Roberto Guzmán

The adsorption of proteins to gas-liquid interfaces

Abstract A kinetic model of protein adsorption to gas-liquid interfaces is described. The model is based on a formalism similar to that used to describe adsorption at gas-solid interfaces, but with proteins we allow for both tight adsorption of a first layer and loose packing of a second layer of proteins. The model is fit to the adsorption isotherm data of Graham and Phillips for the three proteins: β-casein, bovine serum albumin, and lysozyme. We also fit the model to adsorption rate data for the two proteins lysozyme and β-casein. The experimental adsorption rates are considerably faster than those for molecular diffusion alone. The rate of mass transfer of protein to the interface was accordingly modeled by an effective mass-transfer coefficient. The ratio of the mass-transfer coefficients for two different proteins can be predicted by the free-convection correlation of Globe and Dropkin, suggesting that small temperature gradients may have existed in the vessel during the measurements. Finally, we have predicted desorption rates for exchange experiments in which fresh solution replaces bulk protein solution after equilibrium adsorption has been reached. Depending on the residence time of the bulk fluid in the reservoir, our results indicate desorption can be very difficult to measure, particularly at low surface concentrations and even if equilibrium is assumed between the interface and the solution just below the surface. Thus, there appears to be no need to postulate irreversible adsorption of protein to account for the available experimental data on protein adsorption and desorption.

Detachment of captured cancer cells under flow acceleration in a bio-functionalized microchannel

Attachment, deformation and detachment of N-cadherin expressing prostate and breast cancer cell lines in a functionalized microchannel under hydrodynamic loading have been studied. N-cadherin antibodies are immobilized on the microchannel surface to capture the target cancer cells, PC3N and MDA-MB-231-N, from a homogeneous cell suspension. Although difficult, a significant fraction of moving cells can be captured under a low flow rate. More than 90% of the target cells are captured after a certain incubation time under no flow condition. The mechanical response of a captured cancer cell to hydrodynamic flow field is investigated and, in particular, the effect of flow acceleration is examined. The observed cell deformation is dramatic under low acceleration, but is negligible under high acceleration. Consequently, the detachment of captured cells depends on both flow rate and flow acceleration. The flow rate required for cell detachment is a random variable that can be described by a log-normal distribution. Two flow acceleration limits have been identified for proper scaling of the flow rate required to detach captured cells. A time constant for the mechanical response of a captured cell, on the order of 1 min, has been identified for scaling the flow acceleration. Based on these acceleration limits and time constant, an exponential-like empirical model is proposed to predict the flow rate required for cell detachment as a function of flow acceleration.

Biosorption of Cu by Thiobacillus ferrooxidans

Abstract Current technologies for removal and recovery of both toxic and industrial interest metals usually produce wastes with high concentrations of those substances. They are an important source of environmental pollution, specially when they contain heavy metals. This is one of the most important environmental problems, and of the most difficult to solve. So far, there have been a number of studies considering the possibility of removing and recovering heavy metals from diluted solutions. These are due, principally, because of the commercial value of some metals as well as the environmental impact caused by them. The traditional methods for removing have several disadvantages when metals are present in concentrations lower than 100 mg/l. Biosorption, which uses biological materials as adsorbents, has been considered as an alternative method. In this work, several variables that affect the capacity for copper biosorption by T. ferrooxidans have been studied. Particularly, the effect of pH, chemical pretreatment, biomass concentration and temperature have been considered. Results indicate that a capacity as high as 119 mg of Cu/g of dry biomass can be obtained at a temperature of 25 °C.

Mathematical analysis of frontal affinity chromatography in particle and membrane configurations.

The scaleup and optimization of large-scale affinity-chromatographic operations in the recovery, separation and purification of biochemical components is of major industrial importance. The development of mathematical models to describe affinity-chromatographic processes, and the use of these models in computer programs to predict column performance is an engineering approach that can help to attain these bioprocess engineering tasks successfully. Most affinity-chromatographic separations are operated in the frontal mode, using fixed-bed columns. Purely diffusive and perfusion particles and membrane-based affinity chromatography are among the main commercially available technologies for these separations. For a particular application, a basic understanding of the main similarities and differences between particle and membrane frontal affinity chromatography and how these characteristics are reflected in the transport models is of fundamental relevance. This review presents the basic theoretical considerations used in the development of particle and membrane affinity chromatography models that can be applied in the design and operation of large-scale affinity separations in fixed-bed columns. A transport model for column affinity chromatography that considers column dispersion, particle internal convection, external film resistance, finite kinetic rate, plus macropore and micropore resistances is analyzed as a framework for exploring further the mathematical analysis. Such models provide a general realistic description of almost all practical systems. Specific mathematical models that take into account geometric considerations and transport effects have been developed for both particle and membrane affinity chromatography systems. Some of the most common simplified models, based on linear driving-force (LDF) and equilibrium assumptions, are emphasized. Analytical solutions of the corresponding simplified dimensionless affinity models are presented. Particular methods for estimating the parameters that characterize the mass-transfer and adsorption mechanisms in affinity systems are described.

Optimal design of affinity membrane chromatographic columns

Abstract A method for the optimal affinity membrane column design, based in the solution of the Thomas kinetic model for frontal analysis in membrane column adsorption, is presented. The method permits to choose suitable membrane operating conditions, column dimensions and processing time, to maximize the throughput when an operating capacity restriction in the range of 80–95% of the column capacity is used. Two basic design charts were obtained by computer simulation, for residence and processing time calculation, respectively. These charts can be used and manipulated in a wide range of operational conditions, provided that four design specifications related to column axial and radial Peclet numbers, length and pressure drop, are fulfilled. The application of the method was illustrated using experimental data and a simple analytical procedure. The implications of the method and results on the design and optimization of affinity membrane chromatographic columns are discussed.

Purification of plasmid DNA using tangential flow filtration and tandem anion-exchange membrane chromatography

A new bioprocess using mainly membrane operations to obtain purified plasmid DNA from Escherechia coli ferments was developed. The intermediate recovery and purification of the plasmid DNA in cell lysate was conducted using hollow-fiber tangential filtration and tandem anion-exchange membrane chromatography. The purity of the solutions of plasmid DNA obtained during each process stage was investigated. The results show that more than 97% of RNA in the lysate was removed during the process operations and that the plasmid DNA solution purity increased 28-fold. One of the main characteristics of the developed process is to avoid the use of large quantities of precipitating agents such as salts or alcohols. A better understanding of membrane-based technology for the purification of plasmid DNA from clarified E. coli lysate was developed in this research. The convenience of anion-exchange membranes, configured in ready-to-use devices can further simplify large-scale plasmid purification strategies.

Adhesion dynamics of circulating tumor cells under shear flow in a bio-functionalized microchannel

The adhesion dynamics of circulating tumor cells in a bio-functionalized microchannel under hydrodynamic loading is explored experimentally and analyzed theoretically. EpCAM antibodies are immobilized on the microchannel surface to specifically capture EpCAM-expressing target breast cancer cells MDA-MB-231 from a homogeneous cell suspension in shear flow. In the cross-stream direction, gravity is the dominant physical mechanism resulting in continuous interaction between the EpCAM cell receptors and the immobilized surface anti-EpCAM ligands. Depending on the applied shear rate, three dynamic states have been characterized: firm adhesion, rolling adhesion and free rolling. The steady-state velocity under adhesion- and free-rolling conditions as well as the time-dependent velocity in firm adhesion has been characterized experimentally, based on video recordings of target cell motion in functionalized microchannels. A previously reported theoretical model, utilizing a linear spring to represent the specific receptor–ligand bonds, has been adopted to analyze adhesion dynamics including features such as the cell–surface binding force and separation gap. By fitting theoretical predictions to experimental measurements, a unified exponential decay function is proposed to describe the target cell velocity evolution during capture; the fitting parameters, velocity and time scales, depend on the particular cell–surface system.

Breakthrough Performance of Plasmid DNA on Ion-Exchange Membrane Columns

Breakthrough performance of plasmid DNA adsorption on ion‐exchange membrane columns was theoretically and experimentally investigated using batch and fixed‐bed systems. System dispersion curves showed the absence of flow non‐idealities in the experimental arrangement. Breakthrough curves (BTC) were significantly affected by inlet flow rate and solute concentration. In the theoretical analysis, a model was integrated by the serial coupling of the membrane transport model and the system dispersion model. A transport model that considers finite kinetic rate and column dispersed flow was used in the study. A simplex optimization routine, coupled to the solution of the partial differential model equations, was employed to estimate the maximum adsorption capacity constant, the equilibrium desorption constant, and the forward interaction rate constant, which are the parameters of the membrane transport model. The analysis shows that as inlet concentration or flow rate increases, the deviation of the model from the experimental behavior decreases. The BTCs displacement as inlet concentration increases was explained in terms of a greater degree of column saturation reached and more efficient operation accomplished. The degree of column saturation was not influenced by inlet flow rate. It was necessary to consider in the column model the slight variation in the BTC produced by the axial dispersion, in order to accomplish the experimental curve dispersion. Consequently, the design criteria that for Pe > 40 the column axial dispersion can be neglected should be taken with precaution.

Modeling Column Regeneration Effects on Dye-Ligand Affinity Chromatography

The effect of in‐place regeneration of dye−ligand adsorbents on protein adsorption characteristics is presented. Regeneration with chemical treatments and time of exposure determined the protein capacity of the adsorbent, but no effect was observed on its protein binding affinity. Fixed‐bed adsorption of bovine serum albumin and its selectivity with respect to lysozyme was studied. Breakthrough curves were measured for protein adsorption on fixed‐bed columns and analyzed by a simple model to determine the relevant rate constants for the adsorption process. It was found that forward adsorption rate constant increased exponentially with the chemical treatment exposure time. Column linear gradient elution studies showed that adsorbent selectivity decreased with the chemical treatment exposure time due mainly to column loss of adsorption capacity. The implications of the results on the design and optimization of dye−ligand chromatographic processes are discussed.

Kinematics of Specifically Captured Circulating Tumor Cells in Bio-Functionalized Microchannels

The attachment kinematics of cancer cells under hydrodynamic loading in antibody-functionalized microchannels has been studied. Epithelial-cell-adhesion-molecule antibodies are immobilized on the microchannel surface for specific capture of the target cancer cells from homogeneous cell suspensions. The specific interaction between the cancer cell receptors and the immobilized antibodies under static conditions is demonstrated. The capture efficiency of the target cells from homogeneous suspensions under applied hydrodynamic flow field has been investigated, revealing a characteristic shear stress. Applying a lower stress allows the capture of most target cells, while the capture efficiency drops sharply with an increasing shear stress. The captured cells are spatially distributed along the microchannel; both the velocity and the distance travelled by cells prior to capture are measured. The characteristic time and length scales for cell capture are determined, and a log-normal statistical distribution is proposed to describe the observations. Furthermore, a first-order kinetic model for receptor-ligand bond formation provides a rough estimate of the cell adhesion rate constant. Under a low shear stress, the on-rate is much higher than the off-rate, allowing capture of most loaded cells. The off-rate constant increases exponentially with an increasing shear stress, such that above the characteristic stress level, most loaded cells avoid capture.