Effect of protein adsorption on the transport characteristics of asymmetric ultrafiltration membranes.

The effects of bovine serum albumin adsorption on the transport characteristics of asymmetric poly(ether sulfone) ultrafiltration membranes were determined using polydisperse dextrans with gel permeation chromatography. Actual dextran sieving coefficients were evaluated from observed sieving data for both the clean and preadsorbed membranes using a stagnant film model. The flux dependence of the actual dextran sieving coefficients was used to evaluate the intrinsic membrane hindrance factors for convective (i.e., sieving) and diffusive transport for the different molecular weight dextrans using classical membrane transport theory. Protein adsorption caused a reduction in both dextran sieving and diffusion, with the magnitude of the reduction a function of the dextran molecular weight and pore size. The effects of adsorption on the specific pore area and the membrane porosity were then determined using a recent model for solute transport through asymmetric ultrafiltration membranes. The data indicate that protein adsorption occurs preferentially in the larger membrane pores, causing a greater reduction in solute sieving compared to the membrane hydraulic permeability and porosity than would be predicted on the basis of either a simple pore blockage or pore constriction model.     

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.     

Effects of biofilm formation on membrane performance in submerged membrane bioreactors

The effects of biofilm formation on membrane performance were evaluated for a submerged membrane bioreactor (sMBR) system with six different types of micro- and ultrafiltration membranes (working volume=19 l). After operation for 24 h the permeability of the membranes with a larger pore size (microfiltration) decreased to that of the membranes with a much smaller pore size (ultrafiltration). Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) confirmed that biofilms could reduce the influence of the membrane surface properties. The chemical oxygen demand (COD) removal efficiency was 95% for the oily wastewater treatment in the sMBR where the filtration process made an important contribution (47% based on feed COD). Significant enhancement in COD removal occurred at the initial filtration stage because of biofilm formation and the dynamic member role of the biofilm layer. Membranes with various pore sizes had approximately the same permeate quality that was attributed to the biofilm on the membrane surfaces. Nevertheless, the ultrafiltration membranes had 43% more COD removal efficiency than the other applied membranes at the beginning of filtration (before biofilm formation) because of the smaller pore sizes and better sieving.     

Effects of biofilm formation on membrane performance in submerged membrane bioreactors

The effects of biofilm formation on membrane performance were evaluated for a submerged membrane bioreactor (sMBR) system with six different types of micro- and ultrafiltration membranes (working volume = 19 l). After operation for 24 h the permeability of the membranes with a larger pore size (microfiltration) decreased to that of the membranes with a much smaller pore size (ultrafiltration). Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) confirmed that biofilms could reduce the influence of the membrane surface properties. The chemical oxygen demand (COD) removal efficiency was 95% for the oily wastewater treatment in the sMBR where the filtration process made an important contribution (47% based on feed COD). Significant enhancement in COD removal occurred at the initial filtration stage because of biofilm formation and the dynamic member role of the biofilm layer. Membranes with various pore sizes had approximately the same permeate quality that was attributed to the biofilm on the membrane surfaces. Nevertheless, the ultrafiltration membranes had 43% more COD removal efficiency than the other applied membranes at the beginning of filtration (before biofilm formation) because of the smaller pore sizes and better sieving.     

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.     

Use of scanning electron microscopy and energy dispersive X-ray spectroscopy to identify key fouling species during alternating tangential filtration

Alternating tangential flow filtration (ATF) has become one of the primary methods for cell retention and clarification in perfusion bioreactors. However, membrane fouling can cause product sieving losses that limit the performance of these systems. This study used scanning electron microscopy and energy dispersive X-ray spectroscopy to identify the nature and location of foulants on 0.2 μm polyethersulfone hollow fiber membranes after use in industrial Chinese hamster ovary cell perfusion bioreactors for monoclonal antibody production. Membrane fouling was dominated by proteinaceous material, primarily host cell proteins along with some monoclonal antibody. Fouling occurred primarily on the lumen surface with much less protein trapped within the depth of the fiber. Protein deposition was also most pronounced near the inlet/exit of the hollow fibers, which are the regions with the greatest flux (and transmembrane pressure) during the cyclical operation of the ATF. These results provide important insights into the underlying phenomena governing the fouling behavior of ATF systems for continuous bioprocessing.     

Performance characteristics of nanoporous carbon membranes for protein ultrafiltration

Nanoporous carbon membranes could be very attractive for applications of ultrafiltration in the biotechnology industry because of their greater mechanical strength and longer membrane life. The objective of this study was to obtain quantitative data on the performance characteristics of nanoporous carbon membranes formed within a stainless steel support that was first modified by deposition of silica particles within the macroporous support. The nanoporous carbon membrane effectively removed small solutes from a protein solution using diafiltration, with performance comparable to that of commercial polymeric membranes. Protein fouling was evident, although the nanoporous carbon membranes were easily regenerated; cleaning with 0.5 N NaOH at 50 degrees C completely restored the water permeability for multiple cycles. The nanoporous carbon membranes were also compatible with steam sterilization. Significant increases in process flux could be obtained using periodic back-pulsing, with no evidence of any structural alterations in the membrane. These results clearly demonstrate the potential benefits and opportunities for using nanoporous carbon membranes for protein ultrafiltration.     

Separation of macromolecules by ultrafiltration: Removal of poly(ethylene glycol) from human albumin

Mixtures of albumin and poly(ethylene glycol) (PEG) were used to elucidate some of the factors which influence the separation of macromolecules by thin-channel ultrafiltration. Several membranes which readily passed PEG-4000 in the absence of protein were found to exhibit increased rejection of the synthetic polymer when albumin was added to the system. Based on a comparison of filtration flux and net sieving properties, the PM-30 membrane of Amicon was chosen for further characterization. The increased rejection of PEG-4000 in the presence of albumin was independent of albumin concentration between 1 and 100 mg/ml and persisted even after albumin was removed and the system flushed with water. Overnight incubation of the membrane with trypsin restored the original sieving properties, indicating that the ‘permanent’ effects were due to irreversible adsorption to the membrane. By measuring flux over a 10⁶-fold range of albumin concentration it was possible to resolve the effects of protein adsorption, a saturable process which occurs at low protein concentration (<0.01 mg/ml), from the effects of concentration polarization which occur at high protein concentration (>0.1 mg/ml). Only the former process has an effect on the net sieving properties in this system. In spite of the adverse effects of protein adsorption, it was still possible to obtain efficient removal of PEG-4000 from albumin. Exchange of approximately 5 vols. of solvent at room temperature resulted in a 10-fold reduction in the concentration of PEG in the sample, with no loss of albumin, and no formation of albumin dimers.     

Effects of chemical sanitization using NaOH on the properties of polysulfone and polyethersulfone ultrafiltration membranes

Membranes used in bioprocessing applications are typically sanitized before use to insure aseptic operation. However, there is almost no information in the literature on the effects of this preuse sanitization step on the properties of the membrane. Experiments were performed with commercially available hollow fiber polysulfone (PSf) and polyethersulfone (PES) membranes with different nominal molecular weight cutoffs. Data were obtained for the membrane hydraulic permeability, dextran retention coefficients, zeta potential (surface charge), and extent of protein adsorption both before and after sanitization with 0.5 N NaOH at 45°C for 30 min. Changes in chemical composition were examined using ATR‐FT‐IR and XPS. Sanitization caused a large increase in the net negative charge for all membranes. There was a small reduction in hydraulic permeability and a significant increase in dextran retention for the polyethersulfone membranes, consistent with a reduction in the effective pore size. Spectroscopic analyses suggest that this change is likely due to the base‐catalyzed hydrolysis of the lactam ring in polyvinylpyrrolidone (PVP) that is typically is used as a wetting/pore‐forming agent in PSf and PES membranes. Preuse sanitization also appeared to have a small effect on protein adsorption, although the extent of adsorption was quite low for both the virgin and sanitized membranes. The observed changes in membrane properties could have a significant impact on the ultrafiltration performance, demonstrating the importance of standardizing the sanitization procedures even in process development and scale‐down validation studies. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:90–96, 2015     

Effect of solution pH on protein transport through ultrafiltration membranes

Although a number of previous studies have demonstrated that solution pH can have a dramatic effect on protein transport through ultrafiltration membranes, the exact origin of this behavior has been unclear. Experimental data were obtained for the transport of a broad range of proteins with different surface charge and molecular weight. The effective hydrodynamic size of the proteins was evaluated using size‐exclusion chromatography. The membrane charge, both before and after exposure to a given protein, was evaluated using streaming potential measurements. In most cases, the electrostatic interactions were dominated by the distortion of the electrical double layer surrounding the protein, leading to a distinct maximum in protein transmission at the protein isoelectric point. Attractive electrostatic interactions did occur when the protein and membrane had a large opposite charge, causing a second maximum in transmission at a pH between the isoelectric points of the protein and membrane. The sieving data were in good agreement with theoretical calculations based on available models for the partitioning of charged solutes in cylindrical pores. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 27–37, 1999.     

Role of some whey components on mass transfer in ultrafiltration

Ultrafiltration through Carbosep M(4) mineral membrane of protein solutions of decreasing complexity (whey before and after centrifugation or clarification, beta-lactoglobulin) was studied. Mathematical models were used to explain variations in flux with time. Taking into account variations in protein retention and hydraulic resistance of the membrane during ultrafiltration, proteins and lipoproteins were found to be involved not only in the polarization layer (reversible fouling leading to a difference in the osmotic pressure), but also in irreversible fouling by adsorption. Morever, the presence of particles (e.g., inorganic precipitates) in whey explains the build-up of a deposit over and within the membrane which contributes to the decline in flux after 1 h ultrafiltration. The relative importance of these phenomena was quantified.     

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.     

Impact of micro and macroporous TFF membranes on product sieving and chromatography loading for perfusion cell culture

Bioprocess intensification can be achieved through high cell density perfusion cell culture with continuous protein capture integration. Protein passage and cell retention are commonly accomplished using tangential flow filtration systems consisting of microporous membranes. Significant challenges, including low efficiency and decaying product sieving over time, are commonly observed in these cell retention devices. Here, we demonstrate that a macroporous membrane overcomes the product sieving challenges when comparing to several other membrane chemistries and pore sizes within the microporous range. This way, variable chromatography column loading is avoided. The macroporous membrane yielded a 13,000 L/m² volumetric throughput. The membrane's cut-off size results in an increased permeate turbidity due to particles passage, such as cell debris, through pores ranging from 1 to 4 µm. In addition, successful chromatography column plugging mitigation was achieved by employing depth filtration before the chromatographic step. Depth filtration volumetric throughputs were between 600 and 1,000 L/m². Combing a macroporous cell retention device with a depth filter not only provided an alternative to address the challenge of undesired long protein residence times in the bioreactor due to product sieving decay, but also exhibited a throughput increase, making the integration of multicolumn capture chromatography with a perfusion cell culture a more robust process.     

Protein recovery from cell debris using rotary and tangential crossflow microfiltration

Protein recovery from a bacterial lysate was accomplished using microfiltration membranes in a flat crossflow filter and in a cylindrical rotary filter. Severe membrane fouling yielded relatively low long-term permeate flux values of 10(-4)-10(-3) cm/s (where I cm/s = 3.6 x 10(4) L/m(2) - h). The permeate flux was found to be nearly independent of transmembrane pressure and to increase with increasing shear rate and decreasing solids concentration. The flux increased with shear to approximately the one-third power or greater for the flat filter and the one-half power or greater for the rotary filter; the stronger dependence for the rotary filter is thought to result from Taylor vortices enhancing the back transport of debris carried to the membrane surface by the permeate flow. The average protein transmission or sieving coefficient was measured at approximately 0.6, but considerable scatter in the transmission data was observed. The largest sieving coefficients were obtained for dilute suspensions at high shear rate. The rotary filter provided higher fluxes than did the flat filter for dilute suspensions, but not for concentrated suspensions. (c) 1995 John Wiley & Sons, Inc.     

Effect of solution pH on protein transmission and membrane capacity during virus filtration

Although virus filtration is now an integral part of the overall viral clearance strategy for the purification of many commercial therapeutic proteins, there is currently little understanding of the factors controlling the performance of the virus filters. The objective of this study was to examine the effects of solution pH on protein transmission and capacity during virus filtration. Data were obtained using bovine serum albumin as a model protein with Viresolve 180 membranes oriented with both the skin-side up and skin-side down. Membranes were also characterized using dextran sieving measurements both before and after protein filtration. Membrane capacity and protein yield were minimal at the protein isoelectric point, which was due to the greater degree of concentration polarization associated with the smaller protein diffusion coefficient at this pH. In contrast, the actual protein sieving coefficient was maximum at the protein isoelectric point due to the absence of any strong electrostatic exclusion under these conditions. The yield and capacity were both significantly greater when the membrane was oriented with the skin-side down. These results provide important insights into the effects of solution conditions on the performance of virus filtration membranes for protein purification.     

Random-coil model for glomerular sieving of dextran

Dextran has been the most commonly employed test molecule for probing the selectivity of glomerular filtration to macromolecules of varying size. The usual theories for hindered transport of solid spheres through pores have limited utility in interpreting clearance data for dextran or other linear polymers because such polymers in solution more closely resemble random, solvent-filled coils than solid spheres. To provide a model for glomerular filtration of random-coil macromolecules, the equilibrium partitioning of random coils between cylindrical pores and bulk solution was simulated using Monte Carlo calculations, and those results were combined with a hydrodynamic theory for restricted motion of solvent-filled polymer coils in pores. The rates of transport predicted for either neutral random coils or for solid spheres of the same Stokes-Einstein radius were significantly lower than observed transport rates of dextran through the glomerular capillary wall or across synthetic porous membranes. This facilitation of dextran transport was modeled by postulating weak, attractive interactions between dextran monomers and the pore wall. The random-coil model with attractive interactions, modeled using a short-range, square-well potential, was found to adequately represent dextran sieving data in normal rats. Various limitations of this approach are discussed.     

Wide-surface pore microfiltration membrane drastically improves sieving decay in TFF-based perfusion cell culture and streamline chromatography integration for continuous bioprocessing

Although several compelling benefits for bioprocess intensification have been reported, the need for a streamlined integration of perfusion cultures with capture chromatography still remains unmet. Here, a robust solution is established by conducting tangential flow filtration-based perfusion with a wide-surface pore microfiltration membrane. The resulting integrated continuous bioprocess demonstrated negligible retention of antibody, DNA, and host cell proteins in the bioreactor with average sieving coefficients of 98 ± 1%, 124 ± 28%, and 109 ± 27%, respectively. Further discussion regarding the potential membrane fouling mechanisms is also provided by comparing two membranes with different surface pore structures and the same hollow fiber length, total membrane area, and chemistry. A cake-growth profile is reported for the narrower surface pore, 0.65-µm nominal retention perfusion membrane with final antibody sieving coefficients ≤70%. Whereas the sieving coefficient remained ≥85% during 40 culture days for the wide-surface pore, 0.2-µm nominal retention rating membrane. The wide-surface pore structure, confirmed by scanning electron microscopy imaging, minimizes the formation of biomass deposits on the membrane surface and drastically improves product sieving. This study not only offers a robust alternative for integrated continuous bioprocess by eliminating additional filtration steps while overcoming sieving decay, but also provides insight into membranes' fouling mechanism.     

Three-Dimensionally Printed Microfluidic Cross-flow System for Ultrafiltration/Nanofiltration Membrane Performance Testing

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane technology. Understanding the fouling process could lead to optimization and higher efficiency of membrane based filtration. Here we show the design and fabrication of an automated three-dimensionally (3-D) printed microfluidic cross-flow filtration system that can test up to 4 membranes in parallel. The microfluidic cells were printed using multi-material photopolymer 3-D printing technology, which used a transparent hard polymer for the microfluidic cell body and incorporated a thin rubber-like polymer layer, which prevents leakages during operation. The performance of ultrafiltration (UF), and nanofiltration (NF) membranes were tested and membrane fouling could be observed with a model foulant bovine serum albumin (BSA). Feed solutions containing BSA showed flux decline of the membrane. This protocol may be extended to measure fouling or biofouling with many other organic, inorganic or microbial containing solutions. The microfluidic design is especially advantageous for testing materials that are costly or only available in small quantities, for example polysaccharides, proteins, or lipids due to the small surface area of the membrane being tested. This modular system may also be easily expanded for high throughput testing of membranes.      

Evaluation of membranes for use in on-line cell separation during mammalian cell perfusion processes

In this study two microporous hollow fibre membranes were evaluated for their use as cell retention device in continuous perfusion systems. A chemically modified permanent hydrophillic PTFE membrane and a hydrophilized PP membrane were tested. To investigate the filtration characteristics under process conditions each membrane was tested during a long term perfusion cultivation of a hybridoma cell line. In both cultivations the conditions influencing membrane filtration (e.g. transmembrane flux) were kept constant. Filtration behaviour was investigated by monitoring transmembrane pressure and protein permeability. Transmembrane pressure was measured on-line with an autoclavable piezo-resistive pressure sensor. Protein permeability was determined by quantitative evaluation of unreduced, Coomassie stained SDS-PAGE. The membrane fouling process influences the filtration characteristics of both membranes in a different way. After fermentation the PP membrane was blocked by a thick gel layer located in the big outer pores of the asymmetric membrane structure. The hydraulic resistance was higher but the protein permeability was slightly better than of the PTFE membrane. For this reason the PP membrane should be preferred. On the other hand, transmembrane pressure decreases slower when the PTFE membrane is used, which favours this membrane for long term cultivations, especially when low molecular weight proteins (<30 KD) are produced.Abbreviations PP Polypropylene - PTFE Polytetrafluoroethylene     

Membrane fouling in sterile filtration of recombinant human growth hormone

The most troublesome problem encountered during the sterile filtration of protein solutions is membrane fouling. This article presents our study on sterile filtration of a model protein, recombinant human growth hormone (rhGH). Scanning electron microscopy (SEM) analysis shows that 0.22-mum membranes, when used to filter the mannitol-formulated protein solution under a 0.35-bar transmembrane pressure, were plugged to a great extent. When zinc ions were added to induce aggregates, the fouling tendency of rhGH solutions increased with increasing amount and size of the aggregates, indicating that the aggregates present before filtration might be responsible for membrane fouling. However, repeated filtration of the same solution using a fresh filter each time cannot reduce membrane fouling, and all filtrates contain the same trace amount of hGH particulates as the prefiltered solution. Particulate size was determined to be between 0.03 and 0.15 mum by dynamic light scattering. Also, in view of the fact that protein formulations significantly affected the tendency of fouling with the same preexisting aggregates, it is likely that fouling was more attributed to the aggregation taking place in the filter pores during filtration (secondary aggregation) than to the aggregates present before filtration. Adding a surfactant to or increasing the pH of the protein solution improves the filtration, whereas increasing ionic strength slows down the filtration. This result suggests that the balance of the protein's interaction and electrostatic repulsion plays an important role in the protein's fouling tendency. Many factors might change the microenvironment in the pores and disturb this balance. Those considerations and the aggregation tendency of rhGH in the filter pores will be discussed in detail separately. (c) 1996 John Wiley & Sons, Inc.