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There is a constantly growing demand for monoclonal antibodies for therapeutic and diagnostic applications. Due to this trend the development of new and cost-efficient purification methods is of high importance. This study investigates the separation of a monoclonal antibody from model impurity bovine serum albumin using anionic polyelectrolytes. Different types and molecular weights of anionic polyelectrolytes were screened for their applicability as precipitation agents. Polyanetholesulfonic acid was identified as most promising candidate. Furthermore, the influence and interactions of various parameters on antibody precipitation were investigated by means of Design of Experiments. Design of Experiments provided a fast identification of influencing parameters and parameter interactions. The strongest influence on yield and selectivity of precipitation showed the polyelectrolyte concentration and its interaction with the pH value and the sodium chloride and bovine serum albumin concentration. The interactions identified showed, that parameters should be investigated and optimized together, not stand-alone.

We present a feasibility study for an antibody capturing process from clarified cell culture fluid using semi-selective protein precipitation with salt-tolerant copolymers. Protein precipitation is mediated by hydrophobic and electrostatic interactions with the copolymer that can be customized for the respective target. Precipitation yield with different copolymers at ionic strength of 2–22.5 mS cm−1 and pH 5.0–pH 5.7 was evaluated using pure monoclonal antibody solutions. Optimized parameters were used to elucidate yield and purity of various antibodies precipitated at physiological conditions from cell culture fluid of CHO, NS0, and SP2/0 cell culture fluid. Precipitated protein was easily redissolved in small volume, enabling concentrating monoclonal antibodies (mAb) more than 40-fold and up to 100-fold, while residual polymer was removed to >98% using cationic polymer attached to silica flakes. mAb recovery of >90% and host cell protein clearance of >80% were achieved, not requiring any pre-dilution of cell culture fluid. Precipitation showed no impact on mAb binding affinity when compared to non-precipitated mAb. The obtained yield and purity were lower compared to a protein A based purification and loss of mAb was factor 1.5–3.0 higher. Yet, for high titer mAb purification processes being implemented in the future, precipitation is an attractive option due to its ease of scalability and cost-effectiveness. Biotechnol. Bioeng. 2013;110: 2915–2927. © 2013 Wiley Periodicals, Inc.

The effect of low ionic strength leading to reduced polyelectrolyte–protein interactions has been shown by in silico and in vitro experiments, suggesting polyelectrolyte rigidity increasing at low ionic strength, thus leading to reduced interactions with proteins. This contribution elucidates polyelectrolyte–protein precipitation in the 0–2.6-mS cm−1 ionic strength regime with polyelectrolyte rigidity determinations, using viscosimetry at these conditions, also considering protein charge distributions, using different proteins. Precipitation yields increased from 5 to 40 % at low ionic strength to up to 90 % at intermediate ionic strength, depending on protein and polyelectrolyte type, using lysozyme and three different monoclonal antibodies. Comparing precipitation behavior of the monoclonal antibodies, a qualitative correlation between required polyelectrolyte flexibility to enhance protein precipitation and protein average charge as well as hydrophobicity of the antibodies was discovered. Antibodies with lower average charge and less hydrophobicity required more flexible polyelectrolytes to enhance precipitation behavior by allowing interaction of the polyelectrolytes with proteins, attaching to positively charged protein patches while “circumnavigating” negatively charged protein areas. In contrast, antibodies with higher protein average charge showed increasing precipitation yields up to 90 % already at lower ionic strength, associated with then more rigid polyelectrolyte structures. Therefore, designing polyelectrolytes with specific chain flexibility could help to improve precipitation behavior toward specific target proteins in polyelectrolyte-driven purification techniques.

Production of recombinant proteins, e.g. antibodies, requires constant real-time monitoring to optimize yield and quality attributes and to respond to changing production conditions, such as host cell protein (HCP) titers. To date, this monitoring of mammalian cell culture-based processes is done using laborious and time consuming enzyme-linked immunosorbent assays (ELISA), two-dimensional sodium dodecylsulphate polyacrylamide gel electrophoresis, and chromatography-based systems. Measurements are usually performed off-line, requiring regular sample withdrawal associated with increased contamination risk. As information is obtained retrospectively, the reaction time to adapt to process changes is too long, leading to lower yield and higher costs. To address the resulting demand for continuous online-monitoring systems, we present a feasibility study using attenuated total reflection spectroscopy (ATR) to monitor mAb and HCP levels of NS0 cell culture in situ, taking matrix effects into account. Fifty-six NS0 cell culture samples were treated with polyelectrolytes for semi-selective protein precipitation. Additionally, part of the samples was subjected to filtration prior to analysis, to change the background matrix and evaluate effects on chemometric quantification models. General models to quantify HCP and mAb in both filtered and unfiltered matrix showed lower prediction accuracy compared to models designed for a specific matrix. HCP quantification in the range of 2,000–55,000 ng mL−1 using specific models was accurate for most samples, with results within the accepted limit of an ELISA assay. In contrast, mAb prediction was less accurate, predicting mAb in the range of 0.2–1.7 g L−1. As some samples deviated substantially from reference values, further investigations elucidating the suitability of ATR for monitoring are required. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013

Process development in up- and downstream processing requires enhanced, non-time-consuming, and non-expensive monitoring techniques to track product purity, for example, the level of endotoxins, viral particles, and host cell proteins (HCPs). Currently, HCP amounts are measured by laborious and expensive HCP-enzyme-linked immunosorbent assay (ELISA) assays best suited for measuring HCP amounts in the low concentration regime. The measurement of higher HCP amounts using this method requires dilution steps, adding dilution errors to the measurement. In this work we evaluated the suitability of attenuated total reflection spectroscopy for HCP quantification in process development, using clarified cell culture fluid from monoclonal antibody producing Chinese hamster ovary-cells after treatment with different polyelectrolytes for semi-selective clarification. Forty undiluted samples were chosen for multivariate data analysis in the middle infrared range and predicted HCP-values were in good agreement with results obtained by an ELISA-assay, suggesting the suitability of this new method for HCP-quantification. As this method is able to quantify HCP titers ranging from approximately at least 20,000–200,000 ng mL−1, it is suitable especially for monitoring of process development steps with higher HCP concentrations, omitting dilution errors associated with ELISA assays. Biotechnol. Bioeng. 2013; 110: 252–259. © 2012 Wiley Periodicals, Inc.

This work deals with the determination of total sulfite in wine. The determination combines an in-sample hydrogen peroxide oxidation of total sulfite in alkalized wine to sulfate with the separation and quantitation of the latter anion by zone electrophoresis (ZE) on-line coupled with isotachophoresis (ITP) on a column-coupling chip. Sample clean up, integrated into the ITP–ZE separation, eliminated wine matrix in an extent comparable to that provided by a highly selective distillation isolation of sulfite. At the same time, conductivity detection, employed to the detection of sulfate in the ZE stage of the ITP–ZE combination, provided for sulfate the concentration limit of detection corresponding to a 90 μg/l concentration of sulfite in the loaded sample (0.9 μl). Such a detectability allowed a reproducible quantitation of total sulfite when its concentration in wine was 15 mg/l. Formaldehyde binding of free sulfite in wine, included into the pre-column sample preparation, prevented an uncontrolled oxidation of this sulfite form. This step contributed to an unbiased determination of sulfate present in the original wine sample (this determination corrected for the concentration of sulfate determined in the sample after the peroxide oxidation of sulfite to the value equivalent to the total sulfite). The 99–101% recoveries of sulfite, determined for appropriately spiked wine samples, indicate a very good accuracy of the present method. Such a statement also supports excellent agreements of the results of quantitation based on the in-sample peroxide oxidation of the total sulfite (bound sulfite released at a high pH) with those in which this analyte was isolated from wine by distillation (bound sulfite released at a very low pH).

A feasibility study was performed using zone electrophoresis (ZE) coupled on-line with isotachophoresis (ITP) sample pretreatment on a poly(methyl methacrylate) column-coupling chip with integrated conductivity detection for direct determination of drugs in serum. Valproic acid (an antiepileptic drug), having a therapeutic range of 0.35-0.69 mmol/L (50-100 mg/L), was a test analyte while reference serum samples served as proteinaceous matrices. ITP provided in the ITP-ZE combination a multitask sample pretreatment: (1) separation of the analyte from the serum matrix and its concentration into a narrow ITP band, (2) removal of the matrix constituents migrating in the ITP stack from the separation compartment of the chip, (3) ITP stacking of the drug released on a continuous electrophoretic decomposition of the drug-protein complex. A high sample loadability, closely linked with the use of ITP in the first separation stage, made it possible to inject diluted serum samples with the aid of a 0.95 μL sample channel of the chip. Consequently, a 1-2 μmol/L concentration limit of quantitation for valproate from the response of the conductivity detector in the ZE stage of the combination was reached. The drug could be reliably determined in less than 10 minutes also in instances when its concentration in serum was below the lower value of the therapeutic range. 90-94% recoveries of valproate from serum samples were obtained in its direct ITP-ZE determination when the filtration of the diluted serum (a 0.45 pm pore size filter) was the only pre-column sample handling operation. No disturbances attributable to the precipitation of proteins from the loaded samples in the chip channels were detected.

The use of a poly(methylmethacrylate) capillary electrophoresis chip, provided with a high sample load capacity separation system (a 8500 nL separation channel combined with a 500 nL sample injection channel) and a pair of on-chip conductivity detectors, for zone electrophoresis (ZE) determination of oxalate in beer was studied. Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed and electrophoresis was a dominant transport process in the separations performed on the chip. A low pH of the carrier electrolyte (3.8), implemented by aspartic acid and bis-tris propane, provided an adequate selectivity in the separation of oxalate from anionic beer constituents and, at the same time, also a sufficient sensitivity in its conductivity detection. Under our working conditions, this anion could be detected at a 0.5 μmol/L concentration also in samples containing chloride (a major anionic constituent of beer) at a 1800 higher concentration. Such a favorable analyte/matrix concentration ratio made possible accurate and reproducible [typically, 2-5% relative standard deviation (RSD) values of the peak areas of the analyte in dependence on its concentration in the sample] determination of oxalate in 500 nL volumes of 20-50-fold diluted beer samples. Short analysis times (about 200 s), minimum sample preparation, and reproducible migration times of this analyte (0.5-1.0% RSD values) were characteristic for ZE on the chip.

This work deals with zone electrophoresis (ZE) separations of proteins on a poly(methyl methacrylate) chip with integrated conductivity detection. Experiments were performed in the cationic mode of the separation (pH 2.9) with a hydrodynamically closed separation compartment and suppressed electroosmotic flow. The test proteins reached the detector in less than 10 min under these working conditions and their migration times characterized excellent repeatabilities (0.1-0.6% RSD values). The chip-to-chip agreements of the migration times, evaluated from the ZE runs performed on three chips, were within 1.5%. The conductivity detection provided for protein, loaded on the chip at 10-1000 microg/ml concentrations, detection responses were characterized by 1-5% RSD values of their peak areas. Such migration and detection performances made a frame for reproducible baseline separations of a five-constituent mixture (cytochrome c, avidin, conalbumin, human hemoglobin and trypsin inhibitor). On the other hand, a high sample injection channel/separation compartment volume ratio of the chip (500 nl/8500 nl) restricted the resolution of proteins of very close effective mobilities in spite of the fact that in the initial phase of the separation an electric field stacking was applied. A maximum macroconstituent/trace constituent ratio attainable for proteins on the chip was assessed for cytochrome c (quantifiable when its concentration in the loaded sample was 10 microg/ml) and apo-transferrin (containing a trace constituent migrating in the position of cytochrome c detectable when the load of apo-transferrin was 2000 microg/ml). This assessment indicated that a ratio of 1000:1 is attainable with the aid of conductivity detection on the present chip.

The use of capillary zone electrophoresis (CZE) on-line coupled with isotachophoresis (ITP) sample pretreatment (ITP-CZE) on a poly(methylmethacrylate) chip, provided with two separation channels in the column-coupling (CC) arrangement and on-column conductivity detection sensors, to the determination of bromate in drinking water was investigated. Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed and electrophoresis was a dominant transport process in the ITP-CZE separations. A high sample load capacity, linked with the use of ITP in this combination, made possible loading of the samples by a 9.2 microL sample injection channel of the chip. In addition, bromate was concentrated by a factor of 10(3) or more in the ITP stage of the separation and, therefore, its transfer to the CZE stage characterized negligible injection dispersion. This, along with a favorable electric conductivity of the carrier electrolyte solution, contributed to a 20 nmol/L (2.5 ppb) limit of detection for bromate in the CZE stage. Sample cleanup, integrated into the ITP stage, effectively complemented such a detection sensitivity and bromate could be quantified in drinking water matrices when its concentration was 80 nmol/L (10 ppb) or slightly less while the concentrations of anionic macroconstituent (chloride, sulfate, nitrate) in the loaded sample corresponding to a 2 mmol/L (70 ppm) concentration of chloride were still tolerable. The samples containing macroconstituents at higher concentrations required appropriate dilutions and, consequently, bromate in these samples could be directly determined only at proportionally higher concentrations.

The use of a poly(methylmethacrylate) capillary electrophoresis chip, provided with a high sample load capacity separation system (a 8500 nL separation channel coupled to a 500 nL sample injection channel) and a pair of on-chip conductivity detectors, for zone electrophoresis (ZE) determination of oxalate in urine was studied. Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed and electrophoresis was a dominant transport process in the separations performed on the chip. A low pH of the carrier electrolyte (4.0) provided an adequate selectivity in the separation of oxalate from anionic urine constituents and, at the same time, also a sufficient sensitivity in its conductivity detection. Under our working conditions, this anion could be detected at a 8 x 10 - 8 mol/L concentration also in samples containing chloride (a major anionic constituent of urine) at 3.5 x 10 - 3 mol/L concentrations. Such a favorable analyte/matrix concentration ratio (in part, attributable to a transient isotachophoresis stacking in the initial phase of the separation) made possible accurate and reproducible (typically, 2-5% relative standard deviation (RSD) values of the peak areas of the analyte in dependence on its concentration in the sample) determination of oxalate in 500 nL volumes of 20-100-fold diluted urine samples. Short analysis times (about 280 s), no sample pretreatment (not considering urine dilution) and reproducible migration times of this analyte (0.5-1.0% RSD values) were characteristic for ZE on the chip. This work indicates general potentialities of the present chip design in rapid ZE analysis of samples containing the analyte(s) at high ionic matrix/analyte concentration ratios.

Protein precipitation using non-charged and charged polymers is a common method for protein purification, gaining broader interest among manufacturers in downstream processing. While during polymer- surface interactions, the formation of loops, tails and trains has been known for quite a long time, details of polymer conformation and chain length, interacting with the protein during protein precipitation are not fully discovered. Our research presents a more profound understanding of polymer-protein interaction, combining fluorescence and infrared spectroscopic measurements of proteins and polymer standards with well defined chain length to confirm different models of protein-polymer interaction. Lysozyme, chymotrypsinogen A, myoglobin and a monoclonal antibody, all of different molecular weight, isoelectric point and charge distribution at the protein surface, were used for protein-polymer precipitation. The use of polymers of various charge density and chain length showed that the required polymer length per precipitated protein (Ldef) is up to 25-times larger than the diameter of the corresponding protein, depending on the surface charge distribution of the protein, and its isoelectric point, as well as the charge density of the polymer. Our results support proposed mechanisms of polymer wrapping and loop formation for optimal charge neutralization during complexation and imply interaction of several polymer chains per precipitated protein molecule. Electrophoretic light scattering showed a qualitative correlation of the zeta potential of analyzed polymers with their corresponding Ldef values. Comparing protein precipitation behavior of long and short polymer chains, the latter exhibited reduced precipitation efficiency, visible as elevated Ldef.

The use of a poly(methylmethacrylate) chip, provided with two separation channels in the column-coupling (CC) arrangement and on-column conductivity detection sensors, to isotachophoresis (ITP) and ITP-ZE separation and determination of food additives was studied. A group of preservatives and taste intensifying components examined in this study included benzoate, sorbate, p-hydroxybenzoic acid esters (parabens), and glutamate, while various food products and cosmetics represented different matrices (proteins, fat, organic acids, carbohydrates, salts). ITP on the CC chip was found suitable for the determination of glutamate in the food products with only a minimum sample preparation (dilution, filtration). It also provided a rapid and simple procedure for the determination of parabens in cosmetics. On the other hand, ITP experiments with benzoate and sorbate revealed that sample preparations providing high analyte/matrix concentration ratios are essential when these food preservatives are to be determined by ITP on the chip. ITP-ZE combination on the same chip provided a solution to this problem by integrating an efficient ITP sample preparation (concentration of the preservatives and removal of the main part of the matrix), capable of processing μL sample volumes, with a final ZE separation and sensitive detection (low μmol/L limits of detection) of the preservatives. In both ITP and ITP-ZE separations on the CC chip no interference from food matrices was found.

Isotachophoretic (ITP) separation and determination of a group of 13 organic and inorganic acids, currently present in wines, on a poly(methyl methacrylate) chip provided with on-column conductivity detection was a subject of a detailed study performed in this work. Experiments with the ITP electrolyte systems proposed to the separation of anionic constituents present in wine revealed that their separation at a low pH (2.9) provides the best results in terms of the resolution. Using a 94 mm long separation channel of the chip, the acids could be resolved within 10-15 min also in instances when their concentrations corresponded to those at which they typically occur in wines. A procedure suitable to the ITP determination of organic acids responsible for some important organoleptic characteristics of wines (tartaric, lactic, malic and citric acids) was developed. Concentrations of 2-10 mg/l of these acids represented their limits of quantitation for a 0.9 microl volume sample loop on the chip. A maximum sample load on the chip, under the preferred separating conditions, was set by the resolution of malate and citrate. A complete resolution of these constituents in wine samples was reached when their molar concentration ratio was 20:1 or less. ITP analyses of a large series of model and wine samples on the chip showed that qualitative indices [RSH (relative step height) values] of the acids, based on the response of the conductivity detector, reproduced with RSD better than 2% while reproducibilities of the determination of the acids of our interest characterized RSD values better than 3.5%.

The use of a poly(methylmethacrylate) chip, provided with two separation channels in the column-coupling (CC) arrangement and on-column conductivity detection sensors, to electrophoretic separations of a group of inorganic anions (chloride, nitrate, sulfate, nitrite, fluoride and phosphate) that need to be monitored in various environmental matrices was studied. The electrophoretic methods employed in this study included isotachophoresis (ITP) and capillary zone electrophoresis (CZE) with on-line coupled ITP sample pretreatment (ITP-CZE). Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the CC chip were suppressed and electrophoresis was a dominant transport process in the separations performed by these methods. ITP separations on the chip provided rapid resolutions of sub-nmol amounts of the complete group of the studied anions and made possible rapid separations and reproducible quantitations of macroconstituents currently present in water samples (chloride, nitrate and sulfate). However, concentration limits of detection attainable under the employed ITP separating conditions (2-3 x 10(-5) mol/l) were not sufficient for the detection of typical anionic microconstituents in water samples (nitrite, fluoride and phosphate). On the other hand, these anions could be detected at 5-7 x 10(-7) mol/l concentrations by the conductivity detector in the CZE stage of the ITP-CZE combination on the CC chip. A sample clean-up performed in the ITP stage of the combination effectively complemented such a detection sensitivity and nitrite, fluoride and phosphate could be reproducibly quantified also in samples containing the macroconstituents at 10(4) higher concentrations. ITP-CZE analyses of tap, mineral and river water samples showed that the CC chip offers means for rapid and reproducible procedures to the determination of these anions in water (4-6 min analysis times under our working conditions). Here, the ITP sample pretreatment concentrated the analytes and removed nanomol amounts of the macroconstituents from the separation compartment of the chip within 3-4 min. Both the ITP and ITP-CZE procedures required no or only minimum manipulations with water samples before their analyses on the chip. For example, tap water samples were analyzed directly while a short degassing of mineral water (to prevent bubble formation during the separation) and filtration of river water samples (to remove particulates and colloids) were the only operations needed in this respect.

Some basic aspects of capillary electrophoresis (CE) separations on a poly(methyl methacrylate) chip provided with two separation channels in the column-coupling (CC) configuration and on-column conductivity detectors were studied. The CE methods employed in this study included isotachophoresis (ITP), capillary zone electrophoresis (CZE), and CZE with on-line ITP sample pretreatment (ITP-CZE). Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed, and electrophoresis was a dominant transport process in the separations performed by these methods. Very reproducible migration velocities of the separated constituents were typical under such transport conditions, and consequently, test analytes could be quantified by various ITP techniques with 1-2% RSD. The CC configuration of the separation channels provides means for an effective combination of an enhanced load capacity of the separation system with high detection sensitivities for the analytes in concentration-cascade ITP separations. In this way, for example, succinate, acetate, and benzoate could be separated also in instances when they were present in the loaded sample (1.2 microL) at 1 mmol/L concentrations while their limits of detection ranged from 8 to 12 micromol/L concentrations. A well-defined ITP concentration of the analyte(s) combined with an in-column sample cleanup (via an electrophoretically driven removal of the matrix constituents from the separation compartment) can be integrated into the separations performed on the CC chip. These sample pretreatment capabilities were investigated in ITP-CZE separations of model samples in which nitrite, phosphate, and fluoride (each at a 10 micromol/L concentration) accompanied matrix constituents (sulfate and chloride) at considerably higher concentrations. Here, both the concentration of the analytes and cleanup of the sample were included in the ITP separation in the first separation channel while the second separation channel served for the CZE separation of the ITP pretreated sample and the detection of the analytes.

This manuscript describes customization of copolymers to be used for polymer-driven protein purification in bioprocessing. To understand how copolymer customization can be used for fine-tuning, precipitation behavior was analyzed for five target antibodies (mAbs) and BSA as model impurity protein, at ionic strength similar to undiluted cell culture fluid. In contrast to the use of standardized homopolymers, customized copolymers, composed of 2-acrylamido-2-methylpropane sulfonic acid (AMPS) and 4-(acryloylamino)benzoic acid (ABZ), exhibited antibody precipitation yields exceeding 90%. Additionally, copolymer average molecular weight (Mw) was varied and its influence on precipitation yield and contaminant coprecipitation was investigated. Results revealed copolymer composition as the major driving force for precipitation selectivity, which was also dependent on protein hydrophobicity. By adjusting ABZ content and Mw of the precipitant for each of the mAbs, conditions were found that allowed for high precipitation yield and selectivity. These findings may open up new avenues for using polymers in antibody purification processes. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1484–1493, 2013

Technical-scale crystallization of therapeutic proteins may not only allow for a significant cost-reduction in downstream processing, but also enable new applications, e.g., the use of crystal suspensions for subcutaneous drug delivery. In this work, the crystallization of the antigen-binding fragment FabC225 was studied. First, vapor diffusion crystallization conditions from the literature were transferred to 10μL-scale microbatch experiments. A phase diagram was developed in order to identify the crystallization window. The conditions obtained from the microbatch experiments were subsequently transferred to parallelized 5mL-scale stirred-tank crystallizers. This scalable and reproducible agitated crystallization system allowed for an optimization of the crystallization process based on quantitative measurements. The optimized crystallization process resulted in an excellent yield of 99% in less than 2h by increasing the concentration of the crystallization agent ammonium sulfate during the process. The successful scalability of the Fab fragment crystallization process to 100mL-scale crystallizers based on geometric similarity was demonstrated. A favorable crystal size distribution was obtained. Furthermore, a wash step was introduced in order to remove unfavorable low-molecular substances from the crystals.

This work deals with the determination of total sulfite in wine. The determination combines an in-sample hydrogen peroxide oxidation of total sulfite in alkalized wine to sulfate with the separation and quantitation of the latter anion by zone electrophoresis (ZE) on-line coupled with isotachophoresis (ITP) on a column-coupling chip. Sample clean up, integrated into the ITP-ZE separation, eliminated wine matrix in an extent comparable to that provided by a highly selective distillation isolation of sulfite. At the same time, conductivity detection, employed to the detection of sulfate in the ZE stage of the ITP-ZE combination, provided for sulfate the concentration limit of detection corresponding to a 90 microg/l concentration of sulfite in the loaded sample (0.9 microl). Such a detectability allowed a reproducible quantitation of total sulfite when its concentration in wine was 15 mg/l. Formaldehyde binding of free sulfite in wine, included into the pre-column sample preparation, prevented an uncontrolled oxidation of this sulfite form. This step contributed to an unbiased determination of sulfate present in the original wine sample (this determination corrected for the concentration of sulfate determined in the sample after the peroxide oxidation of sulfite to the value equivalent to the total sulfite). The 99-101% recoveries of sulfite, determined for appropriately spiked wine samples, indicate a very good accuracy of the present method. Such a statement also supports excellent agreements of the results of quantitation based on the in-sample peroxide oxidation of the total sulfite (bound sulfite released at a high pH) with those in which this analyte was isolated from wine by distillation (bound sulfite released at a very low pH).

An appropriate combination of separation mechanisms (simultaneous use of differences in pK values, host-guest complexations, and the ionic strength dependences of the actual ionic mobilities) provided zone electrophoresis (ZE) resolution of 22 organic and inorganic acids expected in wines on a polymethylmethacrylate (PMMA) chip with integrated conductivity detection. These separating conditions offered a framework for the ZE determination of organic acids responsible for some important organoleptic characteristics of wines (tartrate, malate, succinate, acetate, citrate, and lactate). The ZE procedure developed in this context is simple and rapid (ca. 10 minutes' analysis time), while affording reproducible migration and quantitation data for the acids. For example, 0.8-2.0% RSD values characterized the migration times of the acids for 25 repeated ZE runs with the same sample carried out in 5 days in the background electrolyte solution prepared freshly on a daily basis, while 3-5% RSD values were typical for the accompanying peak area data. The concentration ranges within which the acids of analytical interest could be determined in one ZE run covered all wine samples included in our study (100-400-fold sample dilutions were needed to work under the conditions corresponding to the validities of the calibration data). 90-110% recoveries of the acids as obtained repeatedly for one of the reference wine samples used in our experiments indicate a good predisposition of the present method to provide accurate analytical results. This statement also supports the results from the determination of the acids in reference wine samples with claimed concentrations of malic (five samples), tartaric (one sample), and lactic (one sample) acids.

This feasibility study deals with the separations of proteins by an on-line combination of zone electrophoresis (ZE) with isotachophoresis (ITP) on a poly(methylmethacrylate) column-coupling (CC) chip with integrated conductivity detection. ITP and ZE provided specific analytical functions while performing the cationic mode of the separation. ITP served, mainly, for concentrations of proteins and its concentrating power was beneficial in reaching a low dispersion transfer (injection) of the proteinous constituents, loaded on the CC chip in a 960 nL volume, into the ZE separation stage. This was complemented by an electrophoretically driven removal of the sample constituents migrating in front of the focused proteins from the separation system before the ZE separation. On the other hand, ZE served as a final separation (destacking) method and it was used under the separating conditions providing the resolutions and sensitive conductivity detections of the test proteins. In this way, ITP and ZE cooperatively contributed to low- or sub-microg/mL concentration detectabilities of proteins and their quantitations at 1-5 microg/mL concentrations. However, a full benefit in concentration detectabilities of proteins, expected from the use of the ITP-ZE combination, was not reached in this work. Small adsorption losses of proteins and detection disturbances in the ZE stage of separation, very likely due to trace constituents concentrated by ITP, appear to set limits in the detection of proteins in our experiments. The ITP-ZE separations were carried out in a hydrodynamically closed separation compartment of the chip with suppressed hydrodynamic and electroosmotic flows of the electrolyte solutions. Such transport conditions, minimizing fluctuations of the migration velocities of the separated constituents, undoubtedly contributed to highly reproducible migrations of the separated proteins (fluctuations of the migration time of a particular protein were typically 0.5% RSD in repeated ITP-ZE runs).

This feasibility study deals with column switching in zone electrophoresis (ZE) separations on a column coupling (CC) chip. The column switching implemented into the ZE separations an on-chip sample clean up applicable for both the multicomponent and high salinity samples. In addition, complemented by different separation mechanisms in the coupled columns (channels), it provided benefits of two-dimensional separations. Properly timed column switching gave column-to-column transfers of the analytes, characterized by 99-102% recoveries, delivered to the second separation stage on the chip the analyte containing fractions contaminated only with minimum amounts of the matrix constituents. A diffusion driven transport of the matrix constituents to the second channel of the chip (due to direct contacts of the electrolyte solutions in the bifurcation region), representing 0.1-0.2% of the loaded sample constituents, was found to accompany the sample clean up performed on the CC chip. This source of potential disturbances to the separation in the second channel, however, is not detectable in a majority of practical situations. With respect to a 900 nl volume of the sample channel on the CC chip, the electric field and isotachophoresis (ITP) stackings were employed to minimize the injection dispersion in the separations and concentrate the analytes. Here, the column switching, removing a major part of the stacker from the separation system, provided a tool effective in a control of the destacking of analytes. Highly reproducible ZE separations as attained in this work also for the chip-to-chip and equipment-to-equipment frames can be ascribed, at least in part, to suppressions of electroosmotic and hydrodynamic flows of the solutions in which the separations were performed.

This work deals with the determination of free sulfite in wine by zone electrophoresis (ZE) with on-line isotachophoresis (ITP) sample pretreatment on a column-coupling (CC) chip with conductivity detection. A rapid pre-column conversion of sulfite to hydroxymethanesulfonate (HMS), to minimize oxidation losses of the analyte, was included into the developed analytical procedure, while ITP and ZE were responsible for specific analytical tasks in the separations performed on the CC chip. ITP, for example, eliminated the sample matrix from the separation compartment and, at the same time, provided a selective concentration of HMS before its transfer to the ZE stage of the separation. On the other hand, ZE served as a final separation (destacking) method and it was used under the separating conditions favoring a sensitive conductivity detection of HMS. In this way, ITP and ZE cooperatively contributed to a 900 μg/l concentration detectability for sulfite as attained for a 60 nl load of wine (a 15-fold wine dilution and the use of a 0.9 μl sample injection channel of the chip) and, consequently, to the determination of free sulfite when this was present in wine at the concentrations as low as 3 mg/l. The separations were carried out in a closed separation compartment of the chip with suppressed hydrodynamic and electroosmotic flows. Such transport conditions, minimizing fluctuations of the migration velocities of the separated constituents, made a frame for precise migration and quantitation data as achieved for HMS in both the model and wine samples. Ninety percent recoveries, as typically obtained for free sulfite in wine samples, indicate promising potentialities of the present method as far as the accuracies of the provided analytical results are concerned.

A microstructured plastic chip contains two connected and independently filled separation channels. The first of them serves for isotachophoresis (ITP) sample pretreatment. The pre-cleaned analytes are transferred to the second channel for a final separation, typically, by zone electrophoresis (ZE). The chip is designed to be run within an automated environment. A broad range of applications was developed in which highly reproducible results were obtained for inorganic, organic and biological trace components.

The use of a poly(methylmethacrylate) chip, provided with a pair of on-line coupled separation channels and on-column conductivity detectors, to isotachophoresis (ITP) separations of optical isomers was investigated. Single-column ITP, ITP in the tandem-coupled columns, and concentration-cascade ITP in the tandem-coupled columns were employed in this investigation using tryptophan enantiomers as model analytes. Although providing a high production rate (about 2 pmol of a pure tryptophan enantiomer separated per second), single-column ITP was found suitable only to the analysis of samples containing the enantiomers at close concentrations. A 94-mm separation path in ITP with the tandem-coupled separation channels made possible a complete resolution of a 1.5 nmol amount of the racemic mixture of the enantiomers. However, this led only to a moderate extension of the concentration range within which the enantiomers could be simultaneously quantified. The best results in this respect were achieved by using a concentration-cascade of the leading anions in the tandem-coupled separation channels. Here, a high production rate, favored in the first separation channel, was followed by the ITP migration of the enantiomers in the second channel under the electrolyte conditions enhancing their detectabilities. In dependence on the migration configuration of the enantiomers, this technique made possible their simultaneous determinations when their ratios in the loaded sample were 35:1 or less (D-tryptophan a major constituent) and 70:1 or less (L-tryptophan a major constituent).

A poly(methylmethacrylate) chip, provided with two separation channels in the column-coupling (CC) arrangement and on-column conductivity detection sensors and intended, mainly, to isotachophoresis (ITP) and ITP-capillary zone electrophoresis (CZE) separations was developed recently. The present work was aimed at assessing its performance relevant to the detection and quantitation of the ITP analytes. Hydrodynamic (HDF) and electroosmotic (EOF) flows of the solution in the separation compartment of the CC chip were suppressed and electrophoresis was a dominant transport process in the ITP separations with model analytes carried out in this context. When the surfaces of the detection electrodes of the conductivity sensors on the chip were appropriately cleaned qualitative indices of the test analytes [relative step heights (RSHs)], provided by a particular detection sensor, agreed within 1% (expressed via RSDs of the RSH values). Their long-term reproducibilities for one sensor, as estimated from 70 ITP runs repeated in 5 days, were 2% or less. Sensor-to-sensor and chip-to-chip fluctuations of the RSH values for the test analytes were 2.5% or less. In addition, experimentally obtained RSH values agreed well with those predicted by the calculations based on the ITP steady-state model. Reproducibilities of the migration velocities attainable on the CC chips with suppressed EOF and HDF, assessed from the migration time measurements of the ITP boundary between well-defined positions on the separation channels of the chips (140 repeated runs on three chips), ranged from 1.4 to 3.3% for the migration times in the range of 100-200 s. Within-day repeatabilities of the time-based zone lengths for the test analytes characterized 2% RSDs, while their day-to-day repeatabilities were less than 5%. Chip-to-chip reproducibilities of the zone lengths, assessed from the data obtained on three chips for 100 ITP runs, were 5-8%.

A convenient peak identification method in stepwise elution was investigated and correlation among the retention times of peaks in ion-exchange chromatography of glycated haemoglobin was assessed. By using a correlation method, accuracy of peak identification among columns with degradation and product deviations can be maintained. The correlative retention time identification procedure is treated theoretically.

A new adjustment calculus is presented to determine the true intraparticle distribution of bound protein within chromatographic beads from confocal fluorescence slice series. The calculus does not require knowledge about optical properties of different chromatographic materials like refractive index and turbidity, but it depends on a parameter which can be adjusted interactively. The algorithm is of complexity O(n) where n is the pixel number. From the reconstructed data we compute the parameters of the protein uptaking process using a model-based approach. It is demonstrated that the protein uptaking rates of the beads strongly dependent on the conditions of the fluid phase influencing the strength of protein surface interaction.

Hepatic-type fatty-acid-binding protein (hFABP) from the cytosol of bovine liver is a 14.4-kDa neutral protein with a blocked N-terminus and a disulfide system located on the surface of the protein. It binds two molecules of fatty acid in one binding site, apparent dissociation constants of the oleic acid/hFABP complex are 0.24 microM and 2.15 microM. Computer analysis of circular dichroic spectra predicts that hFABP contains about 12% alpha-helix, 45% beta-structure, 15% beta-turn and 27% unordered structure. Ellipticities indicative of secondary structure are not affected by fatty acid binding. Cationic amino acid residues of hFABP (1 His, 15 Lys, 2 Arg) were screened for ionic fatty acid/protein interactions. His was excluded, as 1H-NMR analysis of His-C2 and His-C4 protons indicated that binding of oleic acid shifts the pK of His from 6.9 to 7.1 only in hFABP with the disulfide system in the oxidized state; acylation of His with diethylpyrocarbonate does not affect the binding of the fatty acid. Acetylation of Lys reduces binding marginally, whereas modification of Arg with phenylglyoxal lowers the binding activity by 65%. From 1H-NMR investigations, conformational changes within the protein, due to a sort of disaggregation of hFABP upon fatty acid binding, were derived. Most of the proton resonances sharpen up with ligand binding, and some of the methyl resonances shift positions, possibly because they are directly involved in the fatty acid/protein interaction.