Application of membrane-based preferential transport to whole broth processing

Preferential transport in adsorptive membranes can be used to selectively remove biochemicals directly from fermentation broths. During preferential transport, an adsorbing solute is selectively transported across the membrane while nonadsorbing solutes and cells are retained by the membrane. This technique was used to separate lysozyme directly from a feed containing lysozyme, myoglobin, and yeast cells. We found that because the oscillatory flows used in preferential transport involve strokes that are close to symmetric, they are very efficient in alleviating cake formation due to cell deposition on the membrane surface. Theoretical results suggest that, by optimizing process variables, preferential transport can lead to a continuous concentrated stream of the adsorbing protein. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 581-591, 1997.     

Continuous protein separations in a magnetically stabilized fluidized bed using nonmagnetic supports

Continuous protein separations were performed using a magnetically stabilized fluidized bed (MSFB) and a commercially available affinity adsorption resin that contained no magnetically susceptible material. These nonmagnetic materials can be stabilized at relatively low fields (<75 G requiring <30 W) if sufficient magnetically susceptible particles are also present in the stabilized bed. The minimum amount of magnetic particles necessary to stabilize the bed is as low as 20% by volume and is a function of various parameters including the size and density of both particles, the magnetic field strength, and the fluidization velocity. Advantages of these beds for performing separations include true continuous, countercurrent liquid-solids contact, mass-transfer efficiencies nearly equal to that of packed beds, and the ability of handle suspended cells or cell debris. A variety of commercially available affinity, ion-exchange, and adsorptive supports can be used in the bed for continuous separations; results are presented for the adsorption and recovery of lysozyme from an aqueous mixture of lysozyme and myoglobin using an affinity resin.     

Effect of low-molecular-weight proteins on protein (lysozyme) binding to isolated brush-border membranes of rat kidney

Filtered proteins including the low-molecular-weight protein lysozyme are reabsorbed by the proximal tubule via adsorptive endocytosis. This process starts with binding of the protein to the brush-border membrane. The binding of ¹²⁵I-labelled egg-white lysozyme (EC 3.2.1.17) to isolated brush-border membranes of rat kidney and the effect of several low-molecular weight proteins on that binding was determined. The Scatchard plot revealed a one-component binding type with a dissociation constant of 5.3 μM and 53.0 nmol/mg membrane protein for the number of binding sites. The binding of the cationic lysozyme was inhibited competitively by the addition of cationic cytochrome c to the incubation medium, while the neutral myoglobin had no effect. The anionic β-lactoglobulin A inhibited the lysozyme binding in a noncompetitive manner. These data suggest that the binding takes place between positively charged groups of the protein molecule and negative sites on the brush-border membrane, and, the competition between the cationic cytochrome c and the cationic lysozyme for the binding sites may be responsible for the inhibitory effect of cytochrome c on renal lysozyme reabsorption. The binding step at the brush-border membrane appears to be cation-selective.     

Partitioning behavior and enrichment of proteins with reversed micellar extraction: I. forward extraction of proteins from aqueous to reversed micellar phase

Protein extractions using aerosol OT (AOT)-isooctane reverse micelle solutions have been studied to explore the potential for separating and enriching proteins with the reversed micellar extraction. The effects of pH, ionic strength, and different cations of chlorides in a bulk aqueous phase and of AOT concentration in an organic phase on the partitioning of lysozyme and myoglobin and the solubilization of water are presented in detail. The extraction of lysozyme was affected by the concentration of potassium or barium but was almost independent of that of sodium or calcium, whose ionic diameter is smaller than that of potassium and barium. For the extraction of myoglobin, however, the effect of barium concentration was not appreciable. Lysozyme could be enriched into the reversed micellar phase up to 30 times the aqueous feed concentration. (c) 1993 John Wiley & Sons, Inc.     

Lysozyme sensitivity of pantothenate-deficient Lactobacillus plantarum grown with exogenous fatty acids.

A pantothenic acid deficiency in Lactobacillus plantarum reduces lipid synthesis, prevents normal uptake and retention of extracellular amino acids and markedly increases sensitivity of these cells to lysozyme induced lysis. Pantothenate-deficient cells provided with exogenous fatty acids synthesize additional lipids and express nearly normal solute transport activities. The present study has shown that such cells retain a heightened sensitivity to lysozyme induced lysis. These observations indicate that the lysozyme sensitivity of pantothenate-deficient cells is not produced as in indirect effect of membrane lipid depletion, but represents an independent consequence of pantothenate insufficiency.     

Integrated fragmentation of human IgG and purification of Fab using a reactant adsorptive membrane bioreactor separator system

This article discusses an integrated separation–reaction–separation scheme for producing Fab fragment directly from human immunoglobulin G (hIgG) present in serum feed. The novel reactant adsorptive membrane bioreactor separator (or RAMBS) system used in the current study consisted of a stack of microporous adsorptive membranes held within a temperature controlled module. The membrane stack, in the presence of salt, selectively and reversibly adsorbed hIgG by hydrophobic interaction while allowing most other serum proteins to flow through. The bound hIgG was then fragmented by pumping a solution of papain through the reactor at controlled temperature and flow rate. The salt concentration and pH for reaction and separation were systematically optimized using pure hIgG as reactant. The Fab fragment was separated from undigested hIgG and other byproducts such as Fc fragment based on their differences in hydrophobicity. Under optimal conditions, Fab was obtained in the reaction flow through while the other proteins remained bound to the membrane, these being subsequently eluted by lowering the salt concentration. The RAMBS system in addition to being convenient from process integration and intensification points of view also showed higher catalytic efficiency of papain in comparison to that in liquid phase reactions. Biotechnol. Bioeng. 2009; 104: 152–161 © 2009 Wiley Periodicals, Inc.     

Protein crystal growth. Growth kinetics for tetragonal lysozyme crystals

A method for immobilizing protein crystals has been devised for determining face growth rates, and used to investigate the growth kinetics of hen egg white lysozyme crystals. Growth rates were determined at 22 degrees C in 0.1 M sodium acetate, 5% NaCl, pH 4.0, on the visually identified (110) face of tetragonal lysozyme crystals. Protein concentrations ranged from 13 to 57 mg/ml (saturation concentration = 1.7 mg/ml). Growth rate data were fit to the equation R = kappa sigma ri, where R = rate in cm/s; kappa = constant; sigma i = solute growth interface supersaturation; and r = rate dependence upon super-saturation, with the result that kappa = 0.146 X 10(-8) cm/s and r = 2.0. A model of the growth process was developed and the experimental data were used to determine the relative roles of transport and interfacial kinetics in the growth of this crystal. Values for the width of the boundary layer delta, the interfacial concentration Ci, and growth rate R were determined. The model may be used to extrapolate to other growth conditions. The relative role of transport and interfacial kinetics can be expressed by the coefficient gamma = (CB - Ci)/(CB - Cs), when CB is the bulk concentration and Cs the saturation. Values for gamma were found to range from much less than 0.1 for submicron-size crystals to approximately 0.15 for cm sizes. The results indicate that attachment or surface effects are rate-limiting in lysozyme crystal growth in Earth's gravity because solutal convection always provides more transport of solute than can be accommodated by the interface. In order to grow such crystals under transport limiting conditions, it would be necessary to suppress this solutal convection.     

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.     

Influence of chemical chaperones on the properties of lysozyme and the reaction center protein from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The influence of three chemical chaperones: glycerol, 4-hexylresorcinol, and 5-methylresorcinol on the structure, equilibrium fluctuations, and functional activity of the hydrophilic enzyme lysozyme and the transmembrane reaction center (RC) protein from Rb. sphaeroides in a broad range of concentrations has been studied. The chosen chemical chaperones differ strongly in their structure and action on hydrophilic and membrane proteins. The influence of the chemical chaperones (except methylresorcinol) on the structure, dynamics, and functional properties of lysozyme and RC protein are well described in the framework of extended models of preferential hydration and preferential interaction of protein with a chemical chaperone. A molecule of hexylresorcinol consists of a hydrophobic (alkyl radical) and a hydrophilic (aromatic core) moieties; this provides for additional regulation of the functional activity of lysozyme and RC by hexylresorcinol. The influence of methylresorcinol on proteins differs from that of glycerol and hexylresorcinol. Methylresorcinol interacts with the surface of lysozyme directly, not via water hydrogen bonds. This leads to a decrease in the denaturation temperature and an increase in the amplitude of equilibrium fluctuations, allowing it to be a powerful activator. Methylresorcinol interacts with the membrane RC protein only by the condensation of hydration water, which is negligible in this case. Therefore, methylresorcinol does not affect the functional properties of the RC protein. It is concluded that different chaperones at the same concentration as well as one and the same chaperone at different concentrations produce protein 3D structures differing in dynamic and functional characteristics.     

Purification of lysozyme using ultrafiltration

This article examines the separation of lysozyme from chicken egg white by ultrafiltration with 25 kDa and 50 kDa MWCO polysulfone membranes. The effects of pH, system hydrodynamics, feed concentration, and transmembrane pressure on permeate flux, lysozyme transmission, purification factor, and productivity have been discussed. With both types of membranes, higher permeate flux and lysozyme transmission were observed at higher pH. Higher lysozyme purity was generally obtained with the 25 kDa MWCO membrane. Purity of lysozyme decreased when the feed concentration was increased. With the 50 kDa MWCO membrane permeate flux, productivity and the purity of lysozyme were found to increase with increase in transmembrane pressure. The possibility of using a two-step ultrafiltration process for achieving high productivity along with high purity of lysozyme was also investigated.     

Selective displacement chromatography of proteins

In contrast to high molecular weight polyelectrolyte displacers, the efficacy of low molecular weight displacers are dependent on both mobile phase salt and displacer concentration. This sensitivity to the operating conditions opens up the possibility of carrying out selective displacement where the product(s) of interest can be selectively displaced while the low affinity impurities can be desorbed in the induced salt gradient ahead of the displacement train, and the high affinity impurities either retained or desorbed in the displacer zone. This type of displacement combines the operational advantages of step gradient and the high resolution inherent in a true displacement process, in a single operation. Theoretical expressions are presented for establishing selective displacement operating conditions (initial salt concentration, displacer concentration) based on the Steric Mass Action parameters of the displacer and the linear Steric Mass Action parameters of the feed proteins. Experimental results are presented to elucidate the concept of selective displacement in both cation and anion exchange systems. A mixture of alpha-lactalbumin and beta-lactoglobulin A and B has been used for anion-exchange systems; a four-protein mixture consisting of ribonuclease B, bovine and horse heart cytochrome c, and lysozyme has been employed in cation exchange systems. This article also demonstrates that on-line monitoring can be readily employed for the selective displacement process, thus facilitating the scale-up and control of the process. This work sets the stage for the development of robust large scale high resolution separations using selective displacement chromatography. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 119-129, 1997.     

Effect of chemical chaperones on properties of lysozyme and the reaction center protein from Rhodobacter sphaeroides

The influence of three chemical chaperones: glycerol, 4-hexylresorcinol, and 5-methylresorcinol on the structure, equilibrium fluctuations, and the functional activity of the hydrophilic enzyme lysozyme and the transmembrane reaction center (RC) protein from Rb. sphaeroides in a broad range of concentrations has been studied. Selected chemical chaperones are strongly different by the structure and action on hydrophilic and membrane proteins. The influence of the chemical chaperones (except methylresorcinol) on the structure, dynamics, and functional properties of lysozyme and RC protein are well described within the frames of extended models of preferential hydration and preferential interaction of protein with a chemical chaperone. A molecule of hexylresorcinol consists of a hydrophobic (alkyl radical) and a hydrophilic (aromatic nuclus) moieties. This fact provides additional regulation of functional activity of lysozyme and RC by hexylresorcinol. The influence of methylresorcinol on proteins differs from that of glycerol and hexylresorcinol. Methylresorcinol interacts with the surface of lysozyme directly, not via water hydrogen bonds. This leads to a decrease in denaturation temperature T(d), and an increase in the amplitude of equilibrium fluctuation, which allows him to be a powerful activator. Methylresorcinol interacts with the membrane RC protein only by the condensation of hydration water, which is negligible in the case of methylresorcinol. Therefore, methylresorcinol does not effect the functional properties of the RC protein. It was concluded that various chaperones at one and the same concentration and chaperones at different concentrations form diverse 3D structures of proteins, which differ by dynamic and functional characteristics.     

Binding of 125I-labelled albumin by isolated rat renal brush-border membrane vesicles. Evidence for uptake and internalization process

1. In the kidney, filtered proteins are rapidly reabsorbed by the proximal tubule via adsorptive endocytosis. This process starts with the protein binding to the luminal brush-border membrane. 2. The binding of 125I-labelled albumin to rat renal brush-border membrane vesicles and the effect of a low molecular weight protein lysozyme on that binding was assessed by the filtration method. 3. The Scatchard plot revealed a one-component binding-type curve with a dissociation constant Kd of 430.9 nM and 39.6 pmol/mg membrane protein for the number of binding sites. 4. Albumin binding was saturable and reversible, time and temperature dependent and the initial rate enhanced by increasing amounts of lysozyme. 5. The fact that association of albumin with the brush-border membrane vesicles was dependent upon the intravesicular space suggested a double process, binding of the ligand to the membrane surface and its internalization. These data suggest that albumin has a different binding site than that of a low-molecular weight protein lysozyme, with a constant affinity value near physiological loads. That specificity may confer selectivity upon the endocytic uptake process.     

Interactions of lysozyme with Pb(II) using viscosimetry and dilatometry of the equilibrium of dialysis

The conformational hen white egg lysozyme in the binary mixture water/Pb2+ as a function of the binary mixture composition has been studied. Intrinsic viscosity, partial specific volume and preferential adsorption parameter in the 0-0.100 M Pb2+ concentration range has been experimentally measured. The obtained results suggest that the observed conformational transition is a consequence of the interactions established between lysozyme and Pb2+ cation.     

Preparative-scale amino acid separation by thermal parametric pumping on an ion-exchange resin

Thermal parametric pumping was experimentally investigated for the concentration and separation of amino acids. Previous theories of parametric pumping were improved by taking into account dissociation equilibria in the liquid phase. Experiments were carried out with a mixture of glutamic acid, aspartic acid, serine and threonine in a highly acidic solution (HCl). A multi-component equilibrium model was mainy used to simulate the experimental results and to investigate the effect of chloride concentration over a wide range. It is shown that it is always possible to concentrate the amino acids and to separate some of them under certain conditions.     

Optimized recovery of monoclonal antibodies from transgenic goat milk by microfiltration

The Predictive Aggregate Transport Model for microfiltration is used in combination with optimum fluid mechanics and electrostatics to maximize recovery of a heterologous immunoglobulin (IgG) from transgenic goat milk. The optimization algorithm involved varying pH (6.8-9), transmembrane pressure (2-4.5 psi), milk feed concentration (1-2X), membrane module type (linear vs. helical design), and axial velocity (Reynolds number: 830-1170). Operation in the pressure-dependent regime at low uniform transmembrane pressures (approximately 2 psi) using permeate circulation in co-flow, at the pI of the protein (9 in this case) was used to increase IgG recovery from less than 1% to over 95%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and attenuated total reflection Fourier transform infrared spectroscopy of the microfiltration permeate samples confirmed that all the fat globules and most of the casein micelles were retained in the MF membrane whereas a large amount of the target IgG was transported through the membrane. Transmembrane pressure and hence permeation flux was kept low (approximately 15 lmh) to maximize IgG membrane transport and thus recovery, due to a sparse deposit on the membrane which facilitated high solute transport. Next, an analytical method was used to optimize the diafiltration process using the aggregate transport model, experimental target protein sieving coefficients and permeation flux (Baruah and Belfort, 2003). The methodology reported here should be generalizable to the recovery of target proteins found in other complex suspensions of biological origin using the microfiltration process.     

Anesthetic stabilization of protein intermediates: myoglobin and halothane

Halothane, an inhaled anesthetic, destabilizes the folded structure of myoglobin. To determine whether this is due to preferential interactions with less stable folded conformers of myoglobin versus the completely unfolded state, we used photoaffinity labeling, hydrogen exchange, fluorescence spectroscopy, and circular dichroism spectroscopy. Apomyoglobin was used as a model of a less stable conformer of myoglobin. Halothane destabilizes myoglobin and binds with low affinity and stoichiometry but stabilizes and binds with higher affinity to apomyoglobin. The same halothane concentration has no effect on cytochrome c stability. The apomyoglobin/halothane complex is favored at pH 6.5 as compared to pH 4.5 or pH 2.5. Halothane photoincorporates into several sites in apomyoglobin, some allosteric to the heme pocket. Guanidinium unfolding of myoglobin, monitored by CD spectroscopy, shows destabilization at less than 1.3 M Gdm but stabilization at greater than 1.3 M Gdm, consistent with the hypothesis that less stable conformers of myoglobin bind halothane preferentially. We suggest the structural feature underlying preferential binding to less stable conformers is an enlarged cavity volume distribution, since myoglobin has several intermediate-sized cavities, while cytochrome c is more well packed and has no cavities detected by GRASP. Specific binding to less stable intermediates may underlie anesthetic potentiation of protein activity.     

Passive Asymmetric Transport through Biological Membranes

The magnitude of passive diffusional solute transfer through artificial membranes is usually considered to be independent of the direction of the concentration gradient driving force. It can be shown, however, that a composite membrane, having as one component a membrane with a chemical reaction-facilitated diffusion transport mechanism, can result in an asymmetrical flux. An asymmetric flux caused by this type of structural heterogeneity may be one mechanism contributing to the asymmetric properties of biological membranes. Similar vectorial fluxes can be generated in interfacial solute transfer through membranes if hydrodynamic boundary layers occur at the membrane interface and reversible chemical reactions with the permeant species are involved in either phase.     

On-line recovery of large molecules and concentration of small molecules using reciprocating size exclusion chromatography with temperature swing

The reciprocating size exclusion chromatography (RSEC) was operated with swing between two temperatures in a synchronous way with flow direction to recover a large solute on-line from the mixture, in addition to the small solute concentration. The concentration of small solutes in RSEC with a temperature swing was made possible by taking advantage of the temperature-dependent swelling properties of the porous gel. After 7 cycles of frontal mode operation, 76% of Blue Dextran in the feed was recovered and nickel nitrate in the feed reservoir was concentrated by 13%.     

Validation of theoretical framework explaining active solute uptake in dynamically loaded porous media

Solute transport in biological tissues is a fundamental process necessary for cell metabolism. In connective soft tissues, such as articular cartilage, cells are embedded within a dense extracellular matrix that hinders the transport of solutes. However, according to a recent theoretical study (Mauck et al., 2003, J. Biomech. Eng. 125, 602–614), the convective motion of a dynamically loaded porous solid matrix can also impart momentum to solutes, pumping them into the tissue and giving rise to concentrations which exceed those achived under passive diffusion alone. In this study, the theoretical predictions of this model are verified against experimental measurements. The mechanical and transport properties of an agarose–dextran model system were characterized from independent measurements and substituted into the theory to predict solute uptake or desorption under dynamic mechanical loading for various agarose concentrations and dextran molecular weights, as well as different boundary and initial conditions. In every tested case, agreement was observed between experiments and theoretical predictions as assessed by coefficients of determination ranging from R²=0.61 to 0.95. These results provide strong support for the hypothesis that dynamic loading of a deformable porous tissue can produce active transport of solutes via a pumping mechanisms mediated by momentum exchange between the solute and solid matrix.