The Investigation of Protein Diffusion via H-Cell Microfluidics

In this study, we developed a microfluidics method, using a so-called H-cell microfluidics device, for the determination of protein diffusion coefficients at different concentrations, pHs, ionic strengths, and solvent viscosities. Protein transfer takes place in the H-cell channels between two laminarly flowing streams with each containing a different initial protein concentration. The protein diffusion coefficients are calculated based on the measured protein mass transfer, the channel dimensions, and the contact time between the two streams. The diffusion rates of lysozyme, cytochrome c, myoglobin, ovalbumin, bovine serum albumin, and etanercept were investigated. The accuracy of the presented methodology was demonstrated by comparing the measured diffusion coefficients with literature values measured under similar solvent conditions using other techniques. At low pH and ionic strength, the measured lysozyme diffusion coefficient increased with the protein concentration gradient, suggesting stronger and more frequent intermolecular interactions. At comparable concentration gradients, the measured lysozyme diffusion coefficient decreased drastically as a function of increasing ionic strength (from zero onwards) and increasing medium viscosity. Additionally, a particle tracing numerical simulation was performed to achieve a better understanding of the macromolecular displacement in the H-cell microchannels. It was found that particle transfer between the two channels tends to speed up at low ionic strength and high concentration gradient. This confirms the corresponding experimental observation of protein diffusion measured via the H-cell microfluidics.     

Dispersion of protein solutions in short, open capillary tubes as a method for rapid molecular weight determination

A method has been developed by which the molecular weight of proteins and other freely diffusing species can be estimated on the basis of chromatographic peak shapes developed by injection of a sample into an open capillary tube in a liquid chromatography system. In chromatographic peaks obtained from such a system, there are contributions from both convection and diffusion. Thus, peak shape is dependent upon the diffusion coefficient of the molecular species, the flow rate, and the length of the capillary tube. In the work reported here it has been found that for samples of different proteins ranging from 2000 to 14,000 molecular weight, each injected at the same mobile phase flow rate, the ratio (R) of h1, the height of the peak primarily due to convection, to h2, the height of the "makeup" peak, primarily due to diffusion from the capillary wall, is a direct measure of protein molecular weight. Linear plots of R vs molecular weight are obtained under certain conditions.     

Pore network modelling of affinity chromatography: determination of the dynamic profiles of the pore diffusivity of beta-galactosidase and its effect on column performance as the loading of beta-galactosidase onto anti-beta-galactosidase varies with time.

A three-dimensional pore network model for diffusion in porous adsorbent particles was employed in a dynamic adsorption model that simulates the adsorption of a solute in porous particles packed in a chromatographic column. The solution of the combined model yielded the dynamic profiles of the pore diffusion coefficient of beta-galactosidase along the radius of porous adsorbent particles and along the length of the column as the loading of beta-galactosidase onto anti-beta-galactosidase immobilized on the surface of the pores of the particles occurred, and, the dynamic adsorptive capacity of the chromatographic column as a function of the design and operational parameters of the chromatographic system. It was found that for a given column length the dynamic profiles of the pore diffusion coefficient were influenced by (a) the superficial fluid velocity in the column, (b) the diameter of the adsorbent particles, and (c) the pore connectivity of the porous structure of the adsorbent particles. The effect of the magnitude of the pore connectivity on the dynamic profiles of the pore diffusion coefficient of beta-galactosidase increased as the diameter of the adsorbent particles and the superficial fluid velocity in the column increased. The dynamic adsorptive capacity of the column increased as (i) the particle diameter and the superficial fluid velocity in the column decreased, and (ii) the column length and the pore connectivity increased. In preparative affinity chromatography, it is desirable to obtain high throughputs within acceptable pressure gradients, and this may require the employment of larger diameter adsorbent particles. In such a case, longer column lengths satisfying acceptable pressure gradients with adsorbent particles having higher pore connectivity values could provide high dynamic adsorptive capacities. An alternative chromatographic system could be comprised of a long column packed with large particles which have fractal pores (fractal particles) that have high pore connectivities and which allow high intraparticle diffusional and convective flow mass transfer rates providing high throughputs and high dynamic adsorptive capacities. If large scale monoliths could be made to be reproducible and operationally stable, they could also offer an alternative mode of operation that could provide high throughputs and high dynamic adsorptive capacities.     

Hydrodynamics, mass transfer, and yeast culture performance of a column bioreactor with ejector

A bubble column fitted with an ejector has been tested for its physical and biological performance. The axial diffusion coefficient of the liquid phase in the presence of electrolytes and ethanol was measured by a stimulus-response technique with subsequent evaluation by means of a diffusion model. In contrast to ordinary bubble columns, the coefficient of axial mixing is inversely dependent on the superficial air velocity. The liquid velocity acts in an opposite direction to the backmixing flow in the column. The measurement of volumetric oxygen transfer coefficient in the presence of electrolytes and ethanol was performed using a dynamic gassing-in method adapted for a column. The data were correlated with the superficial air and liquid velocities, total power input, and power for aeration and mixing; the economy coefficient of oxygen transfer was used for finding an optimum ratio of power for aeration and pumping. Growth experiments with Candida utilis on ethanol confirmed some of the above results. Biomass productivity of 2.5 g L(-1) h(-1) testifies about a good transfer capability of the column. Columns fitted with pneumatic and/or hydraulic energy input may be promising for aerobic fermentations considering their mass transfer and mixing characteristics.     

Radiotracer measurements of protein mass transfer: kinetics in ion exchange media

We describe a method to measure protein mass transfer kinetics in ion exchange adsorbents for preparative chromatography based on the use of radioactively labeled protein. The method was developed and evaluated using lysozyme as a test protein with the three commercial strong-acid cation exchangers SP-Sepharose-FF, SP-Sepharose-XL, and S-HyperD. Iodination with 125I was used to label the protein, which was added in trace amounts (approximately 0.1%) to an unlabeled protein solution. The solution was recirculated through a shallow bed of the adsorbent particles and the radioactivity accumulated in the bed measured with a gamma-counter as a function of time. Radiotracer-based kinetics measurements were found to be in good agreement with results obtained with a conventional shallow-bed technique, provided that freshly labeled protein solutions were used. The method has advantages in terms of simplicity, ability to deal with adsorption from complex mixtures, and the potential for measurements under tracer diffusion conditions. Kinetics results obtained for the three different stationary phases were generally consistent with previous studies. Protein mass transfer can be described by a pore diffusion model with a nearly salt-independent pore diffusivity for SP-Sepharose-FF and by a homogeneous diffusion model with a saltindependent adsorbed phase diffusivity for S-HyperD. However, it appears that a more complex model, accounting for parallel pore and surface diffusion, is needed to describe protein mass transfer in SP-Sepharose-XL. The modeling results were found to be correlated with the apparent pore sizes determined by inverse SEC.     

Determination of pollutant diffusion coefficients in naturally formed biofilms using a single tube extractive membrane bioreactor

A novel technique has been used to determine the effective diffusion coefficients for 1,1,2-trichloroethane (TCE), a nonreacting tracer, in biofilms growing on the external surface of a silicone rubber membrane tube during degradation of 1,2-dichloroethane (DCE) by Xanthobacter autotrophicus GJ10 and monochlorobenzene (MCB) by Pseudomonas JS150. Experiments were carried out in a single tube extractive membrane bioreactor (STEMB), whose configuration makes it possible to measure the transmembrane flux of substrates. A video imaging technique (VIT) was employed for in situ biofilm thickness measurement and recording. Diffusion coefficients of TCE in the biofilms and TCE mass transfer coefficients in the liquid films adjacent to the biofilms were determined simultaneously using a resistances-in-series diffusion model. It was found that the flux and overall mass transfer coefficient of TCE decrease with increasing biofilm thickness, showing the importance of biofilm diffusion on the mass transfer process. Similar fluxes were observed for the nonreacting tracer (TCE) and the reactive substrates (MCB or DCE), suggesting that membrane-attached biofilm systems can be rate controlled primarily by substrate diffusion. The TCE diffusion coefficient in the JS150 biofilm appeared to be dependent on biofilm thickness, decreasing markedly for biofilm thicknesses of >1 mm. The values of the TCE diffusion coefficients in the JS150 biofilms <1-mm thick are approximately twice those in water and fall to around 30% of the water value for biofilms >1-mm thick. The TCE diffusion coefficients in the GJ10 biofilms were apparently constant at about the water value. The change in the diffusion coefficient for the JS150 biofilms is attributed to the influence of eddy diffusion and convective flow on transport in the thinner (<1-mm thick) biofilms.     

An investigation of bioreactor performance for the production of recombinant protein in immobilized and suspended Escherichia coli

The performance of packed bed (PBR) and modified bubble tank (MBTR) reactors was compared with respect to recombinant protein (β-galactosidase) production by suspended and immobilized E. coli. The MBTR was superior to the PBR due to easy operation and higher protein production. Gas-liquid mass transfer was not affected by the presence of gel beads, and there were no internal or external oxygen diffusion limitations in either reactor. High substrate concentration, small bead size, low cell densities, and similar values of effective diffusion coefficient of oxygen in water and in alginate may have decreased the internal mass transfer limitations.     

A study of mass transfer kinetics in an enantiomeric separation system using a polymeric imprinted stationary phase

Chromatographic data pertaining to the enantioseparation of L- and D-phenylalanine anilide (PA) on a polymeric stationary phase imprinted with L-PA were studied from the viewpoints of phase equilibrium, mass transfer kinetics, and the thermodynamic properties of this enantiomeric separation system. The concentration dependence of the lumped mass transfer rate coefficient (k(m,L)) previously published was analyzed to obtain new information concerning the mass transfer characteristics in this chiral separation system. It was shown that intraparticle diffusion contributed much more to k(m,L) than adsorption/desorption. The positive concentration dependence of k(m,L) seemed to be interpreted by considering that of the surface diffusion coefficient, itself explained by the heterogeneous surface model. The characteristic features of the phase equilibrium, the mass transfer kinetics, and the thermodynamics of the enantiomeric separation system probably result from the adsorption energy distribution on the surface of the imprinted phase having an exponential decay.     

732树脂吸附蛋白质的机理研究

通过树脂吸附水溶液中蛋白质的试验 ,研究了 732树脂对蛋白质吸附过程机理 ,初步分析了动力学行为 ,包括吸附等温线方程、吸附速率方程、总传质系数、树脂内的有效扩散系数等     

Mass transfer resistance of sterile plugs in shaking bioreactors

One of the mass transfer resistances for the gas exchange of shaking flasks is the sterile plug. The gas exchange through the sterile plug is described by an extended model of Henzler and Schedel [Bioprocess Eng. 7 (1991) 123]. Based on this model, a new method was developed to obtain the mass transfer resistance of various sterile closures. It consists of measuring the water evaporation rate of the shaking flask and is therefore very easily applied. Sterile plugs made of cotton, wrapped paper, urethane foam and fibreglass and caps made out of aluminium and silicone have been examined. Instead of the oxygen transfer coefficient (k(O(2))), which is commonly found in the literature, the carbon dioxide diffusion coefficient (D(CO(2))) is used to describe the mass transfer resistance of the sterile plug. The investigation revealed that this resistance is mainly dependent on the neck geometry and to a lesser extent on the plug material and density. The gas exchange of aluminium-caps was not reproducible.     

Parameter estimation of perillyl alcohol in RP-HPLC by moment analysis

Parameter estimations were made for the reversed-phase adsorption of perillyl alcohol (POH), a potent anti-cancer agent, on octadecylsilyl-silica gel (ODS). The average particle diameter of ODS was about 15 μm, and the particles were packed in the column (3.9 × 300 mm). The mobile phase used was a mixture of acetonitrile and water, in which the acetonitrile ranged between 50 and 70 (v/v %). The first absolute moment and the second central moment were determined from the chromatographic elution curves by moment analysis. Experiments were carried out using POH solutions within the linear adsorption range. The fluid-to-particle mass transfer coefficient was estimated using the Wilson-Geankoplis equation. The axial dispersion coefficient and the intraparticle diffusivity were determined from the slope and intercept of a plot ofH vs 1/u ₀, respectively. The contributions of each mass-transfer step were axial dispersion, fluid-toparticle mass transfer, and intraparticle diffusion.     

Identification of the mass transfer mechanisms involved in the transport of human immunoglobulin-G in N,N,N',N'-ethylenediaminetetramethylenephosphonic acid-modified zirconia

Zirconia particles modified with N,N,N',N'-ethylenediaminetetramethylenephosphonic acid (EDTPA), further referred to as r_PEZ, were studied as a support material for use in chromatography. Our previous studies have demonstrated the utility of r_PEZ in the separation of immunoglobulins from biological fluids. In the present study we sought to understand the underlying factors and identify the rate-limiting mechanisms that govern the transport of biomolecules in r_PEZ. Pulse injection techniques were used to elucidate the individual mass transfer parameters. Elution profiles obtained under retained and unretained conditions were approximated by the Gaussian equation and the corresponding HETP contributions were estimated. The dependence of the HETP values on incremental salt concentration in the mobile phase was determined. Resulting data in conjunction with the equations outlined in literature were used to estimate the theoretical number of transfer units for the chromatographic separation process. Our results indicate that surface diffusion probably plays a minor role; however pore diffusion was established to be the rate limiting mechanism for immunoglobulin G adsorption to r_PEZ. The HETP based methodology may be used to estimate the rate limiting mechanisms of mass transfer for any given chromatographic system under appropriate conditions.     

Binding capacity differences for antibodies and Fc-fusion proteins on protein A chromatographic materials

A range of studies were carried out to investigate the underlying reason for differences in dynamic binding capacities observed with various antibodies and Fc-fusion proteins during Protein A chromatography. Dynamic binding capacities were determined for these biomolecules using different protein A stationary phase materials. SEC was carried out to determine the relative sizes of the antibodies and fusion proteins. Pore diffusivities and static binding capacities were also determined on these Protein A resin materials. Trends in the dynamic binding capacities for these molecules did not correlate with differences in pore diffusion coefficients as might be expected for a mass transfer limited system. Instead, dynamic binding capacities were seen to follow the same trends as the static binding capacities and the apparent size of the molecules. Differences in static binding capacities were attributed to be due to differences in steric factor between the molecules. Solution binding stoichiometry studies were employed to estimate intra-Protein A steric effects while binding to the various domains within a Protein A ligand. In addition, steric hindrance was also found to exist between adjacent immobilized Protein A ligands on the chromatographic surface. The combination of intra and inter Protein A steric hindrances can explain differences in binding capacities observed between various antibody and Fc fusion proteins. The effect of Protein A ligand density on these supports was also examined and the results indicate that increasing Protein A ligand density leads to a situation of diminishing returns for binding capacity due to increased steric hindrance on the resin surface. The results presented in this paper show that steric hindrances can dominate over mass transfer effects in causing capacity variation between different molecules on the same stationary phase. This can lead to the development of more cost-efficient chromatographic stationary phases as well as provide information during the selection of Protein A media for preparative purification of monoclonal antibodies and Fc fusion proteins.     

Measurement of Gd-DTPA diffusion through PVA hydrogel using a novel magnetic resonance imaging method.

Polyvinyl alcohol-cryogel (PVA-C) is a hydrogel that is an excellent tissue mimic. In order to characterize mass transfer in this material, as well as to demonstrate in principle the ability to noninvasively measure solute diffusion in tissue, we measured the diffusion coefficient of the magnetic resonance (MR) contrast agent gadolinium diethylene triaminopentaacetic acid (Gd-DTPA) through PVA-C using a clinical MR imager. The method involved filling thick-walled rectangular PVA-C "cups" with known concentrations of Gd-DTPA solutions. Then by using a fast inversion recovery spin echo MR imaging protocol, a signal "null" contour was created in the MR image that corresponded to a second, known concentration of Gd-DTPA. By collecting a series of MR images through the PVA-C wall as a function of time, the displacement of this second known isoconcentration contour could be tracked. Application of Fick's second law of diffusion yielded the diffusion coefficient. Seven separate experiments were performed using various combinations of initial concentrations of Gd-DTPA within the PVA-C cups (3.2, 25.6, or 125 mM) and tracked isoconcentrations contours (0.096, 0.182, or 0.435 mM Gd-DTPA). The experimental results and the predictions of Fick's law were in excellent agreement. The diffusivity of Gd-DTPA through 10% PVA hydrogel was found to be (2.6 +/- 0.04) x 10(-10) m(2)/s (mean +/- s.e.m.). Separate permeability studies showed that the diffusion coefficient of Gd-DTPA through this hydrogel did not change with an applied pressure of up to 7.1 kPa. Accurate measurements could be made within 30 min if suitable Gd-DTPA concentrations were selected. Due to the excellent repeatability and fast data acquisition time, this technique is very promising for future in vivo studies of species transport in tissue.     

Pore‐network modeling of biofilm evolution in porous media

The influence of bacterial biomass on hydraulic properties of porous media (bioclogging) has been explored as a viable means for optimizing subsurface bioremediation and microbial enhanced oil recovery. In this study, we present a pore network simulator for modeling biofilm evolution in porous media including hydrodynamics and nutrient transport based on coupling of advection transport with Fickian diffusion and a reaction term to account for nutrient consumption. Biofilm has non‐zero permeability permitting liquid flow and transport through the biofilm itself. To handle simultaneous mass transfer in both liquid and biofilm in a pore element, a dual‐diffusion mass transfer model is introduced. The influence of nutrient limitation on predicted results is explored. Nutrient concentration in the network is affected by diffusion coefficient for nutrient transfer across biofilm (compared to water/water diffusion coefficient) under advection dominated transport, represented by mass transport Péclet number >1. The model correctly predicts a dependence of rate of biomass accumulation on inlet concentration. Poor network connectivity shows a significantly large reduction of permeability, for a small biomass pore volume. Biotechnol. Bioeng. 2011;108: 2413–2423. © 2011 Wiley Periodicals, Inc.     

Rapid assay for gamma-carboxyglutamic acid in urine and bone by precolumn derivatization and reversed-phase liquid chromatography

A reversed-phase high-performance liquid chromatographic assay for the analysis of gamma-carboxyglutamic acid (Gla) in urine and bone protein hydrolyzates is described. The method employs precolumn derivatization with o-phthalaldehyde and mercaptoethanol. Gla was quantified by reference to an internal standard (beta-carboxyaspartic acid). The "within-run" coefficient of variation of the assay for Gla in urine was between 2.1 and 3.4%, and that for bone protein hydrolyzates was 3.2%. The "between-run" coefficient of variation ranged from 4.1 to 5.5%. There was good agreement between the measurement of urinary Gla by high-performance liquid chromatography and amino acid analyzer. Free Gla could not be detected in serum.     

Self-interaction chromatography of proteins on a microfluidic monolith

A novel miniaturized system has been developed for measuring protein-protein interactions in solution with high efficiency and speed, and minimal use of protein. A chromatographic monolith synthesized in a capillary is used in the method to make interaction measurements by self-interaction chromatography (SIC) in a manner that, compared to column methods, is more efficient as well as more readily practicable even if only small amounts of protein are available. The microfluidic monolith requires much less protein for both column synthesis and the chromatographic measurements than a conventional SIC system, and in addition offers improved mass transfer and hence higher chromatographic efficiency than for previous SIC miniaturization systems. Protein self-interactions for catalase as a model protein, quantified by measurement of second virial coefficients, B(22), were determined by SIC and follow trends that are consistent with previously reported values. Different column derivatization conditions were studied in order to optimize the chromatographic behavior of the microfluidic system for SIC measurements. Chromatographic sensitivity can be further increased by using different column synthesis conditions.     

Targeted purification development enabled by computational biophysical modeling

Chromatographic and non‐chromatographic purification of biopharmaceuticals depend on the interactions between protein molecules and a solid–liquid interface. These interactions are dominated by the protein–surface properties, which are a function of protein sequence, structure, and dynamics. In addition, protein–surface properties are critical for in vivo recognition and activation, thus, purification strategies should strive to preserve structural integrity and retain desired pharmacological efficacy. Other factors such as surface diffusion, pore diffusion, and film mass transfer can impact chromatographic separation and resin design. The key factors that impact non‐chromatographic separations (e.g., solubility, ligand affinity, charges and hydrophobic clusters, and molecular dynamics) are readily amenable to computational modeling and can enhance the understanding of protein chromatographic. Previously published studies have used computational methods such as quantitative structure–activity relationship (QSAR) or quantitative structure–property relationship (QSPR) to identify and rank order affinity ligands based on their potential to effectively bind and separate a desired biopharmaceutical from host cell protein (HCP) and other impurities. The challenge in the application of such an approach is to discern key yet subtle differences in ligands and proteins that influence biologics purification. Using a relatively small molecular weight protein (insulin), this research overcame limitations of previous modeling efforts by utilizing atomic level detail for the modeling of protein–ligand interactions, effectively leveraging and extending previous research on drug target discovery. These principles were applied to the purification of different commercially available insulin variants. The ability of these computational models to correlate directionally with empirical observation is demonstrated for several insulin systems over a range of purification challenges including resolution of subtle product variants (amino acid misincorporations). Broader application of this methodology in bioprocess development may enhance and speed the development of a robust purification platform. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:154–164, 2015     

Modelling of the chromatographic separation of xylose-mannose in ion-exchange resin columns

A mathematical model was proposed for the chromatographic separation of xylose and mannose on an ion-exchange resin in the Pb form: dispersion in the mobile phase, external mass transfer around the particles and internal diffusion were taken into account. Small-scale experiments provided an evaluation of the different parameters. Dispersion in the mobile phase was found to be the predominant phenomenon. The Peclet numbers were calculated by identification in the Laplace domain of the elution profiles. Influence of temperature and initial concentration of the sample were studied.     

The hydrodynamics of a Graesser ("raining bucket") contactor with a reverse micellar phase

A variety of contactor types have been assessed for the liquid-liquid extraction of proteins using reversed micelles; however, many of these contactors suffer from drawbacks such as emulsion formation and poor mass transfer performance. In this study, a small (1.25 L) Graesser "raining bucket" contactor was assessed for use with this system since it has the potential to ameliorate many of these problems. The aim of the work was to evaluate the hydrodynamics of the contactor in order to use this information for future work on mass transfer performance. Hydrodynamic characteristics such as the axial mixing coefficient were determined by residence time distribution studies using a tracer injection method. The effect of rotor speed and flow rate of each phase on axial mixing was investigated, and as a result of its unusual structure, i.e., falling/rising sheet, the interfacial mass transfer area in the Graesser was determined by image analysis. It was found that rotor speed had more influence on the axial mixing coefficient in the aqueous phase than in the reverse micellar phase. The axial mixing coefficient in each phase increased by increasing the flow rate of the same phase. The images obtained in a dropping cell showed that under the conditions of this study (3 rpm, 22 degrees C), the bucket pours one phase through the other in the form of a curtain or sheet. A new image technique was developed to determine the interfacial area of both phases, and it was found that the specific area was 8.6 m(2)/m(3), which was higher than in a spray column but considerably lower than in a RDC or a Graesser run at high rotational speed (50 rpm) without the addition of a surfactant.