A new coiled hollow-fiber module design for enhanced microfiltration performance in biotechnology.

The microfiltration performance of a novel membrane module design with helically wound hollow fibers is compared with that obtained with a standard commercial-type crossflow module containing linear hollow fibers. Cell suspensions (yeast, E. coli, and mammalian cell cultures) commonly clarified in the biotechnology industry are used for this comparison. The effect of variables such as transmembrane pressure, particle suspension concentration, and feed flow rate on membrane performance is evaluated. Normalized permeation fluxes versus flow rate or Dean number behave according to a heat transfer correlation obtained with centrifugal instabilities of the Taylor type. The microfiltration performance of this new module design, which uses secondary flows in helical tubes, is significantly better than an equivalent current commercial crossflow module when filtering suspensions relevant to the biotechnology industry. Flux and capacity improvements of up to 3.2-fold (constant transmembrane pressure operation) and 3.9-fold (constant flux operation), respectively, were obtained with the helical module over those for the linear module.     

A predictive aggregate transport model for microfiltration of combined macromolecular solutions and poly-disperse suspensions: model development

A methodology, called the aggregate transport model, is presented that can a priori predict both the pressure-independent permeation flux and yield of target species for the microfiltration of poly-disperse solutions. The model captures the phenomenon of critical shear rate. Beyond the critical shear rate (expressed as a ratio of shear rate to permeation flux), the transmission of proteins drops sharply as a result of cake classification. The widely reported benefits of operating at uniform transmembrane pressure and constant wall concentration follow from this method. The methodology is general in nature and can be used predictively to obtain an optimal balance between flux and yield of target species during the microfiltration of many commercial poly-disperse suspensions. In the accompanying paper we test this model for microfiltration of transgenic whole goat milk.     

Optimized recovery of monoclonal antibodies from transgenic goat milk by microfiltration

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

A new integrated membrane filtration and chromatographic device

To improve protein separation, a novel integrated device combining membrane filtration and chromatography has been developed. The device basically consists of a hollow fiber filtration module whose shell side is filled with chromatographic resin beads. However, there is an essentially impermeable coated zone near the hollow fiber module outlet. The integrated device enjoys the advantages of both membrane filtration and chromatography; it also allows one to load the chromatographic media directly from the fermentation broth or lysate and separate the adsorbed proteins through the subsequent elution step in a cyclic process. Interfacial polymerization was carried out to coat the bottom section of the hollow fiber membrane; the rest of the hollow fiber membrane remained unaffected. Myoglobin (Mb) and alpha-lactalbumin (alpha-LA) were primarily used as model proteins in a binary mixture; binary mixtures of Mb and bovine serum albumin (BSA) were also investigated. Separation behaviors of binary protein mixtures were studied in devices having either an ultrafiltration (UF) or a microfiltration (MF) membrane. Experimental results show that the breakthrough time and the protein loading capacities were dramatically improved after introducing the impermeable coating in both UF and MF modules. For a synthetic yeast fermentation broth feed, four loading-washing-elution-reequilibration-based cyclic runs for separation of Mb and alpha-LA were performed in the device using a MF membrane with a coated zone without cleaning in between. The Mb and alpha-LA elution profiles for the four consecutive runs were almost superimposable. Due to lower transmembrane flux in this device plus the periodical washing-elution during the chromatographic separation, fouling was not a problem, unlike in conventional microfiltration.     

Dean vortex membrane microfiltration and diafiltration of rBDNF E. coli inclusion bodies

Cross-flow microfiltration (CMF) and diafiltration were used to concentrate and purify recombinant Brain-Derived Neutrophic Factor (rBDNF) inclusion bodies from an E. coli cell suspension and a homogenized E. coli cell suspension (homogenate/lysate). Although these processes have been tested industrially in pilot scale with conventional linear membrane microfiltration modules, their performances were severely limited due to membrane fouling. The purpose of this work was to determine whether Dean vortex microfiltration with controlled centrifugal instabilities (Dean vortices produced in helical flow) could be used to improve filtration performance over that observed with conventional linear cross-flow microfiltration (CMF). For the microfiltration experiments with the feeds containing cell and homogenate suspensions, improvements in flux of about 50 and 70%, respectively, were obtained with the helical module as compared with that obtained with the linear module. For diafiltration with the homogenate suspension as feed, solute transport (as measured by mass) was from 100 to 40% higher after 40 and 100 min, respectively, with the helical module as compared with that obtained with the linear module. In the presence of the neutral surfactant, Tween 20, solute transport for diafiltration was at least 25 times higher during the first 10 min of operation and 100% higher after 300 min with the helical module as compared with that obtained with the linear module. Clearly, improved filtration performance, a purer and more concentrated product, and substantial savings can be expected with the new Dean vortex filters.     

Protein transmission during Dean vortex microfiltration of yeast suspensions.

Substantially higher rates of protein and fluid volume transport for microfiltration of yeast suspensions were possible with improved hydrodynamics using centrifugal fluid instabilities called Dean vortices. Under constant permeate flux operation with suspended yeast cells, a helical module exhibited 19 times the filtration capacity of a linear module. For feed containing both BSA and beer yeast under constant transmembrane pressure with diafiltration, about twice as much protein (BSA and other proteins from cell lysis) was transported out of the feed by the helical module as compared with the linear module. The volumetric permeation flux improvements for the helical over the linear module ranged from 18 to 43% for yeast concentrations up to 4.5 dry wt %.     

Modeling flux in tangential flow filtration using a reverse asymmetric membrane for Chinese hamster ovary cell clarification

Tangential flow filtration is advantageous for bioreactor clarification as the permeate stream could be introduced directly to the subsequent product capture step. However, membrane fouling coupled with high product rejection has limited its use. Here, the performance of a reverse asymmetric hollow fiber membrane where the more open pore structure faces the feed stream and the barrier layer faces the permeate stream has been investigated. The open surface contains pores up to 40 μm in diameter while the tighter barrier layer has an average pore size of 0.4 μm. Filtration of Chinese hamster ovary cell feed streams has been investigated under conditions that could be expected in fed batch operations. The performance of the reverse asymmetric membrane is compared to that of symmetric hollow fiber membranes with nominal pore sizes of 0.2 and 0.65 μm. Laser scanning confocal microscopy was used to observe the locations of particle entrapment. The throughput of the reverse asymmetric membrane is significantly greater than the symmetric membranes. The membrane stabilizes an internal high permeability cake that acts like a depth filter. This stabilized cake can remove particulate matter that would foul the barrier layer if it faced the feed stream. An empirical model has been developed to describe the variation of flux and transmembrane pressure drop during filtration using reverse asymmetric membranes. Our results suggest that using a reverse asymmetric membrane could avoid severe flux decline associated with fouling of the barrier layer during bioreactor clarification.     

Membrane separation of protein precipitates: Studies with cross flow in hollow fibers

Hollow fiber ultrafiltration and microfiltration membranes are examined for the processing of isoelectric soya protein precipitate suspensions. A model based on the various resistances to permeate flux is used to describe membrane performance. The main resistance to permeate flux is due to the interaction between the active membrane and the soluble and precipitated protein; that is, as compared with resistances due to the active membrane itself or the membrane support structure, or arising from concentrated soluble or precipitated protein layers over the membrane surface. Soluble protein rejection and precipitate mean particle diameter are correlated with observed values of this main resistance.In contract to the ultrafiltration of soluble proteins, the flux rates observed when processing protein precipitate suspensions under a similar range of operating conditions do not approach a limiting value with increased transmembrane pressure. At high protein concentrations, greater flux rates may be achieved for precipitated as compared with soluble proteins. The use of a microfiltration membrane does not give further improvement in flux rate; this may be attributed to problems of pore fouling with precipitate particles.     

Bioartificial kidney. II. A convective flow model of a hollow fiber bioartificial renal tubule

An existing model for volume transport in the rat proximal tubule was modified and applied to a bioartificial renal tubule. The predicted volume transport agrees well with experimental data. The volume transport model was coupled to the analytic solutions of flow in the bioartificial renal tubule bioreactor, operated in the open-shell mode with perfusion in both the lumen and surrounding shell. The results suggest that the performance of a multifiber bioreactor can be improved by controlling shell inlet conditions and fiber spacing. These results have important implications for the design and analysis not only for the bioartificial renal tubule bioreactor but also for the general case of hollow fiber bioreactors operated in the open-shell mode with perfusion in both the lumen and surrounding shell.     

Protein loading,elution, and resolution behavior in a novel device that integrates ultrafiltration and chromatographic separation

Hollow fiber membranes and chromatographic resin beads are commonly employed in a variety of bioseparation processes. A new class of integrated separation devices is being studied in which the shell side of a hollow fiber device is filled with adsorbents/chromatographic resin beads. Such devices and the corresponding separation methods integrate feed broth clarification by the microfiltration/ultrafiltration membrane with bioproduct purification by the shell-side resin beads either as an adsorbent or as beads in elution chromatography. A mathematical model has been developed for the prediction of the chromatographic behavior of such an integrated device. Simulations have been done to study the effects of axial dispersion, feed flow rate, water permeation rate, fiber packing density, and void fraction. Numerical solutions were obtained by solving the governing equations. This model can reasonably describe the concentration profiles as well as the breakthrough and elution behaviors in the integrated device.     

Recovery of insect cells using hollow fiber microfiltration

An efficient method was developed for media separation and cell collection for eukaryotic cells growing in suspension. The method is based on tangential flow microfiltration using an open channel arrangement in a hollow fiber configuration. Best results (highest processing flux rate) for polysulfone hollow fibers were obtained using fibers with internal diameter of 0.75 mm, 0.45 mum pore size, and a cell suspension flow at a shear rate of 14000 s(-1) (0.032 L/min per fiber). A flux rate of 500 L/m(2) h can be obtained by maintaining the surface area/cell ratio at 0.05 m(2)/10 L of cells at a concentration of 2.5 x 10(6) cells/mL. Forty liters of infected insect cells can be concentrated 10 times in 20 min without affecting cell viability. (c) 1995 John Wiley & Sons, Inc.     

Murine leukemia virus clearance by flocculation and microfiltration

Clearance of murine leukemia virus from CHO cell suspensions by flocculation and microfiltration was investigated. Murine leukemia virus is a retrovirus that is recommended by the U.S. Food and Drug Administration for validating clearance of retrovirus-like particles. Due to biosafety considerations, an amphotropic murine leukemia virus vector (A-MLV) that is incapable of self-replication was used. Further, A-MLV is incapable of infecting CHO cells, thus ensuring that infection of the CHO cells in the feed did not result in a reduced virus titer in the permeate. The virus vector contains the gene for the enhanced green fluorescent protein (EGFP) to facilitate assaying for infectious virus particles. The virus particles are 80-130 nm in size. The feed streams were flocculated using a cationic polyelectrolyte. Microfiltration was conducted using 0.1 and 0.65 microm pore size hollow fiber membranes. The level of virus clearance in the permeate was determined. For the 0.1 microm pore size membranes a 1,000-fold reduction in the virus titer in the permeate was observed for feed streams consisting of A-MLV, A-MLV plus flocculant, A-MLV plus CHO cells, and A-MLV plus flocculant and CHO cells. While the flocculant had little effect on the level of virus clearance in the permeate for 0.1 microm pore size membranes, it did lead to higher permeate fluxes for the CHO cell feed streams. Virus clearance experiments conducted with 0.65 microm pore size membranes indicate little clearance of A-MLV from the permeate in the absence of flocculant. However, in the presence of flocculant the level of virus clearance in the permeate was similar to that observed for 0.1 microm pore size membranes. The results obtained here indicate that significant clearance of A-MLV is possible during tangential flow microfiltration. Addition of a flocculant is essential if the membrane pore size is greater than the diameter of the virus particles. Flocculation of the feed stream leads to an increase in the permeate flux.     

Crossflow microfiltration of recombinant Escherichia coli lysates after high pressure homogenization

Crossflow membrane filtration was used to process recombinant Escherichia coli cell lysates containing protein inclusion bodies after high pressure homogenization. The number of passes through the high pressure homogenizer changed the viscosities and average particle sizes of the cell lysates. The different cell lysates were processed with a hollow fiber unit containing microfiltration membranes and a plate and frame unit with either ultrafiltration or microfiltration membranes. There were differences in permeate flux and protein transmission for the various membranes with the best performing membranes giving permeate fluxes greater than 60 L m(-2) h(-1) and protein transmissions greater than 90%. For a given membrane, no differences were observed between the cell lysates following homogenization with one, two, and three passes at 83 MPa. The lack of a difference between the three lysates is due to their similarities with respect to the released macromolecules and the presence of small (<0.1 mum) cell debris. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 304-310, 1997.     

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.     

Protein-free human-human hybridoma cultures in an intercalated-spiral alternate-dead-ended hollow fiber bioreactor

Batch cell cultures of a human-human hybridoma line in a convective flow dominant intercalated-spiral altetnate-dead-ended hollow fiber are compared with those using conventional axial-flow hollow fiber bioreactors and a stirred-tank bioreactor. Relatively short-term fed-batch and perfusion cell cultures were also employed for the intercalated-spiral bioreactor. When operating conditions of a batch intercalated-spiral bioreactor were properly chosen, the cell growth and substrate consumption paralleled that of a batch stirred-tank culture. The results verified the premise of the intercalated-spiral hollow fiber bioreactor that nutrient transport limitations can be eliminated when the convective flux through the extracapillary space is sufficiently high.(c) John Wiley & Sons, Inc.     

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     

Evaluation of cell disruption effects on primary recovery of antibody fragments using microscale bioprocessing techniques

Intracellular antibody Fab' fragments periplasmically expressed in Escherichia coli require the release of Fab' from the cells before initial product recovery. This work demonstrates the utility of microscale bioprocessing techniques to evaluate the influence of different cell disruption operations on subsequent solid–liquid separation and product recovery. Initially, the industrial method of Fab' release by thermochemical extraction was established experimentally at the microwell scale and was observed to yield Fab' release consistent with the larger scale process. The influence of two further cell disruption operations, homogenization and sonication, on subsequent Fab' recovery by microfiltration was also examined. The results showed that the heat‐extracted cells give better dead‐end microfiltration performance in terms of permeate flux and specific cake resistance. In contrast, the cell suspensions prepared by homogenization and sonication showed more efficient product release but with lower product purity and poorer microfiltration performance. Having established the various microscale methods the linked sequence was automated on the deck of a laboratory robotic platform and used to show how different conditions during thermochemical extraction impacted on the optimal performance of the linked unit operations. The results illustrate the power of microscale techniques to evaluate crucial unit operation interactions in a bioprocess sequence using only microliter volumes of feed. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

应用中空纤维膜去除无细胞百日咳料液中的脱毒剂和色素

目的应用中空纤维膜洗滤技术去除无细胞百日咳料液中的脱毒剂和色素。方法采用不同浓度的盐溶液作为中空纤维膜洗滤液,通过洗滤过程的水通量衰减程度,洗滤后制品外观和蛋白回收率,原液戊二醛残留量,原液效价和毒力来确定洗滤液。用物理和化学清洗相结合的方式对使用后的膜进行清洗再生,以水通量恢复率对清洗效果进行评估。结果用盐溶液Ⅱ作为洗滤液,在洗滤过程维持跨膜压基本恒定的情况下,中空纤维膜水通量大、且衰减缓慢,洗滤后收集的制品外观呈淡黄色,蛋白颗粒均匀,蛋白回收率高,所制备原液的戊二醛残留量、效价和毒力检定结果均符合《中国药典》三部(2010版)要求。中空纤维膜使用后,用物理和化学清洗相结合的清洗方式使中空纤维膜的水通量恢复率分别达99.06%,98.11%,99.62%。结论中空纤维膜可用于去除无细胞百日咳料液中的脱毒剂和色素。     

Pilot-scale adenovirus seed production through concurrent virus release and concentration by hollow fiber filtration

Recovery of recombinant adenoviruses from infected mammalian cell cultures often requires multiple unit operations such as cell lysis for virus release, microfiltration for clarification, and ultrafiltration for concentration. While development of these multiple unit operations is relatively straightforward, implementation under aseptic conditions in a closed system can be challenging for the production of virus seed at industrial scales. In this study, we have developed a simple, single-step, scaleable process to effectively recover adenoviruses from infected PER.C6 cell cultures for the production of concentrated adenovirus seeds under aseptic conditions. Specifically, hollow fiber tangential flow filtration technology was applied to maximize cell lysis of infected cultures for virus release while simultaneously concentrating the virus to an appropriate level of volume reduction. Hollow fiber filters with small lumen diameter of 0.5 mm were chosen to maximize the wall shear for a highly effective cell lysis and virus release. Cell lysis and virus release were shown to correlate with the exposure time in the hollow fiber cartridge: the shear zone. In most cases, a virus recovery yield of more than 80% and a 15- to 20-fold concentration (or up to 95% volume reduction) was achieved in less than 2 h of processing time. The virus seeds prepared using this process at lab scale and at 300-L scale without clarification have been successfully tested for sterility and potency and used for subsequent infection with consistent virus productivity. This process should enable rapid production of adenovirus seeds with minimal unit operations and high efficiency recovery for adenovirus production at 1000-L scale.