An elegant technique for ex vivo imaging in experimental research-Optical coherence tomography (OCT).

Optical coherence tomography (OCT) is an elegant technology for imaging of tissues and organs and has been established for clinical use for around a decade. Thus, it is used in vivo but can also serve as a valuable ex vivo imaging tool in experimental research. Here, a brief overview is given with a focus on an ex vivo application of OCT. Image and video examples of freshly obtained murine lungs are included. The main advantage of OCT for ex vivo analysis is the non-contact, non-invasive, and non-destructive fast acquisition of a three-dimensional data set with micrometer-resolution.

The use of ion-selective electrodes for evaluating residence time distributions in expanded bed adsorption systems.

The suitability of ion-selective electrodes (ISE) for the determination of residence time distribution (RTD) in turbid, cell-containing fluids was examined. The electrodes were found to give reproducible signals in biomass-containing feedstock with up to 20% wet weight of solids. The enhanced feedstock compatibility of IES, when compared to other tracer sensing devices, allows the study of expanded bed system hydrodynamics under relevant operating conditions. Within the linear range of the corresponding ISE-tracer pair, both examined ISE (Li(+)- or Br(-)-selective) showed to be insensitive against the range of flow rate and pH normally employed during expanded bed adsorption (EBA) of proteins. Analyzing the RTD obtained after a perfect ion tracer pulse in terms of the PDE model (PDE, axially dispersed plug-flow exchanging mass with stagnant zones) gave a quantitative description of the underlying hydrodynamic situation during EBA processing. These data provided a powerful tool to make predictions on the adsorptive global process performance with a defined feedstock type and composition. The link between the hydrodynamic events during feedstock application and the actual process performance was shown when applying intact yeast cell suspensions at different biomass content (up to 7.5% wet weight) and buffer conductivity (5-12 mS) onto an EBA column filled with the adsorbent Streamline Q XL as fluidized phase. On the basis of our experimental results, a guideline for the successful application of the ISE/RTD method to EBA process design is presented.

Isolation of a recombinant formate dehydrogenase by pseudo-affinity expanded bed adsorption.

Formate dehydrogenase (FDH) is an enzyme of industrial interest, which is recombinantly expressed as an intracellular protein in Escherichia coli. In order to establish an efficient and reliable purification protocol, an expanded bed adsorption (EBA) process was developed, starting from the crude bacterial homogenate. EBA process design was performed with the goal of finding operating conditions which, on one hand, allow efficient adsorption of the target protein and which, on the other hand, support the formation of a perfectly classified fluidised bed (expanded bed) in the crude feed solution. A pseudo-affinity ligand (Procion Red HE3B) was used to bind the FDH with high selectivity and reasonable capacity (maximum equilibrium capacity of 30 U/ml). Additionally, a simplified modelling approach, involving small packed beds for generation of process parameters, was employed for defining the operating conditions during sample application. In combination with extended elution studies, a process was set up, which could be scaled up to 7.5 l of adsorbent volume yielding a total amount of 100,000 U of 94% pure FDH per run. On this scale, 19 l of a benzonase-treated E. coli homogenate of 15% wet-weight (pH 7.5, 9 mS/cm conductivity) were loaded to the pseudo-affinity adsorbent (0.25 m sed. bed height, 5 x 10(-4) m/s fluid velocity). After a series of two wash steps, a particle-free eluate pool was obtained with 85% yield of FDH. This excellently demonstrates the suitability of expanded bed adsorption for efficient isolation of proteins by combining solid-liquid separation with adsorptive purification in a single unit operation.

Human chymotrypsinogen B production from Pichia pastoris by integrated development of fermentation and downstream processing. Part 2. Protein recovery.

The purification of human chymotrypsinogen B (hCTRB) after expression and secretion by the yeast Pichia pastoris is described based on two different approaches using integrated initial recovery. Extraction employing aqueous two-phase systems (ATPS) from poly(ethylene glycol) and sodium sulfate allows direct processing of cell containing yeast suspensions of 50% wet weight. The target protein is obtained partially purified in the top phase while cells and cell debris are partitioned to the bottom phase of the system. hCTRB is further purified by adsorption from the top phase to the cation exchanger SP Sepharose Big Beads and elution in a salt step. The single step isolation of hCTRB is possible by expanded bed adsorption (EBA) using a fluidized cation exchanger (Streamline SP XL). A design strategy is shown taking both target protein binding and stable fluidization of the stationary phase in cell containing suspensions into consideration. For the example of hCTRB isolation from cell containing P. pastoris suspensions, a successful use of this strategy is demonstrated. Both initial recovery strategies deliver a product that can be further purified and formulated by ultrafiltration/diafiltration followed by lyophilization, resulting in a homogeneous product. Scale-up to 30-90 L of culture suspension was shown for both methods, resulting in a product of similar quality. Comparing both strategies reveals that the two-step ATPS route is better suited for high cell density cultures, while the single step EBA method is preferred for cultures of moderate cell density. This is due to the fact that application of EBA is restricted to suspensions of 10-12.5% wet weight cell concentration, thus necessitating dilution of the original broth prior to sample application. The data presented show that integrated recovery operations are a valuable alternative to traditional processing for systems that are problematic during initial solid-liquid separation.

Human chymotrypsinogen B production with Pichia pastoris by integrated development of fermentation and downstream processing. Part 1. Fermentation.

Based on an integrated approach of genetic engineering, fermentation process development, and downstream processing, a fermentative chymotrypsinogen B production process using recombinant Pichia pastoris is presented. Making use of the P. pastoris AOX1-promotor, the demand for methanol as the single carbon source as well as an inducer of protein secretion enforced the use of an optimized feeding strategy by help of on-line analysis and an advanced controller algorithm. By using an experimental system of six parallel sparged column bioreactors, proteolytic product degradation could be minimized while also optimizing starting conditions for the following downstream processing. This optimization of process conditions resulted in the production of authentic chymotrypsinogen at a final concentration level of 480 mg.L(-)(1) in the whole broth and a biomass concentration of 150 g.L(-)(1) cell dry weight, thus comprising a space-time yield of 5.2 mg.L(-)(1).h(-)(1). Alternatively to the high cell density fermentation approach, a continuous fermentation process was developed to study the effects of reduced cell density toward oxygen demand, cooling energy, and biomass separation. This development led to a process with a highly increased space-time yield of 25 mg.L(-)(1).h(-)(1) while reducing the cell dry weight concentration from 150 g.L(-)(1) in fed-batch to 65 g.L(-)(1) in continuous cultivation.

Physical properties of detergent-based aqueous two-phase systems.

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.

Minimising biomass/adsorbent interactions in expanded bed adsorption processes: a methodological design approach.

Expanded bed adsorption (EBA) is an integrated technology for the primary recovery of proteins from crude feedstock. Interactions between solid matter in the feed suspension and fluidised adsorbent particles influence bed stability and therefore have a significant impact on protein adsorption in expanded beds. In order to design efficient and reliable EBA processes a strategy is needed, which allows to find operating conditions, where these adverse events do not take place. In this paper a methodological approach is presented, which allows systematic characterisation and minimisation of cell/adsorbent interactions with as little experimental effort as possible. Adsorption of BSA to the anion exchanger Streamline Q XL from a suspension containing S. cerevisiae cells was chosen as a model system with a strong affinity of the biomass towards the stationary phase. Finite bath biomass adsorption experiments were developed as an initial screening method to estimate a potential interference. The adhesiveness of S. cerevisiae to the anion exchanger could be reduced significantly by increasing the conductivity of the feedstock. A biomass pulse response method was used to find optimal operation conditions showing no cell/adsorbent interactions. A good correlation was found between the finite bath test and the pulse experiment for a variety of suspensions (intact yeast cells, E. coli homogenate and hybridoma cells) and adsorbents (Streamline Q XL, DEAE and SP), which allows to predict cell/adsorbent interactions in expanded beds just from finite bath adsorption tests. Under the optimised operating conditions obtained using the prior methods, the stability of the expanded bed was investigated during fluidisation in biomass containing feedstock (up to 15% yeast on wet weight basis) employing residence time distribution analysis and evaluation by an advanced model. Based on these studies threshold values were defined for the individual experiments, which have to be achieved in order to obtain an efficient EBA process. Breakthrough experiments were conducted to characterise the efficiency of BSA adsorption from S. cerevisiae suspensions in EBA mode under varying operating conditions. This allowed to correlate the stability of the expanded bed with its sorption efficiency and therefore could be used to verify the threshold values defined. The approach presented in this work provides a fast and simple way to minimise cell/adsorbent interactions and to define a window of operation for protein purification using EBA.

Fed-batch production of recombinant human calcitonin precursor fusion protein using Staphylococcus carnosus as an expression-secretion system.

A pH-auxostatic fed-batch process was developed for the secretory production of a fusion protein consisting of the pro-part of Staphylococcus hyicus lipase and two synthetic human calcitonin (hCT) precursor repeats under the control of a xylose-inducible promotor from Staphylococcus xylosus. Using glycerol as the energy source and pH-controlled addition of yeast extract resulted in the production of 2000 mg 1(-1) of the fusion protein (420 mg 1(-1) of the recombinant hCT precursor) within 14 h, reaching 45 g 1(-1) cell dry mass with Staphylococcus carnosus in a stirred-tank reactor. Product titer and space-time yield (30 mg calcitonin precursor 1(-1) h(-1)) were thus improved by a factor of 2, and 4.5, respectively, compared to Escherichia coli expression-secretion systems for the production of calcitonin precursors. Two hundred grams of the fusion protein was secreted by the recombinant S. carnosus on a 150-1 scale (scale-up factor of 50) with a minimum use of technical-grade yeast extract (40 mg fusion protein g(-1) yeast extract).

Investigation of mathematical methods for efficient optimisation of aqueous two-phase extraction.

Mathematical strategies were applied to optimise the extraction of recombinant leucine dehydrogenase from E. coli homogenates and endoglucanase 1 from culture filtrates of Trichoderma reesei in polyethylene glycol-phosphate systems. The goal was to test mathematical tools which could facilitate the optimisation procedure in aqueous two-phase systems. A modified simplex approach, the method of steepest ascent and a genetic algorithm were successfully applied and compared. The methods differ in the height of the optimum found, the number of experiments and the time required. The genetic algorithm proved to be an optimisation procedure which can be used well in aqueous two-phase systems. The simplex procedure has to be further improved.

Aqueous two-phase systems containing urea: influence of protein structure on protein partitioning.

During recombinant E. coli fermentation with high-expression levels inclusion bodies are often formed. Aqueous two-phase systems have been successfully used in the presence of urea for the initial recovery step of inclusion bodies from E. coli. Basic studies of the complex interactions are lacking. For a systematic study of protein partitioning in the presence of urea we selected T4-lysozyme mutants with different thermal stability as a model. The stabilization of these variants by phase components was investigated measuring the fluorescence emission of tryptophan residues in the protein. Protein structure was stabilized at pH 7 in the order of S0(4)(2-) >> PEG = Dextran > H(2)O. The conformation of proteins was shown to have a strong influence on the partitioning in aqueous two-phase systems. Tryptophan and its homologuous di- and tripeptdides were partitioned in similar phase systems to normalize for contribution from hydrophobic interactions.

The influence of cell adsorbent interactions on protein adsorption in expanded beds.

Expanded bed adsorption (EBA) is a primary recovery operation allowing the adsorption of proteins directly from unclarified feedstock, e.g. culture suspensions, homogenates or crude extracts. Thus solid-liquid separation is combined with adsorptive purification in a single step. The concept of integration requires that the solid components of the feed solution are regarded as a part of the process, which influences stability, reproducibility, and overall performance. This aspect is investigated here at the example of the influence of presence and concentration of intact yeast cells (S. cerevisiae) on the adsorption of model proteins (hen egg white lysozyme and bovine serum albumin) to various stationary phases (cation and anion-exchange, hydrophobic interaction, immobilised metal affinity). The interaction of the cells with the adsorbents is determined qualitatively and quantitatively by a pulse response method as well as by a finite bath technique under different operating conditions. The consequence of these interactions for the stability of expanded beds in suspensions of varying cell concentration is measured by residence time distributions (RTDs) after tracer pulse injection (NaBr, LiCl). Analysis of the measured RTD by the PDE model allows the calculation of the fraction of perfectly fluidised bed (phi), a parameter which may be regarded as a critical quantity for the estimation of the quality of fluidisation of adsorbents in cell containing suspensions. The correlation between bed stability and performance is made by analysing the breakthrough of model proteins during adsorption from unclarified yeast culture broth. A clear relationship is found between the degree of cell/adsorbent interaction, bed stability in terms of the phi parameter, and the sorption efficiency. Only beds characterised by a phi value larger than 0.8 in the presence of cells will show a conserved performance compared to adsorption from cell free solutions. A drop in phi, which is due to interactions of the fluidised adsorbent particles with cells from the feed, will directly result in a reduced breakthrough efficiency. The data presented highlight the importance of including the potential interaction of solid feedstock components and the expanded adsorbents into the design of EBA processes, as the interrelation found here is a key factor for the overall performance of EBA as a truly integrated operation.

Visualizing two-component protein diffusion in porous adsorbents by confocal scanning laser microscopy.

The use of confocal scanning laser microscopy (CSLM) has recently been described for the visualization of intraparticle protein profiles during single-protein finite bath uptake experiments. By coupling of fluorescent molecules to proteins the penetration of porous media by labeled macromolecules could be detected by scanning single adsorbent particles for fluorescence emission after laser excitation. Thus the internal protein distribution profile, which is a central element in modeling of protein transport in porous adsorbents, became experimentally accessible. Results from the simultaneous visualization of two proteins by this technology are shown here. The use of two different fluorescent dyes for protein labeling and two independent detectors in the CSLM allowed for the first time ever the direct observation of a two-component diffusion process within a porous stationary phase. The finite bath uptake of human immunoglobulin G (hIgG) and bovine serum albumin (BSA) to two different ion exchange adsorbents (SP Sepharose Fast Flow and Source 30S) and to an affinity adsorbent (Protein A Sepharose) was measured using Cy5 and Oregon Green as labels. Single adsorbent particles were scanned for intensity distribution of fluorescence emission from the two fluorophors. The intraparticle profiles obtained from the confocal images were translated into a relative protein concentration thus allowing the calculation of protein uptake kinetics from direct measurement in the stationary phase. The confocal technique may prove to be a very powerful means of data generation for modeling of multi-component mass transfer phenomena in protein adsorption.

The influence of complex biological feedstock on the fluidization and bed stability in expanded bed adsorption

The stability of expanded bed adsorption systems (EBA) was studied in biomass containing culture broth by residence time distribution (RTD) experiments, using pulse inputs of fluorescent molecules as tracers. Different commercial adsorbents (Streamline DEAE, SP, Phenyl, Chelating, and AC) were tested at various biomass concentrations (2.5-12 %, wet weight) of whole (Saccharomyces cerevisiae) yeast, yeast cell homogenate, and Escherichia coli homogenate. Analyzing the RTD according to the PDE model (PDE: axially dispersed plug-flow exchanging mass with stagnant zones) allowed the calculation of three parameters: the number of transfer units for mass exchange between mobile and stagnant fraction (N), the Peclet number for overall axial dispersion (P), and the mobile fraction of the liquid in axially dispersed plug flow (varphi). When fluidization was performed in particle-free buffer the normalized response signal (after perfect input pulse) was symmetric (N:0; P: 50-100; varphi: 1), thus, demonstrating the formation of a homogeneous fluidized (expanded) bed. Upon application of suspended biomass the RTD was skewed, depending on the adsorbent used and the type and level of biomass present in the sample. This situation leads to three different characteristic pictures: the well-fluidized system (N: >/= 7-10; P: >/= 40; varphi: 0.80-0.90), the system exhibiting bottom channeling (N: < 1-2; P: >/= 40; varphi: 0.5-0.7) and, the system where extensive agglomeration develops (N: 4-7; P: 20-40; varphi: < 0.5). These results demonstrate that changes in the hydrodynamics of EBA already take place in the presence of moderate concentrations of biomass. Furthermore, those changes can be quantitatively described mainly in terms of the fraction of stagnant zones in the system, which are formed due to the interaction of biomass and adsorbent. The technique described here can be used to evaluate a certain combination of adsorbent and biomass with regard to its suitability for expanded bed adsorption from whole broth. Copyright 1999 John Wiley & Sons, Inc. Biotechol Bioeng 64: 484-496, 1999.

Aqueous two-phase systems containing urea: influence on phase separation and stabilization of protein conformation by phase components.

During recombinant Escherichia coli fermentation with high expression levels, inclusion bodies are often formed. Aqueous two-phase systems have been used in the presence of urea for the initial recovery steps. To investigate phase behavior of such systems we determined phase diagrams of poly(ethylene glycol) (PEG)/sodium sulfate/urea/water and PEG/dextran T-500 (DEX)/urea/phosphate buffer/water at different concentrations of urea and different molecular weight of PEG. PEG/Na2SO4 aqueous two-phase systems could be obtained including up to 30% w/w urea at 25 degrees C and PEG/dextran T-500 up to 35% w/w urea. The binodial was displaced toward higher concentrations with increasing urea concentrations. The partition coefficient of urea was near unity. An unstable mutant of T4-lysozyme with an amino acid replacement in the core (V149T) was used to analyze the effect of phase components on the conformation of the enzyme. We showed that partitioning of tryptophan was not dependent on the concentration of urea in the phase system.

Investigations on protein adsorption to agarose-dextran composite media.

A new stationary phase for protein purification was investigated with regard to its performance during capture of selected model proteins. The commercially available matrix consists of a porous agarose backbone, to which dextran is covalently attached. The dextran carries ion-exchange ligands, thus providing a binding space of high ligand density. Breakthrough of various proteins during frontal application to packed beds was measured and the experiments were analyzed in terms of equilibrium and breakthrough capacity. A significant increase of static capacity, as compared with conventional porous matrices, was found. Good dynamic properties allowed utilization of a high percentage of the equilibrium capacity at 10% breakthrough. For all proteins, a decreasing ratio of breakthrough to equilibrium capacity was detected with increasing feed concentration. This observation suggested a significant contribution of solid diffusion to the transport of proteins into the adsorbent particles. The specific architecture of the stationary phase, where the agarose base structure is derivatized with ion-exchange ligand-bearing dextran, may lead to this behavior.

Cell/adsorbent interactions in expanded bed adsorption of proteins.

Expanded bed adsorption (EBA) is an integrated technology for the primary recovery of proteins from unclarified feedstock. A method is presented which allows a qualitative and quantitative understanding of the main mechanisms governing the interaction of biomass with fluidized resins. A pulse response technique was used to determine the adsorption of various cell types (yeast, Gram positive and Gram negative bacteria, mammalian cells and yeast homogenate) to a range of commercially available matrices for EBA. Cells and cell debris were found to interact with the ligands of agarose based resins mainly by electrostatic forces. From the adsorbents investigated the anion exchange matrix showed the most severe interactions, while cation exchange and affinity adsorbents appeared to be less affected. Within the range of biologic systems under study E. coli cells had the lowest tendency of binding to all matrices while hybridoma cells attached to all the adsorbents except the protein A affinity matrix. The method presented may be employed for screening of suitable biomass/adsorbent combinations, which yield a robust and reliable initial capture step by expanded bed adsorption from unclarified feedstock.

Visualising intraparticle protein transport in porous adsorbents by confocal microscopy.

Confocal scanning microscopy was used to study protein uptake to porous adsorbents during batch experiments in a finite bath. By coupling of a fluorescent dye to the protein molecules the penetration of single adsorbent particles at different times during batch uptake could be observed visually. Intensity profiles of the protein distribution within a single particle were obtained by horizontal scanning. After integration of the profiles the overall fluorescence within a bead could be calculated. Relating the overall fluorescence at different incubation times to the value at equilibrium allowed the construction of the fractional approach to equilibrium versus time. These data were compared to uptake curves, which had been obtained by measurements of the protein concentration in the supernatant and an excellent agreement of the curves was detected. The procedure was performed for two different proteins (lysozyme and human IgG) on two different media for protein adsorption (SP Sepharose Fast Flow and SP Sepharose XL; Pharmacia Biotech) and in all cases it could be shown, that the results from the direct measurements by confocal microscopy correspond very well to the data obtained from the indirect measurements in the fluid phase.

Pegylated-liposomal doxorubicin versus doxorubicin, bleomycin, and vincristine in the treatment of AIDS-related Kaposi's sarcoma: results of a randomized phase III clinical trial.

PURPOSE: Kaposi's sarcoma (KS), the most common neoplasm in patients with AIDS, is a significant clinical problem for which current therapies are frequently unsatisfactory. We conducted a randomized phase III clinical trial to compare the efficacy and toxicities of a new form of therapy, pegylated-liposomal doxorubicin, with standard combination chemotherapy in patients with advanced AIDS-related KS (AIDS-KS). PATIENTS AND METHODS: Two hundred fifty-eight patients with advanced AIDS-KS were randomly assigned to receive either pegylated-liposomal doxorubicin (20 mg/m2) or the combination of doxorubicin (20 mg/m2), bleomycin (10 mg/m2) and vincristine (1 mg) (ABV) every 14 days for six cycles. Standard response criteria, toxicity criteria, and predefined indicators of clinical benefit were examined to evaluate outcomes. RESULTS: Among 133 patients randomized to receive pegylated-liposomal doxorubicin, one achieved a complete clinical response and 60 achieved a partial response for an overall response rate of 45.9% (95% confidence interval [CI], 37% to 54%). Among 125 patients randomized to receive ABV, 31 achieved a partial response (24.8%; 95% confidence interval [CI], 17% to 32%). This difference was statistically significant (P < .001). In addition to objective responses, prospectively defined clinical benefits and toxicity outcomes also favored pegylated-liposomal doxorubicin. CONCLUSION: Pegylated-liposomal doxorubicin is more effective and less toxic than the standard combination chemotherapy regimen ABV for treatment of AIDS-KS.

Analysis of hybridoma cell culture processes by SDS/gel capillary electrophoresis and matrix-assisted laser desorption ionization-time-of-flight MS.

SDS/gel capillary electrophoresis (SDS/gel CE) and matrix-assisted laser desorption ionization-time-of-flight MS (MALDI-TOF-MS) were employed to analyse changes in the culture broth during batch and continuous cultivation of hybridoma cells. The stability of IgG was analysed by SDS/gel CE and capillary zone electrophoresis (CZE). The results obtained by the new analytical procedures reflect the changes in the cultivation conditions very well, indicating that these tools can be used to follow animal cell culture processes. A new technique to collect fractions of very small volumes during the CZE separation is described, and the successful off-line coupling of CZE and MALDI-TOF-MS for the analysis of biotechnological processes is demonstrated.

The influence of particle size distribution and operating conditions on the adsorption performance in fluidized beds.

The influence of matrix properties and operating conditions on the performance in fluidized-bed adsorption has been studied using Streamline diethyl-aminoethyl (DEAE), an ion exchange matrix based on quartz-weighted agarose, and bovine serum albumin (BSA) as a model protein. Three different particle size fractions (120-160 microm, 120-300 microm, and 250-300 microm) were investigated. Dispersion in the liquid phase was reduced when particles with a wide size distribution were fluidized compared to narrow particle size distributions. When the mean particle diameter was reduced, the breakthrough capacities during frontal adsorption were enlarged due to a shorter diffusion path length within the matrix. At small particle diameters the effect of film mass transfer became more relevant to the adsorption performance in comparison to larger particles. Therefore matrices designed for fluidized-bed adsorption should have small particle diameter and increased mean particle density to ensure small diffusion path length in the particle and a high interstitial velocity to improve film mass transfer. Studies on the influence of sedimented matrix height on axial mixing showed an increased Bodenstein number with increasing bed length. Higher breakthrough capacities were also found for longer adsorbent beds due to reduced dispersion and improved fluid and particle side mass transfer. With increasing bed height the influence of flow rate on breakthrough capacity was reduced. For a settled bed height of 50 cm breakthrough capacities of 80% of the equilibrium capacity for flow rates varying from 3 to 9 cm/min could be achieved.