Biomedical subjects

Andrew L Zydney

Publications and source records attributed to Andrew L Zydney.

17 recordsLinked to original sources

PubMed2006

Ultrafiltration characteristics of pegylated proteins.

There is growing clinical interest in the use of pegylated recombinant proteins with enhanced stability, half-life, and bioavailability. The objective of this study was to develop a quantitative understanding of the ultrafiltration characteristics of a series of pegylated proteins with different degrees of pegylation. Sieving data were compared with available theoretical models and with corresponding results for the partition coefficient in size exclusion chromatography (SEC). The sieving coefficients of the pegylated proteins depended not only on the protein size and the total molecular weight of the polyethylene glycol (PEG) but also on the number of PEG chains. This is in sharp contrast to the partition coefficient in SEC, which was uniquely determined by the total molecular weight of the PEG and protein. This difference is due to the deformation and/or elongation of the PEG chains caused by the convective flow into the membrane pores, an effect that is not present in SEC. These results provide important insights into the transport and separation characteristics of pegylated proteins.

Chromatography, Gel↗

PubMed2005

Controlling protein transport in ultrafiltration using small charged ligands.

Previous studies have demonstrated that protein transport during ultrafiltration can be strongly influenced by solution pH and ionic strength. The objective of this study was to examine the possibility of controlling protein transmission using a small, highly charged ligand that selectively binds to the protein of interest. Experiments were performed using bovine serum albumin and the dye Cibacron Blue. Protein sieving data were obtained with essentially neutral and negatively charged versions of a composite regenerated cellulose membrane to examine the effects of electrostatic interactions. The addition of only 1 g/L of Cibacron Blue to an 8 g/L BSA solution reduced the BSA sieving coefficient through the negatively-charged membrane by more than two orders of magnitude, with this effect being largely eliminated at high salt and with the neutral membrane. Protein sieving data were in good agreement with model calculations based on the partitioning of a charged sphere in a charged pore accounting for the change in net protein charge due to ligand binding and the increase in solution ionic strength due to the free ligand in solution.

Cellulose↗

PubMed2005

Effect of peracetic acid reprocessing on the transport characteristics of polysulfone hemodialyzers.

Peracetic acid is used extensively for reprocessing hemodialyzers, despite several indications that reprocessing alters the dialyzer transport characteristics. The objective of this study was to obtain quantitative data for the effects of peracetic acid reprocessing on the clearance and sieving coefficients of urea, vitamin B12, and polydisperse dextrans using Fresenius F80A polysulfone dialyzers. Reprocessing restored the urea and vitamin B12 clearance to close to their original values. However, the reprocessed dialyzers had substantially lower clearance of the larger molecular weight dextrans, which was attributed to reductions in the effective pore size caused by residual plasma proteins within the membrane. Storage in peracetic acid provided some additional removal of residual proteins, although the clearance and sieving coefficients of the larger dextrans remained well below their original values. Peracetic acid caused no degradation of the membrane polymer, in sharp contrast to results obtained with bleach reprocessing.

Biocompatible Materials↗

PubMed2004

Optimization of ultrafiltration/diafiltration processes for partially bound impurities.

Ultrafiltration and diafiltration processes are used extensively for removal of a variety of small impurities from biological products. There has, however, been no experimental or theoretical analysis of the effects of impurity- product binding on the rate of impurity removal during these processes. Model calculations were performed to account for the effects of equilibrium binding between a small impurity and a large (retained) product on impurity clearance. Experiments were performed using D-tryptophan and bovine serum albumin as a model system. The results clearly demonstrate that binding interactions can dramatically reduce the rate of small impurity removal, leading to large increases in the required number of diavolumes. The optimal product concentration for performing the diafiltration shifts to lower product concentrations in the presence of strong binding interactions. Approximate analytical expressions for the impurity removal were developed which can provide a guide for the design and optimization of industrial ultrafiltration/diafiltration processes.

Complex Mixtures↗

PubMed2004

Retention of small charged impurities during ultrafiltration.

Ultrafiltration is used to remove small impurities from a variety of processing streams. However, the clearance of small charged impurities may be inadequate due to electrostatic exclusion by the charged ultrafiltration membranes, an effect that has been largely unappreciated. Ultrafiltration experiments were performed to evaluate the transmission of several model impurities with different electrical charge through ultrafiltration membranes having different surface charge characteristics. Highly charged impurities are strongly rejected by charged cellulose and polyethersulfone membranes even though these solutes are much smaller than the membrane pore size. These effects could be eliminated by using high ionic strength solutions to shield the electrostatic interactions. The sieving data are in good agreement with model calculations based on the partitioning of charged spheres into charged cylindrical pores. Guidelines are developed for estimating conditions needed to obtain effective removal of small charged impurities through charged ultrafiltration membranes.

Buffers↗

PubMed2004

Use of protein charge ladders to study electrostatic interactions during protein ultrafiltration.

Recent studies have demonstrated the importance of electrostatic interactions in membrane systems, but there is still controversy about the underlying phenomena. Protein charge ladders, consisting of a set of chemical derivatives of a given protein that differ by single charge groups, were used to quantify the electrostatic interactions during protein ultrafiltration. Myoglobin charge ladders were generated by acylation, with the different derivatives analyzed simultaneously by capillary electrophoresis. Filtration experiments were performed using polyethersulfone and composite regenerated cellulose membranes, with the membrane charge determined from the streaming potential. As expected, the rejection increased as the protein became more heavily charged due to the increase in electrostatic repulsion. However, the transmission of the weakly charged myoglobin species increased dramatically at very low ionic strength. This increase in transmission was attributed to a shift in pH within the pore caused by hydrogen ion partitioning into the charged membrane. The sieving data were in good agreement with theoretical calculations accounting for the effects of this pH shift on the electrostatic interactions.

Computer Simulation↗

PubMed2004

Effect of electrostatic, hydrodynamic, and Brownian forces on particle trajectories and sieving in normal flow filtration.

Particle deposition and fouling are critical factors governing the performance of microfiltration and ultrafiltration systems. Particle trajectories were evaluated by numerical integration of the Langevin equation, accounting for the combined effects of electrostatic repulsion, enhanced hydrodynamic drag, and Brownian diffusion. In the absence of Brownian forces, particles are unable to enter the membrane pores unless the drag associated with the filtration velocity can overcome the electrostatic repulsion. Brownian forces significantly alter this behavior, allowing some particles to enter the pore even at low filtration velocities. The average particle transmission, evaluated from the probability of having a particle enter the pore, increases with increasing filtration velocity due to the greater hydrodynamic drag force on the particle. These results provide important insights into particle behavior in membrane systems.

Journal Article↗

PubMed2003

Effect of membrane morphology on system capacity during normal flow microfiltration.

Understanding the effects of membrane fouling on system capacity is critical for the successful design and scale-up of microfiltration systems. The underlying morphology and structure of the microfiltration membrane can have a significant effect on system capacity by altering the rate and extent of fouling. Experimental data were obtained for system capacity during protein microfiltration using several model membranes with both homogeneous and composite structures. Data were compared with predictions of a new model that can account for both pore blockage and cake formation, and also includes the effects of membrane morphology on internal flow profiles within the membrane. Membranes with highly interconnected pores have a significantly higher capacity due to the reduction in flux decline arising from the fluid flow under and around any surface blockage. The model calculations are in good agreement with the flux decline data, allowing far more accurate predictions of system capacity than for the commonly used V(max) analysis.

Animals↗

PubMed2003

Separation of alpha-lactalbumin and beta-lactoglobulin using membrane ultrafiltration.

There is considerable commercial interest in the preparation of individual whey proteins as high-value food additives, nutraceuticals, and therapeutics. This study examined the use of membrane filtration for the separation of alpha-lactalbumin and beta-lactoglobulin. Stirred cell filtration experiments were performed using both cellulosic and polyethersulfone membranes to determine the optimal pH, ionic strength, and filtration conditions. Selectivities of greater than 55 could be achieved at pH 5.5 and 50 mM ionic strength using a 30-kD cellulose membrane. A diafiltration process was then designed for the protein separation. A 16-diavolume filtration yielded beta-lactoglobulin as the retentate product with a purification factor of 100 and recovery of 90%. The alpha-lactalbumin was recovered in the filtrate with a purification factor of more than 10 and nearly 99% yield. Model calculations were in good agreement with the experimental data.

Cellulose↗

PubMed2002

Application of a pore-blockage--cake-filtration model to protein fouling during microfiltration.

Although protein fouling is a critical factor governing the performance of microfiltration systems, there have been relatively few studies comparing the fouling behavior of different proteins. Flux-decline data were obtained for the filtration of bovine serum albumin, lysozyme, pepsin, immunoglobulin G, and myoglobin through polycarbonate track-etch membranes. The data were analyzed using a recently developed model that accounts for simultaneous pore blockage and cake formation. The model was in very good agreement with the data for all five proteins, demonstrating the general applicability of this new theoretical framework. The initial fouling due to pore blockage is directly related to the concentration of protein aggregates in solution, which was measured independently by quasi-elastic light scattering. The results provide important insights into the mechanisms of protein fouling during microfiltration.

Adsorption↗

PubMed2002

Affinity ultrafiltration: effects of ligand binding on selectivity and process optimization.

The effective design of affinity ultrafiltration processes using a selective macroligand requires a detailed understanding of the effects of ligand-binding interactions on product yield and purification. Theoretical calculations were performed to evaluate the performance of affinity diafiltration separations with both competitive and independent binding interactions for the product and impurity. The intrinsic selectivity for independent binding decreased during the diafiltration due to the increase in fractional impurity binding as the impurity is selectively removed. The opposite behavior was seen for competitive binding because the strongly bound product displaces the impurity from the binding sites. Purification-yield diagrams were used to examine the effects of affinity-ligand concentration and binding constants on the separation. Model calculations were in excellent agreement with experimental data for the separation of tryptophan isomers using bovine serum albumin as the steroselective macroligand. Simulations with a fixed number of diavolumes show a clear optimum in product yield and purification factor at an intermediate ligand concentration due to the competing effects of the intrinsic selectivity and the rate of impurity removal. These results provide an appropriate framework for the design and optimization of affinity ultrafiltration systems.

Binding, Competitive↗

PubMedDate not supplied

Separation of protein charge variants by ultrafiltration.

The removal of product variants that form during downstream processing remains a challenge in the purification of recombinant therapeutic proteins. We examined the feasibility of separating variants with slightly different net charge using high-performance membrane ultrafiltration. A myoglobin variant was formed by reaction of the lysine epsilon-amino group with succinic anhydride. Sieving data were obtained over a range of solution conditions using commercial polyethersulfone ultrafiltration membranes. Maximum selectivity of about 7-fold was obtained at very low conductivity due to the strong electrostatic repulsion of the more negatively charged variant. Protein separations were performed by diafiltration. A two-stage process generated solutions of the normal myoglobin (in the permeate) and the charge variant (in the retentate), both at greater than 9-fold purification and 90% yield. These results provide the first demonstration that membrane systems can be used to separate proteins that differ by only a single charged amino acid residue.

Complex Mixtures↗

PubMedDate not supplied

Clarification of yeast cell suspensions by depth filtration.

Depth filtration can be very attractive for initial clarification because of low capital costs and ease of operation. However, there is currently no fundamental understanding of the effects of the filter pore size and morphology on the overall capacity and filtrate quality. The objective of this study was to examine the flux, capacity, and filtrate turbidity of a series of depth filters with different pore size ratings and multilayer structures for the filtration of yeast cell suspensions. Data were analyzed using available fouling models to obtain insights into the flux decline mechanisms. Filters with small pore size provide high filtrate quality at low capacity, with the reverse being true for the larger pore sizes. The multilayer structure of commercial depth filters leads to improved performance, although the choice of layer properties is critical. The highest capacity was achieved using a multilayer filter in which the upper layer allows significant yeast cell penetration into the filter matrix but still protects the retentive layer that is needed for a high quality filtrate.

Cell Culture Techniques↗

PubMedDate not supplied

Effect of membrane charge on flow and protein transport during ultrafiltration.

Although several recent studies have demonstrated the importance of electrostatic interactions in ultrafiltration, there have been few quantitative studies of the effects of membrane charge density on protein transport and membrane hydraulic permeability. Data were obtained using a series of charge-modified cellulose membranes, with the surface charge density controlled by varying the extent of addition of a quaternary amine functionality. The membrane charge was evaluated from streaming potential measurements. Protein transmission decreased by a factor of 100 as the membrane zeta potential increased from 0.3 to 6.6 mV. The protein sieving data were in good agreement with a partitioning model accounting for electrostatic effects, while the hydraulic permeability data were consistent with a flow model accounting for the effects of counter-electroosmosis. The results provide the first quantitative analysis of the effects of membrane charge density on the performance of ultrafiltration membranes.

Cytochromes c↗

PubMedDate not supplied

Protein fouling of virus filtration membranes: effects of membrane orientation and operating conditions.

The capacity of virus filters used in the purification of therapeutic proteins is determined by the rate and extent of membrane fouling. Current virus filtration membranes have a complex multilayer structure that can be used with either the skin-side up or with the skin-side facing away from the feed, but there is currently no quantitative understanding of the effects of membrane orientation or operating conditions on the filtration performance. Experiments were performed using Millipore's Viresolve 180 membrane under both constant pressure and constant flux operation with sulfhydryl-modified BSA used as a model protein. The capacity with the skin-side up was greater during operation with constant flux and at low transmembrane pressures, with the flux decline or pressure rise due primarily to osmotic pressure effects. In contrast, data obtained with the skin-side down showed a slower, steady increase in total resistance with the cumulative filtrate volume, with minimal contribution from osmotic pressure. The capacity with the skin-side down was significantly greater than that with the skin-side up, reflecting the different fouling mechanisms in the different membrane orientations. These results provide important insights for the design and operation of virus filtration membranes.

Membranes, Artificial↗

PubMedDate not supplied

Effect of bleach reprocessing upon the clearance characteristics and surface charge of polysulfone hemodialyzers.

The effects of reprocessing upon hemodialysis have been debated since the introduction of this practice. The objective of this study was to develop a quantitative understanding of the effects of bleach reprocessing upon Fresenius polysulfone dialyzers. Data were obtained for the clearance of urea, vitamin B12, and polydisperse dextrans before and after reprocessing, both with and without exposure to plasma in a simulated dialysis session. Bleach reprocessing of the native dialyzer caused a significant increase in solute clearance because of the removal of polyvinylpyrrolidone from the membrane copolymer. This was confirmed by zeta potential measurements that showed a substantial increase in the negative charge on the membrane surface. Exposure to plasma proteins during hemodialysis effectively protected the dialyzer from chemical degradation by the bleach, with only small changes in solute clearance after the optimal reprocessing cycle. However, inadequate exposure to bleach was unable to restore the original solute clearance while over cleaning with bleach caused an increase in clearance. Considerable care must be taken in the proper design of reprocessing cycles for hemodialysis membranes.

Dextrans↗

PubMedDate not supplied

Phosphate clearance for bleach reprocessed polysulfone hemodialyzers: effects of electrostatic interactions.

Hyperphosphatemia is a major problem for hemodialysis patients due to inadequate phosphate clearance during dialysis. The objective of this study was to understand the effects of bleach reprocessing on phosphate clearance. Data were obtained using Fresenius polysulfone dialyzers exposed to bleach in an in vitro dialysis. The phosphate clearance decreased slightly after a single reprocessing cycle, with more than a 10% reduction observed after 15 cycles. Streaming potential measurements showed that bleach reprocessing caused an increase in the negative charge on the polysulfone membrane, suggesting that the change in phosphate clearance was due to an increase in electrostatic repulsion. The changes in clearance and surface charge were consistent with theoretical calculations of the effects of electrostatic interactions on solute diffusion. These results provide the first evidence that bleach reprocessing can reduce phosphate clearance due to electrostatic interactions between the phosphate anion and negatively charged dialysis membranes.

Equipment Reuse↗