Publications by authors named "Dorota Antos"

36 Publications

Influence of the geometry of extra column volumes on band broadening in a chromatographic system. Predictions by computational fluid dynamics.

J Chromatogr A 2021 Sep 14;1653:462410. Epub 2021 Jul 14.

Department of Chemical and Process Engineering, Rzeszów University of Technology, Rzeszów, Poland. Electronic address:

A computational fluid dynamics method was used for prediction of flow behavior and band profiles of small- and macro-molecule compounds eluting in extra-column volumes (ECV) of an Äkta chromatographic system. The model compounds were: acetone, bovine serum albumin and an antibody. The construction of ECV was approximated by different types of geometries, starting from the simplest two-dimensional (2D) arrangement consisting of a straight capillary tube, and ending with a three-dimensional system (3D), which accounted for the flow path curvature of individual elements of ECV, including: injection loop capillary, multi-way valve, connecting capillary and detector cell. The accuracy of the model predictions depended on the flow path length and the eluent flowrate. 2D-geometry models reproduced pretty well the shapes of band profiles recorded at the lowest eluent flowrate used, but they failed for increased flowrates. The 3D-geometry model was found to be sufficiently accurate for all conditions investigated. It was exploited to analyze band broadening in the individual ECV elements. The simulation results revealed that the flow behavior in the injection loop capillaries strongly influenced the shape of band profiles, particularly at higher eluent velocities. This was attributed to the formation of Dean vertices triggered by centrifugal forces in curved parts of the eluent flow path.
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http://dx.doi.org/10.1016/j.chroma.2021.462410DOI Listing
September 2021

A case study of the mechanism of unfolding and aggregation of a monoclonal antibody in ion exchange chromatography.

J Chromatogr A 2021 Jan 11;1636:461687. Epub 2020 Nov 11.

Department of Chemical and Process Engineering, Rzeszów University of Technology, Powstańców Warszawy Ave. 6, 35-959 Rzeszów, Poland. Electronic address:

A mechanistic model for describing unfolding of a monoclonal antibody (mAb) in ion exchange chromatography has been developed. The model reproduced retention behavior characteristic for conformational changes of antibodies upon adsorption, including: multi-peak elution, aggregate formation, and recovery reduction. Two competitive paths in the adsorption mechanism of the unfolded protein were assumed: refolding in the adsorbed phase to the native form followed by its desorption, or direct desorption followed by instantaneous aggregation in the liquid phase. The reduction in recovery of the eluted protein was attributed to spreading of the unfolded protein on the adsorbent surface, which enhanced the binding affinity. The model was formulated based on the analysis of retention behavior of a model mAb that was eluted in pH gradients on a strong cation exchange resin. The pH profile was found to be distorted in the presence of the protein, which was ascribed to dissociation of ionizable groups of the protein in the adsorbed phase. Since the protein retention was strongly pH dependent, that phenomenon was also accounted for in mathematical modeling. A series of independent experiments was designed to evaluate the model parameters that quantified the process thermodynamics and kinetics: the Henry constants of the native, unfolded, spread and aggregated forms of the protein along with underlying kinetic coefficients. The model was efficient in reproducing the retention pattern of the protein and the aggregate content in eluting band profiles. After proper calibration, the model can potentially be used to quantify protein unfolding and elution in other ion exchange systems.
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http://dx.doi.org/10.1016/j.chroma.2020.461687DOI Listing
January 2021

A high-throughput method for fast detecting unfolding of monoclonal antibodies on cation exchange resins.

J Chromatogr A 2020 Dec 11;1634:461688. Epub 2020 Nov 11.

Department of Chemical and Process Engineering, Rzeszów University of Technology, Rzeszów, Poland. Electronic address:

A fast method for assessing the stability of monoclonal antibodies (mAbs) adsorbed on ion exchange resins has been developed. The method exploited a real time polymerase chain reaction equipment to determine the temperature of protein phase transition, i.e., the so called melting temperature, based on differential scanning fluorimetry. Changes to the melting temperature were screened under various adsorption conditions and correlated with the protein stability upon adsorption. The method was tested for two different mAbs bound to various types of strong cation exchangers at different pH and loading concentrations. The mAbs destabilized upon adsorption due to strong binding, which manifested itself in aggregate formation and recovery reduction. The phenomenon depended on the resin type and binding conditions. However, regardless of the process conditions and resins used, drop in the melting temperatures to a critical value of about 30° could serve as an indicator of destructive changes in the protein structure in the adsorbed phase. The measurements were simultaneously accomplished for a number of samples with very small material consumption. Therefore, the method may be applied for screening resins and operating variables for a given mAb to exclude conditions that induce structure destabilization and aggregation.
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http://dx.doi.org/10.1016/j.chroma.2020.461688DOI Listing
December 2020

Effects of negative and positive cooperative adsorption of proteins on hydrophobic interaction chromatography media.

J Chromatogr A 2020 Aug 6;1625:461309. Epub 2020 Jun 6.

Department of Chemical and Process Engineering, Rzeszów University of Technology, Powstańców Warszawy Ave. 6, 35-959 Rzeszów, Poland. Electronic address:

The adsorption behavior of the model proteins: alpha-Lactalbumin, Bovine Serum Albumin, Lysozyme, and a monoclonal antibody, in single component and in binary mixtures, was investigated on two different hydrophobic interaction chromatography resins using both static and dynamic methods. A kinetic model of the adsorption process was developed, which accounted for protein unfolding and intermolecular interactions in the adsorbed phase. The latter incorporated positive cooperative interactions, resulting from preferred and multilayer adsorption on the adsorbent surface, as well as negative cooperative interactions attributed to exclusion effects due to size exclusion and repulsion. Cooperative adsorption resulted in negative or positive deviations from the Langmuir model for both single and multicomponent isotherms. The model was used to assess possible contributions of different adsorption mechanisms of proteins and their structurally different forms to the overall adsorption pattern, as well as to simulate chromatographic band profiles under different loading conditions. For proteins with unstable structure, the overall adsorption isotherm was dominated by binding of unfolded species at low surface coverage and by positive cooperative adsorption at high surface coverage. Furthermore, regardless of structural stability, exclusion effects influenced strongly adsorption equilibrium, particularly at low surface coverages. In case of chromatographic elution, i.e. under dynamic conditions, unfolding, negative cooperative adsorption, and kinetic effects governed the retention behavior and determined peak shapes, whereas the effect of positive cooperative adsorption was negligible.
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http://dx.doi.org/10.1016/j.chroma.2020.461309DOI Listing
August 2020

Mechanism of nutrition activity of a microgranule fertilizer fortified with proteins.

BMC Plant Biol 2020 Mar 24;20(1):126. Epub 2020 Mar 24.

Department of Chemical and Process Engineering, Rzeszow University of Technology, Powstańców Warszawy Ave. 6, 35-959, Rzeszów, Poland.

Background: A microgranule fertilizer was designed for localized fertilization of soil with controlled release of nutrients. The microgranule matrix was fortified with proteins, which were obtained from food industry byproducts or waste, i.e., whey protein from milk serum, soy protein from soy isolate and egg white protein from chicken egg white powder. The mechanism of the protein decomposition and migration of micro and macromolecule compounds through two different model soil systems was investigated. The potential of the protein fortified fertilizer for localized fertilization of the potted maize seeds was evaluated.

Results: The study revealed that proteins slowly diffused through soil with simultaneous degradation, which was accompanied with release of ammonia ions. The highest concentration of proteins and degradation products was found in a close vicinity of the microgranule. The microgranules were used as a local fertilizer for maize seeds in the pot experiments. The experiments confirmed statistically significant improvement in root density of maize plant compared to control group.

Conclusions: Byproducts or waste of food industry, such as the milk serum and soy can be used as a source of proteins that degrade in soil without a pretreatment. The degradation is accompanied with formation of ammonium ions, which can be utilized by plants as a nitrogen source. The fertilizer microgranule should be placed in a close vicinity to the plant seed, since the maximum of the protein concentration and ammonia ions is reached at a very close distance from the microgranule.
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http://dx.doi.org/10.1186/s12870-020-02340-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7092569PMC
March 2020

Scale up of a chromatographic capture step for a clarified bacterial homogenate - Influence of mass transport limitation and competitive adsorption of impurities.

J Chromatogr A 2020 May 8;1618:460856. Epub 2020 Jan 8.

Department of Chemical and Process Engineering, Rzeszów University of Technology, Powstańców Warszawy Ave. 6, 35-959 Rzeszów, Poland. Electronic address:

A model-based approach for scaling up chromatographic capture step was developed. The purification of human basic fibroblast growth factor protein 2 (FGF2) from an E. coli homogenate on a cation exchange resin was selected as a case study. Non-ideal effects accompanying the capture operation were examined, including: reduction in the protein diffusivity in the presence of the homogenate, competitive adsorption between FGF2 and undefined impurities, and flow behavior in external column volumes. The viscosity of the homogenate was measured as a function of dilution degree and shear stress, and its contribution to the diffusivity reduction was quantified. A dynamic model was formulated which accounted for underlying kinetic and thermodynamic dependencies. The model parameters were determined for a lab scale system using a small 2-mL column. The model was successfully used to scale up the capture operation from the lab scale column to a preparative bench scale column of about 1 L volume.
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http://dx.doi.org/10.1016/j.chroma.2020.460856DOI Listing
May 2020

Effect of flow behavior in extra-column volumes on the retention pattern of proteins in a small column.

J Chromatogr A 2019 Aug 2;1598:154-162. Epub 2019 Apr 2.

Department of Chemical and Process Engineering, Powstańców Warszawy Ave. 6, 35-959, Rzeszów, Poland. Electronic address:

Experimental and theoretical analysis of deformation of band profiles in extra-column volumes (ECV) was performed, and its influence on the retention pattern of proteins in a small chromatographic column was quantified. Several macromolecule and small-molecule compounds, and their mixtures were eluted from a chromatographic system in the absence and presence of the column. The peak deformation in ECV was attributed to non-uniform velocity distribution in the radial direction in connecting capillaries. The phenomenon enhanced with increasing molecular weight of the model compound, when radial diffusion dominated the mechanism of band spreading. The band shape was also affected by the geometry of the injection system used, i.e., an injection loop capillary or a superloop. The phenomenon vanished for a small molecule compound, for which plug flow conditions could be established. The difference in flow behaviour of the macromolecule and small-molecule compounds caused them to migrate with different velocities in ECV, which resulted in partial separation of their bands. The ECV effect influenced the retention behaviour of macromolecules in a small column; it caused tailing of peaks and asymmetry of breakthrough curves. To describe the elution profiles in ECV and in the column, a mathematical model was used which accounted for non-ideality of the flow pattern. The model reproduced accurately band profiles of macromolecules within a range of relatively low velocities, typical however for protein chromatography.
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http://dx.doi.org/10.1016/j.chroma.2019.03.060DOI Listing
August 2019

Retention Behavior of Polyethylene Glycol and Its Influence on Protein Elution on Hydrophobic Interaction Chromatography Media.

Chromatographia 2018 7;81(12):1641-1648. Epub 2018 Nov 7.

Faculty of Chemistry, Rzeszow University of Technology, al. Powstańców Warszawy 12, 35-959 Rzeszow, Poland.

The retention behavior of polyethylene glycol (PEG) on different types of hydrophobic interaction chromatography (HIC) resins containing butyl, octyl, and phenyl ligands was analyzed. An incomplete elution or splitting of the polymer peak into two parts was observed, where the first one was eluted at the dead time of the column, whereas the second one was strongly retained. The phenomenon was attributed to conformation changes of the polymer upon its adsorption on hydrophobic surface. The effect enhanced with increasing molecular weight of the polymer and hydrophobicity of the HIC media. Addition of PEG to the mobile phase reduced binding of proteins to HIC resins, which was demonstrated with two model systems: lysozyme (LYZ) and immunoglobulin G (IgG), and their mixtures. In case of LYZ, the presence of PEG caused reduction in the protein retention, whereas for IgG-a decrease in efficiency of the protein capture. The effect depended on the adsorption pattern of PEG; it was pronounced in the systems in which conformational changes of the polymer were suggested to occur.
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http://dx.doi.org/10.1007/s10337-018-3635-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6267512PMC
November 2018

Prediction tool for loading, isocratic elution, gradient elution and scaling up of ion exchange chromatography of proteins.

J Chromatogr A 2018 Sep 22;1566:89-101. Epub 2018 Jun 22.

Department of Chemical and Process Engineering, Powstańców Warszawy Ave. 6, 35-959 Rzeszów, Poland.

An efficient mathematical tool for the design and scaling up of protein chromatography is suggested, in which the model parameters can be determined quickly over a wide operating space without large material investments. The design method is based on mathematical modelling of column dynamics and moment analysis. The accuracy of the dynamic models that are most frequently used for simulations of chromatographic processes is analyzed, and possible errors that can be generated using the moment analysis are indicated. The so-called transport dispersive model was eventually employed for the process simulations. The model was modified to account for the protein dispersion in void volumes of chromatographic systems. The manner of the model calibration was suggested, which was based on a few chromatographic runs and verified over a wide space of the operating parameters, including composition and flow rate of the mobile phase, column dimensions, residence time, and mass loading. The model system for the study was ion-exchange chromatography. The analysis was performed based on the elution profiles of basic fibroblast growth factor 2 and lysozyme, on two different IEX media.
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http://dx.doi.org/10.1016/j.chroma.2018.06.057DOI Listing
September 2018

Effect of mass overloading on binding and elution of unstable proteins in hydrophobic interaction chromatography.

J Chromatogr A 2017 Apr 1;1492:79-88. Epub 2017 Mar 1.

Department of Chemical and Process Engineering, Rzeszów University of Technology, Powstańców Warszawy Ave. 6, 35-959, Rzeszów, Poland. Electronic address:

Adsorption behavior of unstable proteins, i.e., bovine serum albumin and α-lactalbumin, has been studied on a hydrophobic interaction chromatography medium under mass overloading conditions at different kosmotropic salt concentrations in the mobile phase. A mechanistic model has been formulated and used to describe kinetics and thermodynamics of protein interactions with the adsorbent surface. The model assumed two-site binding adsorption and reversible protein unfolding, which allowed predicting the inhibition of protein unfolding at high column loadings. A simplified procedure for the determination of model parameters has been developed, which was based on the inverse method. The model was successfully used to reproduce the pattern of chromatographic elution as well as the course of breakthrough curves. The model formulation was supported by Nano Differential Scanning Fluorimetry measurements, which were exploited to determine the protein stability in the liquid and adsorbed phases at different column loadings and salt concentrations.
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http://dx.doi.org/10.1016/j.chroma.2017.02.073DOI Listing
April 2017

A shortcut method for evaluation of protein deposition onto the membrane surface in crossflow ultrafiltration.

Eng Life Sci 2017 Apr 24;17(4):370-381. Epub 2016 Oct 24.

Department of Chemical and Process Engineering Rzeszów University of Technology Rzeszów Poland.

In this study, a procedure for quantifying the surface deposition of proteins in crossflow ultrafiltration has been developed. The procedure consists of determining the protein adsorption behavior onto the membrane surface from a few dynamic measurements performed in a nonfiltration and a filtration mode, and evaluating the concentration polarization (CP) layer thickness based on the adsorption data. To predict the interdependence between the protein adsorption and CP, a simplified mathematical model has been formulated. The model was used to assess the protein adsorption and thus yield reduction in the ultrafiltration process at different protein concentration in the solution. As a case study, ultrafiltration of aqueous solutions of BSA and lysozyme (LYZ) was examined on a polyethersulfone membrane with the molecular weight cutoff of 10 or 100 kDa. The protein concentration in the solutions varied within a relatively low concentration range, i.e. below 10 mg mL, characteristic for solvent exchange between sequential operations of protein purification by chromatography and extraction. Both proteins markedly differed in the mechanism of surface deposition; for BSA hydrophobic interactions were suggested to be dominant, whereas in case of LYZ electrostatic interactions contributed the most to the deposition mechanism. The effect of additives of the protein solutions, i.e. inorganic salts, PEG, and urea depended on the adsorption mechanism and was also specific for each protein. Nevertheless, the proposed procedure performed well in the evaluation of surface deposition and yield reduction, regardless of the protein type and its solvent environment.
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http://dx.doi.org/10.1002/elsc.201500159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6999320PMC
April 2017

Protein separation in carousel multicolumn setup. Performance analysis and experimental validation.

J Chromatogr A 2016 Aug 28;1460:40-50. Epub 2016 Jun 28.

Department of Chemical and Process Engineering, Rzeszów University of Technology, Powstańców Warszawy Ave. 6, 35-959 Rzeszów, Poland. Electronic address:

To overcome limitations of periodic separations of proteins in batch chromatographic columns Carousel Multi-Column Setup (CMS) has been recently suggested and theoretically analyzed in a previous study (R. Bochenek, W. Marek, W. Piątkowski, D. Antos, J. Chromatogr. A, 1301 (2013) 60-72). In this system, feed and mobile phase streams are subsequently delivered through parallel columns to mimic their countercurrent movement with respect to the fluid flow. All fluxes in the system are synchronized to ensure continuous feed delivery, which however causes reduction in the size of the operating window compared to batchwise-operating systems. In this study to improve the performance of CMS, additional process variables have been considered, such as the flow rate gradient and feed concentration. Though altering both variables allowed improving the separation selectivity and extending the operating window, the feed concentration appeared to be the most influential parameter affecting the process performance. Moreover, a procedure for practical realization of protein separations in CMS has been developed, including hints about the process design, configuration of columns and detectors, and use of pumps. As the case study, the separation of a ternary mixture of proteins, i.e., cytochrome C, lysozyme and immunoglobulin G, on hydrophobic interaction columns was used. A target product was a protein with intermediate adsorption strength that was isolated out of a more and less strongly adsorbed compound.
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http://dx.doi.org/10.1016/j.chroma.2016.06.080DOI Listing
August 2016

Center-cut separation of intermediately adsorbing target component by 8-zone simulated moving bed chromatography with internal recycle.

J Chromatogr A 2016 Jul 4;1453:19-33. Epub 2016 May 4.

Max Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany; Institute of Process Engineering, Faculty of Process and Systems Engineering, Otto von Guericke University Magdeburg, 39106 Magdeburg, Germany. Electronic address:

An 8-zone simulated moving bed chromatography with internal recycle (8ZSMB-IR) has been designed for center-cut separation, that is, for isolating an intermediately adsorbed component out of a multicomponent mixture. The system consists of two integrated subunits and operates in a fully continuous manner. In the first subunit the feed mixture is split into two fractions containing either a single component or a binary mixture. The binary mixture is recycled through the internal raffinate or extract port into the second subunit, where the target product is isolated. Additionally, the solvent is also recycled internally. For a case study, the separation of a ternary mixture of cycloketones as a model system under weakly non-linear isotherm conditions has been investigated. A few novel configurations of the 8ZSMB-IR unit including the arrangement of the internal recycle of extract, raffinate and solvent streams between two subunits have been examined with respect to various performance indicators for the process realization. The unit performed best with the developed configuration when the internal raffinate stream was recycled and the solvent recycling loop was closed between the last and the first zone of the first subunit. That configuration has further been analyzed experimentally and numerically. On the basis of the results a strategy for determining reliable operating conditions for the 8ZSMB-IR process has been developed. The procedure exploited a model of the process dynamics, which was implemented to refine the isotherm coefficients and to quantify the mixing effect of the liquid stream inside the recycling loops. The upgraded model with the adjusted parameters has been validated based on experimental data and successfully applied for optimizing the operating conditions of the separation.
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http://dx.doi.org/10.1016/j.chroma.2016.04.083DOI Listing
July 2016

Overcoming solubility limits in overloaded gradient hydrophobic interaction chromatography.

J Chromatogr A 2015 Mar 30;1386:1-12. Epub 2015 Jan 30.

Department of Chemical and Process Engineering, Rzeszow University of Technology, Rzeszow, Poland. Electronic address:

The impact of the solubility limits on the performance of gradient protein chromatography has been studied. As a case study elution of model protein, i.e., lysozyme, on hydrophobic interaction media has been selected. A dependence of the protein solubility and crystallization kinetics on the content of cosmotropic salt in the mobile phase has been determined. Moreover, adsorption properties of the protein versus the mobile phase composition have been quantified. A model of chromatographic column dynamics has been developed which incorporated the mass transport kinetics accompanying both adsorption and crystallization processes. The model was used to study the influence of operating parameters such as flowrate and concentration loading on the solubility pattern inside the column and the separation performance. The analysis performed indicated existence of supersaturation regions for which, due to slow kinetics of crystallization, chromatographic process could be performed under conditions of strong concentration overloading while avoiding undesirable effects of flow blockage in chromatographic systems.
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http://dx.doi.org/10.1016/j.chroma.2015.01.069DOI Listing
March 2015

Adsorption behavior of proteins on temperature-responsive resins.

J Chromatogr A 2014 Jan 25;1324:181-9. Epub 2013 Nov 25.

Department of Chemical and Process Engineering, Rzeszów University of Technology, Powstańców Warszawy Ave. 6, 35-959 Rzeszów, Poland. Electronic address:

The adsorption behavior of proteins on thermo-responsible resins based on poly(N-isopropylacrylamide) and its copolymer containing an anionic co-monomer has been investigated. The influence of the polymer composition, i.e., the content of the co-monomer and crosslinker on the thermo-sensitivity of the protein adsorption has been quantified. The properties of ungrafted polymer as well grafted onto the agarose matrix have been analyzed and compared. Batch and dynamic (column) experiments have been performed to measure the adsorption equilibrium of proteins and to quantify the phase transition process. As model proteins lysozyme, lactoferrin, α-chymotrypsinogen A and ovalbumin have been used. The adsorption process was found to be governed by ionic interactions between the negatively charged surface of resin and the protein, which enabled separation of proteins differing in electrostatic charge. The interactions enhanced with increase of temperature. Decrease of temperature facilitated desorption of proteins and reduced the salt usage in the desorption buffer. Grafted polymers exhibited markedly higher mechanical stability and, however, weaker temperature response compared to the ungrafted ones.
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http://dx.doi.org/10.1016/j.chroma.2013.11.040DOI Listing
January 2014

Isolation of monoclonal antibody from a Chinese hamster ovary supernatant. II: dynamics of the integrated separation on ion exchange and hydrophobic interaction chromatography media.

J Chromatogr A 2013 Aug 4;1305:64-75. Epub 2013 Jul 4.

Chemical and Process Engineering Department, Rzeszów University of Technology, Powstańców Warszawy Avenue, 6, 35-959 Rzeszow, Poland.

Dynamics of the purification process of a CHO derived monoclonal antibody by ion exchange chromatography (IEC), hydrophobic interaction chromatography (HIC) and their integration has been investigated. To quantify the adsorption behavior of the target protein (IgG1) and impurities contained in the supernatant, their elution course on IEC and HIC columns has been analyzed versus pH and/or the salt concentration in the mobile phase. A short-cut method has been proposed for mathematical modeling and determining underlying kinetic and thermodynamic parameters. The accuracy of the model predictions has been verified by comparing the simulated and experimental band profiles recorded in both chromatographic processes. After verification, the model was used to optimize operating conditions for the column loading and chromatographic elution in the integrated process IEC/HIC. Two alternative loading techniques based on the upstream and downstream feed dilution were taken into account in the optimization routine. In the first one the feed stream was diluted with the loading buffer prior to the column loading, while in the latter one the feed dilution was realized inside the column using the multiple-injection technique. It was shown that the downstream dilution allowed significant reduction of the contact time between the protein and the loading buffer.
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http://dx.doi.org/10.1016/j.chroma.2013.06.076DOI Listing
August 2013

Isolation of monoclonal antibody from a Chinese hamster ovary supernatant. I: assessment of different separation concepts.

J Chromatogr A 2013 Aug 4;1305:55-63. Epub 2013 Jul 4.

Department of Chemical and Process Engineering, Rzeszów University of Technology, Powstańców Warszawy Ave., 6, 35-959 Rzeszow, Poland.

The performance of different non-affinity purification techniques commonly used for isolating CHO derived monoclonal antibodies has been investigated. Ion exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), aqueous-two-phase extraction (ATPE) and their integration has been evaluated in terms of yield and purity of the product obtained. The integration of chromatographic techniques comprised two steps, in which the CHO supernatant was directly injected into the IEC column to capture monoclonal IgG1 and then the isolated fraction was processed using the HIC column. To reduce the influence of the feed media on retention of the target protein, the feed mixture was on-column diluted by use of the multi-injection technique. In the coupled process of extraction and chromatography the ATPE operation was used for the pre-purification of the supernatant as well as for buffer exchange. The bottom ATPE phase containing the target protein was further purified on the HIC column without feed dilution. The influence of operating conditions on the effectiveness of different purification processes has been evaluated. The best performance with respect to the product purity was achieved for the coupled process of IEC and HIC. The experimental data acquired were exploited in subsequent investigations for determining underlying kinetic parameters and for the process prediction and optimization.
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http://dx.doi.org/10.1016/j.chroma.2013.06.077DOI Listing
August 2013

Evaluating the performance of different multicolumn setups for chromatographic separation of proteins on hydrophobic interaction chromatography media by a numerical study.

J Chromatogr A 2013 Aug 28;1301:60-72. Epub 2013 May 28.

Department of Chemical and Process Engineering, Rzeszow University of Technology, Powstancow Warszawy Avenue 6, 35-959 Rzeszow, Poland.

A theoretical study has been performed on the effectiveness of isolating a target component out of a multi-component protein mixture using different arrangements of chromatographic columns. Three continuous systems have been considered which were able to perform solvent gradient separations, such as: open loop simulated moving bed, countercurrent solvent gradient purification and carousel multicolumn setup. The performance of the continuous processes was examined with respect to productivity, yield and eluent consumption and compared to a single-column batch system. As a case study separation of a ternary mixture of proteins on HIC media has been selected. Two separation problems have been analyzed referring to the situation when the target component was the most strongly adsorbed as well as when it exhibited intermediate adsorption strength. A mathematical model has been used to simulate the process dynamics and to optimize operating conditions for the separation. The numerical study indicated that batch column arrangements can outperform SMB-based configurations regarding all performance indicators considered, which has been attributed to solvent mixing in the recycled streams and distortion of the gradient shape in SMB units. It has been concluded that the performance of complex multicolumn systems should be verified vs. batch column operations prior to the realization of the separation process.
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http://dx.doi.org/10.1016/j.chroma.2013.05.053DOI Listing
August 2013

Theoretical study of using simulated moving bed chromatography to separate intermediately eluting target compounds.

J Chromatogr A 2012 Aug 8;1253:58-70. Epub 2012 Jul 8.

Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

This study deals with the separation of ternary mixtures based on Simulated Moving Bed chromatography to isolate target components with intermediate adsorption strength. To overcome the limitations of conventional SMB systems, which are designed for binary separations and unable to perform center-cut separations, several modifications have been proposed. The purpose of this study was to provide a theoretical comparison of several of advanced SMB configurations capable to separate ternary mixtures. Emphasis is given to those techniques, which have already been used in practice, and to those having potential for future industrial application. SMB cascades connected in series via the extract or raffinate ports of the first unit are analyzed and compared are as well as an integrated 8-zone SMB unit with internal recycle. Additionally, the commercialized pseudo SMB process (JO process) was evaluated. The performance of these modified SMB systems was investigated based on the assumption of linear adsorption isotherms for all three components considering three separation problems characterized by different separation difficulty. Besides the influence of separation factors, the concentrations of the impurities in the feed mixture and the purity requirements for the target product were studied systematically.
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http://dx.doi.org/10.1016/j.chroma.2012.06.096DOI Listing
August 2012

Multiple-injection technique for isolating a target protein from multicomponent mixtures.

J Chromatogr A 2011 Aug 22;1218(32):5423-33. Epub 2011 Feb 22.

Chemical and Process Engineering Department, Rzeszow University of Technology, W. Pola Str., 2, 35-959 Rzeszow, Poland.

An integrated chromatographic process comprising ion exchange (IEC) and hydrophobic interaction chromatography (HIC) for isolating a target protein form multicomponent mixtures has been analyzed. The model mixture contained immunoglobulin G that was the key product of the separation process, cytochrome C and ovalbumin. The adsorption characteristics and the mass transport kinetics of the model proteins have been determined along with their dependencies on the operating variables such as pH, temperature and the salt concentration for IEC as well as HIC media. Limitations of the process efficiency resulting from kinetic effects, solubility constraints and the necessity of the mobile phase exchange between chromatographic steps have been discussed. To improve the performance of the integrated process the multiple-injection technique has been suggested. This technique consisted in loading feed mixtures dissolved in a good solvent onto the column by several small-volume injections under conditions of strong protein adsorption. It allowed diminishing interactions between the sample-solvent and protein and elimination of undesired effects such as band splitting and band broadening. For the process design and optimization a dynamic model has been used accounting for thermodynamics and kinetics of the process. The optimization results indicated superiority of the multiple-injection technique over standard isocratic injections in terms of the process yield and productivity.
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http://dx.doi.org/10.1016/j.chroma.2011.02.039DOI Listing
August 2011

Influence of the sample-solvent on protein retention, mass transfer and unfolding kinetics in hydrophobic interaction chromatography.

J Chromatogr A 2010 Apr 25;1217(17):2812-20. Epub 2010 Feb 25.

Chemical and Process Engineering Department, Rzeszow University of Technology, W. Pola Str., 2, 35-959 Rzeszow, Poland.

Typical mobile phase employed in hydrophobic interaction chromatography contains cosmotropic salts, which promote retention and simultaneously reduce the protein solubility in the mobile phase. To increase mass overloading in the separation process the protein can be dissolved in a sample-solvent with concentration of salt lower than that in the mobile phase or in salt free solutions. However, this methodology may cause band splitting and band deformation, which results in yield losses. In this study, these phenomena were analyzed based on the retention behavior of two model proteins, i.e., lysozyme and bovine serum albumin. Retention of these proteins was accompanied by strong band broadening originated from slow rates of mass transfer and/or of adsorption-desorption process involving the protein conformational changes. The mass transport resistances and unfolding kinetics were found to contribute to the sample-solvent effects. To avoid band deformations the process variables such as the salt concentration and temperature were adjusted in such a way that complete resolution between band profile of the sample-solvent and the protein was achieved. For the process simulation a dynamic model, which accounted for underlying kinetics was used. General guidelines of the process design were developed.
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http://dx.doi.org/10.1016/j.chroma.2010.02.043DOI Listing
April 2010

Effects of thermal heterogeneity in hydrophobic interaction chromatography.

J Chromatogr A 2009 Sep 11;1216(39):6716-27. Epub 2009 Aug 11.

Chemical and Process Engineering Department, Rzeszow University of Technology, W. Pola Str., 2, 35-959 Rzeszow, Poland.

Manipulating temperature and salt concentration can have a powerful effect on the separation effectiveness in hydrophobic interaction chromatography (HIC). However, use of temperature as an operating variable in large-scale applications may involve undesirable consequences such as radial heterogeneity of the column temperature. In this study non-ideal effects of heat transfer in HIC columns were analyzed. The radial temperature gradients were measured by thermocouples immersed in a bed packed into a preparative column. The column wall was either thermostatted by a water jacket or left under ambient conditions. The influence of ineffective column thermostatting and of heat losses on the radial temperature profiles was demonstrated and predicted by a model of heat dispersion in a packed bed. To analyze possible positive or negative effects of thermal heterogeneity on band propagation, non-isothermal chromatographic elution of a model protein (alpha-chymotrypsinogen A) was recorded under salt gradient conditions as well as at constant salt concentration. To predict temperature and concentration profiles a model of the column dynamics was used. The model accounted for kinetics of mass and heat transfer. A good agreement between experimental and simulated profiles was achieved. It was shown that by proper selection of the process conditions undesirable temperature effects can be avoided or controlled.
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http://dx.doi.org/10.1016/j.chroma.2009.08.007DOI Listing
September 2009

Altering efficiency of hydrophobic interaction chromatography by combined salt and temperature effects.

J Chromatogr A 2009 Dec 21;1216(50):8712-21. Epub 2009 Apr 21.

Chemical and Process Engineering Department, Rzeszow University of Technology, W. Pola Street, 2, 35-959 Rzeszow, Poland.

The coupled effect of salt concentration and temperature on the retention behavior of proteins in hydrophobic interaction chromatography has been studied. The retention data of four model proteins, i.e., myoglobin, lysozyme, alpha-chymotrypsinogen and bovine serum albumin, have been acquired by isocratic experiments of chromatographic elution within the temperature range 5-25 degrees C at different ammonium sulphate concentrations in the mobile phase. The retention dependencies quantified as functions of the salt concentration and temperature have been exploited in designing the process of gradient elution. The propagation velocity of proteins under conditions of the step gradient of salt and temperature has been determined by use of the equilibrium theory. To evaluate kinetic effects accompanying the band propagation the transport-dispersive model has been employed. It has been shown that altering the propagation of the salt and temperature waves in a proper manner allows improving the separation efficiency. Moreover, manipulation of specific kinetics effects can also be exploited in protein separations.
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http://dx.doi.org/10.1016/j.chroma.2009.04.046DOI Listing
December 2009

Adsorption behaviour of sugars versus their activity in single and multicomponent liquid solutions.

J Chromatogr A 2009 Dec 21;1216(50):8697-704. Epub 2009 Jan 21.

Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

In this work the activity of three carbohydrates (sucrose, glucose and fructose) in highly concentrated aqueous solutions was studied along with its effect on the adsorption behaviour of the investigated compounds. Activities of individual sugars in aqueous solutions of single solute as well as in binary mixtures were quantified on the basis of solubility properties. Solid-liquid equilibria of sugars were correlated with the NRTL (nonrandom, two liquid) model of activity coefficient formulation. Activities of individual sugars were incorporated into the single component adsorption isotherm model, which reproduced accurately the course of the adsorption equilibria of sugars in aqueous solutions obtained experimentally in previous work using an ion-exchange resin. Activities of sugars determined in binary solute systems along with the single component isotherms were used to predict competitive adsorption equilibria. To calculate adsorbed phase concentrations of individual sugars in binary mixtures the adsorbed solution theory was adopted. The isotherm shapes calculated were compared to the data of competitive adsorption from the former study and found to be able to describe these experimental results.
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http://dx.doi.org/10.1016/j.chroma.2009.01.043DOI Listing
December 2009

Behavior of adsorbed and fluid phases versus retention properties of amino acids on the teicoplanin chiral selector.

J Chromatogr A 2008 May 22;1192(1):130-8. Epub 2008 Mar 22.

Chemical and Process Engineering Department, Rzeszów University of Technology, Rzeszów, Poland.

The relationship between adsorption equilibria of two amino acids, i.e., l,d-threonine and l,d-methionine on the teicoplanin chiral selector and their phase behavior has been analyzed. The experimental and numerical methods have been proposed to determine activity coefficients of amino acids in different solvent systems. The procedure was based on the analysis of solubility properties of the amino acids in aqueous solutions of methanol, ethanol and propanol-2-ol used as the mobile phases in chromatographic elution. The solubility measured in mixed alcohol-water solutions was correlated with the non-random-two-liquid (NRTL) model for the activity coefficients. The values of activity coefficients were incorporated into the adsorption isotherm equation, which allowed the analysis of retention properties of the amino acids versus their fluid phase behavior. For the investigation the experimental data of adsorption equilibria of amino acids as well as of the mobile phase constituents acquired in a previous work were exploited. The composition of both the mobile and the adsorbed phases was found to affect the retention properties of the amino acids. For water-rich mobile phases the activity in the adsorbed phase determined the retention mechanism, while for the alcohol-rich systems activity in the mobile phase was predominant.
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http://dx.doi.org/10.1016/j.chroma.2008.03.056DOI Listing
May 2008

Protein separations with induced pH gradients using cation-exchange chromatographic columns containing weak acid groups.

J Chromatogr A 2008 Feb 26;1181(1-2):83-94. Epub 2007 Dec 26.

Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904-4741, USA.

The behavior of chromatographic columns packed with resins containing both weak and strong cation-exchange groups is investigated in order to obtain protein separations by means of internally generated pH gradients in response to step changes in buffer composition. A local equilibrium model is developed to predict pH transitions using non-adsorbed buffers, i.e. containing neutral and negatively charged buffering species, based exclusively on the resin titration curve. In agreement with experimental results, the model predicts practical, fairly linear gradients between pH 5 and 7, which are formed using suitable mixtures of acetate and phosphate buffers. The separation of mixtures of ovalbumin, albumin, and transferrin is used as a model system, but, unlike most previous work, we consider preparative conditions. Near baseline resolution is obtained with protein loads as high as 10mg/mL and mobile phase velocities at high as 460 cm/h using porous, 70-microm diameter particles. The peaks obtained with this approach are much sharper than could be obtained isocratically or using externally generated, unretained gradients as a result of the peak compression caused by the axial pH gradient formed along the column. Moreover, separation is obtained at very low ionic strengths (2-3 mS/cm). The effects of flow velocity, mobile phase composition, time of injection, and protein load on retention and elution pH are investigated systematically demonstrating a range of ways in which the separation can be controlled and optimized.
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http://dx.doi.org/10.1016/j.chroma.2007.12.054DOI Listing
February 2008

Influence of preferential adsorption of mobile phase on retention behavior of amino acids on the teicoplanin chiral selector.

J Chromatogr A 2007 Nov;1173(1-2):58-70

Chemical and Process Engineering Department, Rzeszów University of Technology, Rzeszów, Poland.

The adsorption behavior of two amino acids, i.e., l,d-threonine and l,d-methionine has been investigated on the chiral stationary phase (CSP)column packed with teicoplanin bonded to a silica support. The study has been performed under non-linear conditions of adsorption isotherm for various types of organic modifiers (methanol, ethanol, propan-2-ol and acetonitrile) in the reversed-phase mode. A heterogeneous adsorption mechanism of amino acids has been identified that was strongly affected by the nature of organic modifier. Generally, isotherm non-linearity and retention decreased with decrease of the modifier content in the mobile phase exhibiting a minimum at water-rich mobile phases. These trends were suggested to result from a combined effect of the mobile as well as the adsorbed phase composition. To determine the composition of the adsorbed phase the excess adsorption of modifiers in aqueous solutions has been measured and their binary adsorption equilibria have been quantified and compared. Strongly non-ideal behavior of solvents in the mobile phase and the adsorbed phase has been accounted for by activity coefficients. The fraction of the modifiers in the adsorbed phase decreased in the sequence: methanol, ethanol, propan-2-ol and acetonitrile.
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http://dx.doi.org/10.1016/j.chroma.2007.09.076DOI Listing
November 2007

Effect on separation of injecting samples in a solvent different from the mobile phase.

J Chromatogr A 2007 Aug 12;1162(1):62-73. Epub 2007 Feb 12.

Chemical Process Engineering, Otto-von-Guericke-University, Universitätsplatz 2, 39106 Magdeburg, Germany.

The separation of compounds possessing low solubility in the mobile phase could be improved by applying stronger solvents for dissolving the feed. A model system has been investigated with ethanol-water as the mobile phase, Chirobiotic T as the stationary phase and D,L-threonine as the sample dissolved in pure water. The adsorption isotherms of D,L-threonine were determined as a function of the water content by means of a peak fitting method. Optimal injection conditions of elution chromatography were determined by process simulation. Finally, the optima obtained were verified with experimental data.
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http://dx.doi.org/10.1016/j.chroma.2007.02.010DOI Listing
August 2007

Deformation of gradient shape as a result of preferential adsorption of solvents in mixed mobile phases.

J Chromatogr A 2006 Sep 11;1127(1-2):187-99. Epub 2006 Jul 11.

Rzeszów University of Technology, Chemical Engineering and Process Control Department, al. Powstańców Warszawy 6, 35-959 Rzeszów, Poland.

Gradient elution has been studied in typical normal and reversed-phase systems. Deformations of gradient profiles have been evidenced as a result of preferential adsorption of modifiers of the mobile phase. This phenomenon was pronounced in the normal-phase system, for which gradient profiles deviated significantly from those programmed. This influenced the retention and shapes of band profiles of the eluting solute. Hence, in order to predict gradient propagation correctly the adsorption equilibrium of modifiers has been quantified. Moreover, at low modifier content, deformations of band profiles of the solute has been registered as a result of the competitive adsorption in the system solute-modifier. This effect has been predicted by a competitive adsorption model. For the reversed-phase systems the influence of the modifier adsorption on gradient propagation was insignificant for typical mobile phases investigated. Therefore, the work has been focused on gradient predictions in the normal-phase system.
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http://dx.doi.org/10.1016/j.chroma.2006.06.018DOI Listing
September 2006

Effect of temperature on competitive adsorption of the solute and the organic solvent in reversed-phase liquid chromatography.

J Chromatogr A 2006 Jan 15;1103(2):284-95. Epub 2005 Dec 15.

Department of Chemical and Process Engineering, Rzeszów University of Technology, 6 Powstańców Warszawy Avenue, 35-959 Rzeszów, Poland.

In analysis of the temperature effect on chromatographic separations the influence of the adsorption of organic solvent on the retention properties of solute is generally not taken into account. In fact, adsorption behavior of solutes is strongly affected by competitive adsorption of organic solvents, which is temperature dependent. In this work changes of adsorption equilibrium of an organic solvent as well as a solute with temperature have been analyzed. Data of the excess adsorption of methanol from aqueous solutions on octadecyl-bonded silica have been acquired at different temperature. Experiments have been performed over a relatively narrow temperature range corresponding to typical chromatographic conditions, i.e., 10-50 degrees C. The competitive adsorption equilibria of model solutes (i.e., two homologous compounds: cyclopentanone and cyclohexanone) have been measured at different temperature and composition of the mobile phase. Temperature alterations to the retention properties were found to result from combined effects of changes in adsorption behavior of the organic solvent and of the solute. The influence of temperature on the separation selectivity has been considered.
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http://dx.doi.org/10.1016/j.chroma.2005.11.038DOI Listing
January 2006
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