Properties of microfiltration membranes: the effects of adsorption and shear on the recovery of an enzyme

An experimental study of the interaction of the enzyme yeast alcohol dehydrogenase (YADH) with microfiltration membranes has been carried out. Most measurements were made with capillary pore inorganic membranes (Anopore) with some comparative measurements being made with polymeric membranes of low protein affinity (Durapore). It has been shown that the prolonged exposure of the enzyme to the inorganic membrane under low-shear conditions (slow recycle) resulted in a loss of enzyme activity. Under filtration conditions, the membrane permeation rate decreased continuously with time. This decrease could be quantified using the standard blocking filtration law, which describes a decrease in pore volume due to deposition of enzyme on the walls of the pore. No significant loss in activity of permeating enzyme occurred under solution conditions where the enzyme was stable. However, a significant loss of such activity occurred under solution conditions where the enzyme was slightly unstable. The experiments indicate that the likely mechanism for activity loss is a membrane/enzyme interaction resulting from a shear induced deformation of the enzyme structure. Two conclusions of practical importance are drawn from the work. (c) 1992 John Wiley & Sons, Inc.     

Effect of protein fouling on filtrate flux and virus breakthrough behaviors during virus filtration process

Virus filtration process is used to ensure viral safety in the biopharmaceutical downstream processes with high virus removal capacity (i.e., >4 log₁₀). However, it is still constrained by protein fouling, which results in reduced filtration capacity and possible virus breakthrough. This study investigated the effects of protein fouling on filtrate flux and virus breakthrough using commercial membranes that had different symmetricity, nominal pore size, and pore size gradients. Flux decay tendency due to protein fouling was influenced by hydrodynamic drag force and protein concentration. As the results of prediction with the classical fouling model, standard blocking was suitable for most virus filters. Undesired virus breakthrough was observed in the membranes having relatively a large pore diameter of the retentive region. The study found that elevated levels of protein solution reduced virus removal performance. However, the impact of prefouled membranes was minimal. These findings shed light on the factors that influence protein fouling during the virus filtration process of biopharmaceutical production.     

Removal versus fragmentation of amyloid-forming precursors via membrane filtration

The presence of even minute amounts of protein aggregates in solution can significantly alter the kinetics of amyloid formation. Removal of such pre-existing aggregates is critical for reproducible analysis of amyloid formation. Here we examine the effects of membrane filtration on insulin fibrillization. We find that filtration of insulin with large pore membranes (≥ 100 nm) generally slows fibril formation relative to unfiltered solutions by removing pre-aggregated protein. Unexpectedly, filtration with small pore membranes (< 100 nm) showed no beneficial effect and, in some cases, accelerated insulin fibril formation. This effect may be due to fragmentation of pre-existing aggregates during filtration through small pore membranes, which can increase the number of amyloid-forming precursors. These findings reveal the complexity of removing protein aggregates via filtration and suggest optimal filtration protocols for conducting fibril formation analysis of insulin and similar amyloidogenic proteins.     

Transmission of poly(dT60) single-stranded DNA through polycarbonate track-etched ultrafiltration membranes

The objective of this study was to examine membrane filtration of a single stranded DNA (ssDNA) with 60 thymine nucleotides, and to elucidate the variables controlling its transmission across track-etched porous membranes. Dead end filtration measurements were performed using different pore size membranes (10, 15, and 30 nm) at different transmembrane pressures in solutions with ionic strength ranging from 0 to 1000 mM NaCl. The diffusivity of the ssDNA was determined using fluorescence recovery after photobleaching, yielding hydrodynamic radii ranging from 1.6 to 2.8 nm, with values decreasing with increasing solution ionic strength. Despite the small ssDNA/membrane pore size, nearly 100% rejection was observed for measurements performed with the 10 and 15 nm pore size membranes under low-ionic strength conditions. These high rejections can be attributed to strong repulsive electrostatic ssDNA-membrane interactions. With increasing ionic strength, electrostatic interactions as well as the effective size of the ssDNA decreases and the flexibility of the ssDNA increases, leading to a reduction in ssDNA rejection. A design of experiments approach was used to plan filtration experiments that adequately covered the variable space with a manageable number of experiments. The results yielded an empirical expression relating ssDNA rejection to pore size, solution ionic strength and transmembrane pressure. There was evidence of flow induced elongation at high-transmembrane pressures in the 30 nm pore size membranes, but not in the smaller pore size membranes. These results are consistent with critical flux estimates developed using a free draining model for the ssDNA.     

Properties of microfiltration membranes: Mechanisms of flux loss in the recovery of an enzyme

The transmission and rate of filtration of the enzyme yeast alcohol dehydrogenase (YADH) has been studied at capillary pore microfiltration membranes. Photon correlation spectroscopy (PCS) with nanometer resolution showed that the enzyme existed as discreate molecules only for a narrow range of pH and ionic strength. Under such conditions, the transmission of the enzyme was high. However, the rate of filtration still decreased continuously with time. Analyssis of the time dependence of the rate of filtration indicated that this decrease was due to in-pore enzyme deposition at low concentration ("standard blocking model") and suface depositon at high concentration ("cake filtration model"). Use of atomic force microscopy (AFM) gave unequivocal and quantitative confirmation of these inferences. The work shows the great advantage of using advanced physical characterization techniques, both for the identification of the optimum conditions for filtration (PCS) and for the elucidation of mechanisms giving rise to inefficiencies in the filtration process (AFM). (c) 1995 John Wiley & Sons, Inc.     

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.     

Prefiltration enhances performance of sterile filtration for glycoconjugate vaccines

Recent studies have reported very low capacity during sterile filtration of glycoconjugate vaccines due to rapid fouling of the sterile filter. The objective of this study was to explore the potential for significantly increasing the capacity of the sterile filter through the use of an appropriate prefilter. Data were obtained using prefilters with different pore size and chemistry, with the sterile filtration performed at constant filtrate flux using 0.22 μm nominal pore size Durapore® polyvinylidene difluoride membranes. Prefiltration through 5 μm pore size Durapore® or Nylon prefilters nearly eliminated the fouling of the sterile filter, leading to more than a 100-fold reduction in the rate of pressure increase for the sterile filter. This dramatic improvement in sterile filter performance was due to the removal of large components (greater than 1 μm in size) as confirmed by dynamic light scattering. These results demonstrate the potential of using large pore size prefilters to significantly enhance the performance of the sterile filtration process for the production of important glycoconjugate vaccines.     

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.     

The effects of buffer condition on the fouling behavior of MVM virus filtration of an Fc-fusion protein

A combined pore blockage and cake filtration model was applied to the virus filtration of an Fc-fusion protein using the three commercially available filters, F-1, F-2, and F-3 in a range of buffer conditions including sodium-phosphate and tris-acetate buffers with and without 200 mM NaCl at pH 7.5. The fouling behaviors of the three filters for the feed solutions spiked with minute virus of mice were described well by this combined model for all the solution conditions. This suggests that fouling of the virus filters is dominated by the pore blockage mechanism during the initial stage of the filtration and transformed to the cake filtration mechanism during the later stage of the filtration. Both flux and transmembrane resistance can be described well by this model. The pore blockage rate and the rate of increase of protein layer resistance over blocked pores are found to be affected by membrane properties as well as the solution conditions resulting from the modulation of interactions between virus, protein, and membrane by the solution conditions.     

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.     

Recovery of alkaline proteases by membrane filtration

This work describes the recovery of an extracellular alkaline protease from fermentation broths of a Bacillus sp ATCC 21536, at pH=10.0 using ultrafiltration (MWCO 100,000) and microfiltration (0.1 m) membranes in hollow fiber devices. The influence of membrane pore size and polymeric material and membrane filtration performance was studied. High protein recoveries and high average flux rates were obtained with polysulfone membranes. A decrease of concentration polarization was obtained, simultaneously with enhancement of filtration flux rate and enzyme recovery by using submicron sized charged particles. These polymers lead to flocculation and adsorption of whole cells and soluble factors from the fermentation broth. The best results were obtaiend by combination of cationic (0.1%) and anionic (0.04%) polymers.     

The processing of a plasmid-based gene from E. Coli. Primary recovery by filtration

We describe the primary recovery of plasmid DNA from alkaline lysis mixtures using a nutsche filter operated under pressure. Six different filter cloths constructed of polypropylene, polyester and stainless steel were tested, with pore sizes ranging from 5–160?μm. Both pore size and the material of the filter membranes employed in filtration experiments exerted considerable impact on the purity and yield of the plasmid DNA. The greatest degree of solids extrusion, shearing of chromosomal DNA and subsequent contamination of the filtrate was observed with the 160?μm polyester filter. The best compromise was obtained with a 5?μm polypropylene cloth. For an alkaline lysis mixture containing 101?g wet weight solids per litre, filtration through this cloth proceeded at an average rate of 22.5?cm?h^?1. Virtually complete removal of solids (99.4%) and protein (96.8%) was achieved, with a 8.2-fold purification of plasmid DNA at the expense of a 33% loss in yield. The filtration performance of this membrane was further modified by precoating with diatomaceous earths of different permeabilities (0.07–1.2?darcies). The finest filter aid resulted in very pure plasmid DNA (65%), complete suspended solids removal and ?1), and some losses of plasmid DNA, due to adsorption on to the diatomaceous earth, were also observed (5.7%).     

Factors in the Membrane Filtration of Enteroviruses

The filtration of two species of enteroviruses through membranes of porosity ranging from 50 to 220 mμ was studied. It was shown that extensive or total losses of virus may attend filtration at these porosities, apparently owing to adsorption of the virus to the membrane matrix. This could be minimized by the incorporation of serum into the virus suspension at the time of filtration, or by pretreating the membrane with serum or with a gelatin solution. It was also found that the first few drops of filtrate, even under optimal conditions, were likely to be virus-free, so that the filtration of too small a volume of virus suspension would result in a relatively great loss of titer. The degree to which these factors were critical was found to decrease with increasing pore diameter.     

Crossflow filtration of yeast broth cultivated in molasses

A broth of yeast cells cultivated in molasses was crossfiltered with a thin-channel module. The permeation flux gradually decreased at a constant cell concentration. The flux was much lower than that obtained for yeast broth cultivated in yeast extract, polypeptone, and dextrose (YPD) medium during the filtration. The flux did not depend on the membrane pore size (0.45 to 5 mum). The steady-state flux was one-twentieth that calculated for a cake filtration mode from the amount of cake per unit filtration area and the specific resistance of the cake measured in a dead-end filtration apparatus. The lower flux was due to small particles (most of which were less than 1 mum in diameter) in the molasses. The mehanism of crossflow filtration of broths of yeast cells cultivated in molasses was clarified by analysis of the change in flux with time and observations with scanning electron microscopy. At the initial stage of crossflow filtration the yeast cells and particles from the molasses were deposited on the membrane to form the molasses were deposited on the membrane to form a cake in a similar way to dead-end filtration. After the deposition of cells onto the membrane ceased, the fine particles from molasses formed a thin layer, which had higher resistance than the cake formed next to the membrane. The backwashing method was effective to increase the flux. The flux increased low when the pore size was 0.45 to 0.08 mum, but using larger pores of 3 to 5 mum it returned almost to the bases line. (c) 1994 John Wiley & Sons, Inc.     

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.     

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.     

Virus filtration of high‐concentration monoclonal antibody solutions

The ability to process high‐concentration monoclonal antibody solutions (> 10 g/L) through small‐pore membranes typically used for virus removal can improve current antibody purification processes by eliminating the need for feed stream dilution, and by reducing filter area, cycle‐time, and costs. In this work, we present the screening of virus filters of varying configurations and materials of construction using MAb solutions with a concentration range of 4–20 g/L. For our MAbs of interest—two different humanized IgG1s—flux decay was not observed up to a filter loading of 200 L/m² with a regenerated cellulose hollow fiber virus removal filter. In contrast, PVDF and PES flat sheet disc membranes were plugged by solutions of these same MAbs with concentrations >4 g/L well before 50 L/m². These results were obtained with purified feed streams containing <2% aggregates, as measured by size exclusion chromatography, where the majority of the aggregate likely was composed of dimers. Differences in filtration flux performance between the two MAbs under similar operating conditions indicate the sensitivity of the system to small differences in protein structure, presumably due to the impact of these differences on nonspecific interactions between the protein and the membrane; these differences cannot be anticipated based on protein pI alone. Virus clearance data with two model viruses (XMuLV and MMV) confirm the ability of hollow fiber membranes with 19 ± 2 nm pore size to achieve at least 3–4 LRV, independent of MAb concentration, over the range examined. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Adsorption characteristics of an immobilized metal affinity membrane.

An immobilized metal affinity (IMA) hollow-fiber membrane was prepared by radiation-induced graft polymerization of glycidyl methacrylate (GMA) onto a porous polyethylene hollow fiber, followed by chemical conversion of the produced epoxide group into an iminodiacetate (IDA) group and its chelation with copper(II) ion. The IDA hollow fiber, whose degree of GMA grafting was 120%, was found to retain 0.42 mol of Cu ion/kg of dry weight of the resulting IMA hollow fiber. The pure water flux of the affinity membrane was 0.90 m/h at a filtration pressure of 1 x 10(5) Pa. The 0.1 g/L L-histidyl-L-leucine (His-Leu) solution permeated across the IMA hollow fiber, whose inner diameter and thickness were 0.78 and 0.365 mm, respectively, at a prescribed filtration pressure ranging from 0.2 x 10(5) to 1.0 x 10(5) Pa. The adsorption of His-Leu during permeation of the solution showed that the overall adsorption rate was independent of the filtration pressure, i.e., the residence time, because of the negligible diffusional resistance of His-Leu to the pseudobioaffinity ligand located on the pore surface of the membrane. No deterioration in the adsorption capacity was observed after five cycles of His-Leu adsorption, its elution, and reimmobilization of copper. The adsorption isotherm of bovine serum albumin (BSA) on the IMA hollow fiber was measured and compared with that for the conventional agarose-based bead containing the IDA-Cu ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Viability of an ectomycorrhizal fungus during cross-flow filtration

Factors affecting the viability and infectivity of an ectomycorrhizal fungus during moderate concentration by cross-flow filtration were determined. Mycelial suspensions were concentrated with three commercial membrane filters (Prostak Millipore Co., M14 Tech-Sep Co. and Ceraflo Norton Co.) under aseptic conditions. Medium components may reduce the filtration rate due to their low solubility. An antifoam agent did not reduce the average flux rates as much as did the malt extract. Clear unobstructed channels (I.D. 6mm) of the tubular modules (Tech-Sep) gave the best results both in terms of performance (filtration rate) and cell viability. Shear stresses caused by pumping and flow through narrow retentate channels were probably responsible for lowering viability and infectivity. There was no linear relationship between permeate fluxes and cell concentration. There is an optimum pore size both in terms of performance (filtration rate) and cell viability. Physical blockage of large pores by hyphae could explain lower permeate flux rates than those obtained with lower pore sizes membranes.     

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.