mimic more closely the scaffolding of proteins, and thus provide more protein-like selectivity. Chromatographic results revealed a more than 7-fold improvement in polymers imprinted using the new monomer versus a traditionally formulated polymer imprinted with methacrylic acid as the functional monomer.
Buoyancy-induced mixing occurs during expanded bed adsorption processes when the feed stream entering the bottom of the system has a lower density than that of the fluid above it. In the absence of a headspace, mixing in the expanded bed can be modeled as a single, well-mixed vessel, with first-order dynamics. In the presence of a headspace, the system exhibits second-order dynamics for the densities typically encountered in protein chromatography, and can be modeled as two well-mixed vessels (the expanded bed and the headspace) arranged in series. In this paper, the mixing dynamics of the expanded bed are described and a mathematical model of the system is presented. Experimental measurements of density changes during the dilution of sucrose and salt solutions in a STREAMLINE 25 column are presented. These show excellent agreement with predictions using the model. A number of strategies for wash and elution in expanded mode, both in the presence and absence of headspace, are discussed.
A molecularly imprinted polymer which recognises the mycotoxin ochratoxin A was prepared using the mimic N-(4-chloro-1-hydroxy-2-naphthoylamido)-(L) -phenylalanine as a template. The polymer was obtained by dissolving the template, methacrylic acid and ethylendimethacrylate in chloroform and polymerising the mixture by thermal treatment at 60 °C. The monolith obtained was crushed, sieved to 30–90 μm and extensively washed till the template could no longer be found in the washing solution. The binding properties towards the template, ochratoxin A and several related molecules were measured by eluting with acetonitrile and chloroform a HPLC column packed with the imprinted polymer. The experimental results show that the polymer recognises not only the template well, but also the ochratoxin A. The specific molecular recognition effect is due to hydrogen bond interactions but in order to assure the full recognition effect adjunctive steric factors are necessary. The magnitude of these interactions can be controlled by the use of limited amounts of acetic acid in the mobile phase.
This is a review discussing the production and properties of cryogels (from the Greek κριoσ (kryos) meaning frost or ice), immobilization of ligands in cryogels and the application of affinity cryogels in bioseparation. Cryotropic gel formation proceeds in a non-frozen liquid microphase existing in the macroscopically frozen sample. Due to the cryoconcentration of gel precursors in the non-frozen liquid microphase, cryogelation is characterised by a decrease in the critical concentration of gelation and an increase in gelation rates compared with traditional gelation at temperatures above freezing point.
Clavulanic acid (CA) is a beta-lactamase inhibitor produced by strains of Streptomyces clavuligerus . Nowadays, the combination of CA with amoxycillin is the most successful example of the use of a beta-lactam antibiotic sensitive to beta-lactamases together with an inhibitor of these enzymes. Clavulanic acid is purified from fermentation broth by a series of steps consisting mainly of two-phase separation processes such as liquid–liquid extraction, adsorption or ion-exchange chromatography, among others. Amberlite IRA 400, a strong anion-exchange resin, has a very high adsorption capacity for CA (Mayer et al. 1997). This resin can be pre-treated with NaCl (chloride cycle), to remove selectively only those anions, which are able to displace chloride from the resin or with NaOH (hydroxyl cycle), to remove all species of anions. In order to decide the best operating conditions for CA recovery by ion-exchange resins and then to construct a model of this separation process, batch experiments were conducted using Amberlite IRA 400 in the chloride cycle. These runs were carried out in a 200 ml stirred tank, at two different initial solution pH, 6.2 and 4.0; the temperature was maintained at 10 °C and 20 °C during adsorption and 30 °C during the desorption step. It was possible, on the basis of these batch results, to model the separation process, including the adsorption kinetics, equilibrium data and mass transfer limitations.
The adsorption and desorption pattern of alkaline protease was studied using different aliphatic and aromatic hydrophobic ligands. Overall, higher adsorption was obtained on ligands coupled to 6% cross-linked gel than the 4% gel. The highest adsorption was obtained on butyl (94%) and phenyl (98.4%) of 6% cross-linked gel. The adsorption was dependent on concentration and nature of the ligand. In a single-step operation, almost 20-fold purification with 40% yield of the enzyme was obtained using all the optimized experimental parameters.
The purification of human IgG3 subclass out of IgG (Immunoglobulin-G) was studied using protein A-Sepharose affinity chromatography. The effect of operational parameters such as flow rate, ionic strength, pH and size of sample was investigated, and the process was scaled-up 10-fold. The use of 0.5 m NaCl in the loading buffer had a dramatic effect in the purity of IgG3 recovered in the flowthrough fraction (values in the order of 97% were consistently obtained). This was attributed to a more effective binding of IgG subclasses 1, 2 and 4 to protein A (well known classical mechanism based in Fc fragment) and in some extent to a decrease in the binding of subclass 3 to protein A by the alternative mechanism based in the Fab fragment. The increase in residence time also increased in a relevant way the purity of IgG3. This is attributed to an increased effectiveness of the mechanisms mentioned above. The recovery yields in the IgG3 rich fraction were in the range 21–32% and are possibly a consequence of binding to protein A by the alternative mechanism and also due to deactivation during processing.
The isolation and preliminary characterization of a trypsin-like protease with high activity at 20 °C is described. This protease was isolated from Antarctic krill ( Euphasia superba ) by a two-step chromatography process and the use of zymogram analyses. The protease has a molecular weight of 30 kDa and a p I of 4.1. Its specific activity at 20 °C on BAPNA is 0.5 U/mg.
In this review article the function of the binding site monomers in the molecular imprinting procedure is discussed. Especially, new developments towards stoichiometric noncovalent interactions are highlighted. In stoichiometric noncovalent interactions template and binding site monomer in an 1:1 molar ratio are nearly completely bound to each other. This is only possible if the association constants are considerably high ( K ass < 900 M −1 ). Using this type of interaction in molecular imprinting no excess of binding sites is necessary and binding sites are only located inside the imprinted cavity. Since all cavities can be reloaded these polymers show high capacity (e.g., for preparative application) and are especially suited for the synthesis of catalytically active imprinted polymers. Discussed are binding site interactions based on amidines (and guanidines), multiple hydrogen bonding, charge-transfer interactions, and host–guest inclusion. The systematic investigation of the underlying binding reaction is described in detail. With low-molecular weight model substances the thermodynamics of the association can be conveniently investigated, e.g., by NMR spectroscopy.
Expanded bed adsorption (EBA) was examined as the initial capture/purification step in the purification of monoclonal antibodies from Chinese hamster ovary (CHO) cultures. Two process alternatives each using EBA were compared to a conventional Protein A process without EBA. One alternative used Protein A affinity EBA followed by packed-bed cation and anion-exchange steps. The other alternative used cation-exchange EBA as the capture step followed by packed-bed Protein A and anion-exchange steps. The process using Protein A EBA produced comparable purity (host cell protein, DNA, Protein A, antibody aggregate) to the conventional process. However, the Protein A EBA column showed a significant decrease in dynamic capacity with a limited number of cycles. The process using cation EBA achieved comparable levels of host cell proteins (HCP) and DNA but not antibody aggregate or leached Protein A compared to the conventional process.
