Bernd Stanislawski

Isotachophoresis and isotachophoresis : zone electrophoresis separations of inorganic anions present in water samples on a planar chip with column-coupling separation channels and conductivity detection

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.

Isotachophoresis and isotachophoresis‐zone electrophoresis of food additives on a chip with column‐coupling separation channels

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.

Determination of organic acids and inorganic anions in wine by isotachophoresis on a planar chip.

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%.

Determination of bromate in drinking water by zone electrophoresis-isotachophoresis on a column-coupling chip with conductivity detection.

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 νL sample injection channel of the chip. In addition, bromate was concentrated by a factor of 103 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.

Isotachophoresis separations of enantiomers on a planar chip with coupled separation channels

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).

Conductivity detection and quantitation of isotachophoretic analytes on a planar chip with on-line coupled separation channels

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%.

Determination of oxalate in urine by zone electrophoresis on a chip with conductivity detection.

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 8×10–8 mol/L concentration also in samples containing chloride (a major anionic constituent of urine) at 3.5×10–3 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.

Zone electrophoresis of proteins on a poly(methyl methacrylate) chip with conductivity detection

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.

Host cell protein quantification by fourier transform mid infrared spectroscopy (FT-MIR)†

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.

Feasibility study of semi‐selective protein precipitation with salt‐tolerant copolymers for industrial purification of therapeutic antibodies

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.