Small molecule clearance in ultrafiltration/diafiltration in relation to protein interactions:Study of citrate binding to a Fab.

Ultrafiltration/diafiltration (UFDF) is commonly utilized in the purification of recombinant proteins to concentrate and buffer exchange the product. It is often the final step in the purification process, placing the protein in its final formulation and clearing small molecules introduced in upstream purification steps. This article presents a case study of reduced small molecule clearance in ultrafiltration/diafiltration of an antigen-binding fragment of a monoclonal antibody. Citrate, a commonly utilized small molecule in downstream processes, is shown to have reduced clearance due to specific interactions with the protein product. The study presents process solutions and utilizes a simple model to characterize clearance of small molecules which exhibit interactions with product protein.

Water permeability of high-flux dialyzer membranes after Renalin reprocessing.

Dialysis with high-flux membranes is widely used, in part, because they are thought to increase the removal of middle molecules when compared with low-flux membranes. Dialyzer reprocessing; however, is thought to alter middle molecule clearance. Renalin, a mixture of germicidal agents, has widespread use in dialyzer reprocessing. We determined the effect of Renalin reprocessing on the water permeability of three different dialyzers of Fresenius (F80A and 200A) and Gambro (17R) manufacture using the dead-end filtration method. Two hundred and seventeen, predominantly used but some new, dialyzers were evaluated. Water permeability of the used, but not the new, dialyzers fell abruptly and dramatically with reprocessing. The permeability fell almost 70% in the F80A dialyzer after three reprocessing procedures with similar, but somewhat slower declines, seen in the other two dialyzers. We conclude that there is a decline in water permeability seen in Renalin reprocessed dialyzers. This factor and the associated change in solute clearance and ultrafiltration characteristics should be considered in assessing the effectiveness of dialyzer reprocessing.

Determination of effective protein charge by capillary electrophoresis: effects of charge regulation in the analysis of charge ladders.

Protein charge ladders are an effective tool for measuring protein charge and studying electrostatic interactions. However, previous analyses have neglected the effects of charge regulation, the alteration in the extent of amino acid ionization associated with differences between the pH at the protein surface and in the bulk solution. Experimental data were obtained with charge ladders constructed from bovine carbonic anhydrase. The protein charge for each element in the ladder was calculated from the protein electrophoretic mobility as measured by capillary electrophoresis using the hindrance factor for a hard sphere with equivalent hydrodynamic radius. The protein charge was also evaluated theoretically from the amino acid sequence by assuming a Boltzmann distribution in the hydrogen ion concentration. The calculations were in excellent agreement with the data, demonstrating the importance of charge regulation on the net protein charge. These results have important implications for the use of charge ladders to evaluate effective protein charge in solution.

Effect of ion binding on protein transport through ultrafiltration membranes.

Electrostatic interactions can have a significant impact on protein transmission through semipermeable membranes. Experimental data for the transport of bovine serum albumin (BSA) through a polyethersulfone ultrafiltration membrane were obtained in different salt solutions over a range of pH and salt concentrations. Net BSA charge under the same conditions was evaluated from mobility data measured by capillary electrophoresis. The results show that specific ionic composition, in addition to solution pH and ionic strength, can strongly affect the rate of protein transport through semipermeable ultrafiltration membranes. The effects of different ions on BSA sieving are due primarily to differences in ion binding to the protein, which leads to significant differences in the net protein charge at a given pH and ionic strength. This effect could be described in terms of an effective protein radius, which accounts for the electrostatic exclusion of the charged protein from the membrane pores. These results provide important insights into the nature of the electrostatic interactions in membrane systems.

Protein-membrane interactions during hemodialysis: effects on solute transport.

Although several previous studies have shown that plasma protein adsorption can reduce solute clearance during hemodialysis, there is currently no quantitative understanding of the factors that govern the extent of these protein-membrane interactions. In this study, quantitative data were obtained for the clearance of urea, vitamin B12, and polydisperse dextrans using polyacrylonitrile (AN69) and cellulose triacetate dialyzers before and after exposure to human plasma in a simulated dialysis session. Contact with plasma had little effect on clearance of urea and vitamin B12, but caused more than an order of magnitude reduction in clearance for solutes with molecular weights > 10,000. These data were analyzed using a two layer model in which contact with plasma was assumed to cause a thin protein layer to form on the surface of the membrane. The protein layer had an effective pore size of approximately 12 A, and was approximately 1 microm thick, as determined by a hydrodynamic analysis of the clearance data, and from independent estimates based on changes in fiber bundle volume and ultrafiltration coefficient. The thickness of the protein layer increased with increasing dialysis time, ranging from 0.25 microm after 40 min to 0.86 microm after 180 min. These results provide important insights into the effects of contact with plasma on solute clearance during hemodialysis.

Solute washout experiments for characterizing mass transport in hollow fiber immunoisolation membranes.

The transport characteristics of immunoisolation membranes can have a critical effect on the design of hybrid artificial organs and cell therapies. However, it has been difficult to quantitatively evaluate the desired transport properties of different hollow fiber membranes due to bulk mass transfer limitations in the fiber lumen and annular space. An attractive alternative to existing methodologies is to use the rate of solute removal or "washout" from the annular space during constant flow perfusion through the fiber lumen. Experimental washout curves were obtained for glucose and a 10 kD dextran in two different hollow fiber devices. Data were analyzed using a theoretical model which accounts for convective and diffusive transport in the lumen, membrane, and annular space. The model was in good agreement with the experimental results and provided an accurate measure of the effective membrane diffusion coefficient for both small and large solutes. This approach should prove useful in theoretical analyses of solute transport and performance of hollow fiber artificial organs.

Measurement of protein charge and ion binding using capillary electrophoresis.

A new technique is described for the rapid and accurate measurement of electrophoretic mobilities of proteins in different solution environments using capillary electrophoresis. Data were obtained at different pH using surface-modified capillaries to reduce nonspecific protein adsorption and using hydrodynamic mobilization to improve reproducibility and overall accuracy. The net protein charge and extent of anion binding were evaluated from the mobility data obtained in different pH and ionic environments for bovine serum albumin. The results were in good agreement with titration data obtained using ion-selective electrodes and mobility data obtained using free solution electrophoresis. The method requires extremely small amounts of protein (picogram quantities and nanoliter volumes) and is easily automated, making it very suitable for protein characterization and for initial screening of possible separation techniques.

Electrostatic effects on protein partitioning in size-exclusion chromatography and membrane ultrafiltration.

Although size-exclusion chromatography and membrane ultrafiltration are generally viewed as size-based separation processes, there is considerable evidence for the importance of electrostatic interactions. Experimental studies of size-exclusion chromatography and membrane ultrafiltration were performed in parallel using both neutral dextrans and charged proteins. Data for protein retention time and membrane sieving clearly indicate that the effective protein size increases with decreasing ionic strength due to the reduction in electrostatic shielding. These results were quantified using available theoretical models for the partitioning of charged solutes. The data clearly demonstrate the similarity of the electrostatic interactions and partitioning effects in size-exclusion chromatography and membrane ultrafiltration.

Analysis of protein fouling during ultrafiltration using a two-layer membrane model.

Protein fouling can significantly alter both the flux and retention characteristics of ultrafiltration membranes. There has, however, been considerable controversy over the nature of this fouling layer. In this study, hydraulic permeability and dextran sieving data were obtained both before and after albumin adsorption and/or filtration using polyethersulfone ultrafiltration membranes. The dextran molecular weight distributions were analyzed by gel permeation chromatography to evaluate the sieving characteristics over a broad range of solute size. Protein fouling caused a significant reduction in the dextran sieving coefficients, with very different effects seen for the diffusive and convective contributions to dextran transport. The changes in dextran sieving coefficients and diffusive permeabilities were analyzed using a two-layer membrane model in which a distinct protein layer is assumed to form on the upstream surface of the membrane. The data suggest that the protein layer formed during filtration was more tightly packed than that formed by simple static adsorption. Hydrodynamic calculations indicated that the pore size of the protein layer remained relatively constant throughout the adsorption or filtration, but the thickness of this layer increased with increasing exposure time. These results provide important insights into the nature of protein fouling during ultrafiltration and its effects on membrane transport.

Protein fouling during microfiltration: comparative behavior of different model proteins.

Recent studies of protein fouling have provided considerable insight into both the underlying fouling mechanisms and the mathematical description of the flux decline. However, most of the data have been obtained with a single model protein, making it difficult to generalize the results to commercially relevant process streams. Experiments were thus performed using a range of proteins with different physicochemical characteristics to determine the relationship between the protein structure and fouling behavior. Fouling in these systems occurred by two distinct mechanisms: deposition of large protein aggregates and chemical attachment of native proteins to the growing deposit. The chemical attachment generally occurred via the formation of intermolecular disulfide linkages involving a free sulfhydryl group in the native protein. Proteins without a free sulfhydryl group were typically unable to form these intermolecular linkages. The quasi-steady flux for the different proteins was proportional to the square of the protein surface charge density, consistent with a model in which protein deposition occurs when the drag force on the proteins associated with the convective filtrate flow is sufficient to overcome electrostatic repulsive interactions. These results clearly demonstrate the importance of the protein structure, charge, and reactivity in determining the rate and extent of protein fouling during microfiltration.

High performance tangential flow filtration.

Conventional tangential flow filtration (TFF) has traditionally been limited to separation of solutes that differ by about ten-fold in size. Wide pore-size distributions, membrane fouling, and concentration polarization phenomena have commonly been cited as reasons for this limitation. The use of TFF in the biotechnology industry has therefore been restricted to cell-protein, virus-protein, and protein-buffer separations. A multi-disciplinary team with industrial and academic members was formed to overcome these limitations and enable protein-protein separations using High Performance TFF (HPTFF) systems. Pore-size distributions have been improved with the development of new membrane formulation and casting techniques. Membrane fouling has been controlled by operating in the transmembrane pressure-dependent regime of the filtrate flux curve and by carefully controlling fluid dynamic start-up conditions. Concentration polarization was exploited to enhance, rather than limit, the resolution of solutes. Concentration polarization has also been controlled by operating a co-current filtrate stream that maintains transmembrane pressure constant along the length of the TFF module. High yields and purification factors were obtained even with small differences in protein sieving. IgG-BSA and BSA monomer-oligomer mixtures have successfully been separated with these systems. HPTFF technology provides a competitive purification tool to complement chromatographic processing of proteins.

Protein fractionation using electrostatic interactions in membranel filtration.

One of the critical factors limiting the development of membrane systems for protein fractionation has been the poor selectivity that has generally been obtained with these membrane devices. We have demonstrated that it is possible to dramatically improve the selectivity of available membrane systems by exploiting the different electrostatic interactions between the two proteins and the membrane. The separation factor for the albumin-hemoglobin system could be increased to more than 70 simply by reducing the salt concentration and adjusting the pH to around 7 (near the isoelectric point of hemoglobin). This very high selectivity was a direct result of the strong electrostatic exclusion of the charged albumin from the membrane pores under these conditions. This high selectivity makes it possible to very effectively separate these albumin-hemoglobin mixtures using membrane filtration, and this was demonstrated experimentally using both a simple batch filtration process and a continuous diafiltration system. The hemoglobin recovery in the diafiltration experiment was greater than 70% after a 3-diavolume filtration, with the Hb purification factor being around 100 under these conditions. These results clearly demonstrate the potential of membrane systems for the fractionation of proteins even with very similar molecular weights.

Effects of intermolecular thiol-disulfide interchange reactions on bsa fouling during microfiltration.

Several studies have shown that one of the critical factors governing protein fouling of microfiltration membranes is the presence of denaturedand/or aggregated protein in the bulk solutions. Experiments were performed to evaluate the role of intermolecular disulfide interchange reactionson protein aggregation and membrane fouling during stirred cell microfiltration of bovine serum albumin (BSA). The flux decline during BSA filtration was quite dramatic due to the formation of a protein deposit thatfully covered the membrane pores. This flux decline could be completely eliminated by capping the free sulfhydryl group present on the BSA with eithera carboxymethyl or cysteinyl group, demonstrating the critical importance of this free thiol in the intermolecular aggregation reactions and, in turn, protein fouling. BSA aggregation during storage could be reduced by the addition of metal chelators (EDTA and citrate) or dithiothreitol, orby storage at lower pH (7.0) these solutions all had a significantly lower rate of fouling upon subsequent filtration. This behavior is completely consistent with the known chemistry of the thiol-disulfide interchange reaction, demonstrating that an understanding of these intermolecular (aggregation) reactions can provide a rational framework for the analysis and control of protein fouling in these membrane systems. (c) 1994 John Wiley & Sons, Inc.

A two layer model for the effects of blood contact on membrane transport in artificial organs.

The performance of many artificial organs can be strongly affected by the transport characteristics of the semi-permeable membranes used in these devices, but there is little data on the effects of blood contact on membrane transport properties. Experimental data were obtained for the solute flux through cellulosic, polyacrylonitrile, and polyethersulfone membranes using polydispersed dextrans. Blood contact had a very large effect on diffusive solute transport through the asymmetric polyethersulfone membranes, but only a small effect on diffusion through the symmetric AN69 and Cuprophan membranes. In contrast, blood contact caused a similar reduction in convective solute transport (sieving) through both the polyethersulfone and AN69 membranes. Convective transport through the blood contacted membranes was also dependent on the flow direction, with greater transport obtained when the membrane was oriented with the blood contacted surface downstream. These data were analyzed using a two layer membrane model consisting of an upper layer of blood cells and proteins adsorbed to the surface of the native membrane. This model accurately accounted for the different effects of blood contact on convection and diffusion, as well as the observed asymmetry in convective solute transport. These results have important implications for the analysis of solute transport in artificial organs.

Diffusive and convective solute transport through hemodialysis membranes: a hydrodynamic analysis.

Recent clinical studies have shown that the overall effectiveness of hemodialysis is determined by both the convective and diffusive transport of a wide range of different molecular weight solutes. In this study, transport data were obtained for vitamin B12 and for polydisperse dextrans with a wide range of molecular weights using flat sheet Cuprophan and AN69 polyacrylonitrile membranes. The flux dependence of the actual sieving coefficient was described using classical membrane transport theory, allowing accurate measurements of both the diffusive and convective contributions to the overall solute transport through the porous structure of these dialysis membranes. Asymptotic membrane sieving coefficients and hindered diffusivities were in good agreement with a hydrodynamic model that accounts for the membrane pore size distribution through an expression for the solute partition coefficient in a random porous medium. This model provides an accurate quantitative description of both solute diffusion and convection through hemodialysis membranes, which is critical for the effective design and operation of hemodialyzers.

Effect of solution pH and ionic strength on the separation of albumin from immunoglobulins (IgG) by selective filtration.

Although protein fractionation by selective membrane filtration has numerous potential applications in both the downstream processing of fermentation broths and the purification of plasma proteins, the selectivity for proteins with only moderately different molecular weights has generally been quite poor. We have obtained experimental data for the transport of bovine serum albumin (BSA) and immunoglobulins (IgG) through 100,000 and 300,000 molecular weight cutoff polyethersulfone membranes in a stirred ultrafiltration device at different solution pH and ionic strength. The selectivity was a complex function of the flux due to the simultaneous convective and diffusive solute transport through the membrane and the bulk mass transfer limitations in the stirred cell. Under phsioligical conditions (pH 7.0 and 0.15 M NaCI) the maximum selectivity for the BSA-IgG separation was only about 2.0 due primarily to the effects of protein adsorption. In contrast, BSA-IgG selectivities as high as 50 were obtained with the same membranes when the protein solution was at pH 4.8 and 0.0015 M NaCl. This enhanced selectivity was a direct result of the electrosatatic contributions to both bulk and membrane transport. The membrane selectivity could actually be reversed, with higher passage of the larger IgG molecules, by using a 300,000 molecular weight cutoff membrane at pH 7.4 and an ionic strength of 0.0015 M NaCl. These results clearly demonstrate that the effectiveness of selective protein filtration can be dramatically altered by appropriately controlling electrostatic interactions through changes in pH and/or ionic strength. (c) 1994 John Wiley & Sons, Inc.

Intermolecular electrostatic interactions and their effect on flux and protein deposition during protein filtration.

Although membrane filtration is used extensively to process protein solutions containing a variety of electrolytes, there is currently little fundamental understanding of the effect of the solution environment (and in particular, the solution pH) on the filtrate flux in these systems. We have obtained data for the flux and sieving coefficients during the batch (stirred cell) filtration of solutions of bovine serum albumin, immunoglobulins, hemoglobin, ribonuclease A, and lysozyme through 0.16-micron microfiltration membranes at different pH values. The flux declined significantly for all five proteins due to the formation of a protein deposit on the upper surface of the membrane. The quasi-steady ultrafiltrate fluxes at the individual protein isoelectric pH's were essentially identical, despite the large differences in molecular weight and physicochemical characteristics of these proteins. The flux increased at pH's away from the isoelectric point, with the data well-correlated with the protein surface charge density. These results were explained in terms of a simple physical model in which the protein deposit continues to grow, and thus the flux continues to decline, until the drag force on the proteins associated with the filtrate flow is no longer able to overcome the intermolecular repulsive interactions between the proteins in the bulk solution and those in the protein deposit on the surface of the membrane.

Effect of blood contact on the transport properties of hemodialysis membranes: a two-layer membrane model.

The effects of blood-membrane interactions on hydraulic permeability, sieving coefficients, and diffusive permeabilities of Cuprophan and AN69 polyacrylonitrile hemodialysis membranes were studied using polydisperse dextrans (MW 5,000-20,000). Blood contact had no effect on the transport characteristics of the Cuprophan, but it caused a significant reduction in all of the transport parameters for the AN69 membrane due to the formation of a layer of adsorbed plasma proteins on the upper surface of the membrane. The transport characteristics of the membrane after exposure to blood are described using a two-layer membrane model, with this model able to accurately account for the different effects of blood contact on the sieving coefficients and diffusive permeabilities as well as the observed asymmetry in solute transport through the blood-contacted membrane. These results have important implications for solute transport during clinical hemodialysis.

Effect of solution environment on the permeability of red blood cells.

The cell membrane permeability governs the rate of solute transport into and out of the cell, significantly affecting the cell's metabolic processes, viability, and potential usefulness in both biotechnological applications and physiological systems. Most previous studies of the cell membrane permeability have neglected the possible effects of suspending medium on membrane transport, even though there is extensive experimental evidence that suspending phase composition can significantly affect other properties related to the cell membrane (e.g., cell deformability, fragility, and aggregation rate). This study examined the effects of suspending phase composition (both proteins and electrolytes) on the permeability of human red blood cells to the metabolites creatinine and uric acid. Data were obtained using a stirred ultrafiltration device with direct cell- and proteinfree sampling through a semipermeable membrane. Both the uric acid and creatinine permeabilities were strongly affected by the suspending phase composition, with the permeabilities in different buffer solutions varying by as much as a factor of three. The predominant factors affecting the permeability were the presence (or absence) of chloride, phosphate/adenine, and proteins, although the magnitude and even the direction of these effects were significantly different for creatinine and uric acid transport. The dramatic differences in behavior for uric acid and creatinine reflect the different transport mechanisms for these solutes, with uric acid transported by a carrier-mediated mechanism and creatinine transported by passive diffusion through the lipid bilayer. These results provide important insights into the effects of solution environment on cell membrane transport and other cell membrane-mediated properties.