Rotating cylindrical filters used in perfusion cultures: CFD simulations and experiments

The particle and fluid dynamics in a rotating cylindrical filtration (RCF) system used for animal cell retention in perfusion processes was studied. A validated CFD model was used and the results gave numerical evidence of phenomena that had been earlier claimed, but not proven for this kind of application under turbulent and high mesh permeability conditions, such as bidirectional radial exchange flow (EF) through the filter mesh and particle (cells) lateral migration. Taylor vortices were shown to cause EF 10‐100 times higher than perfusion flow, indicating that EF is the main drag source, at least in early stages of RCF operation. Particle lateral migration caused a cell concentration reduction (CCR) near the filter surface of approximately 10%, contributing significantly to cell separation in RCF systems and giving evidence that the mesh sieving effect is not the sole phenomenon underlying cell retention in RCF systems. Filter rotation rate was shown to significantly affect both EF and CCR. A higher separation efficiency (measured experimentally at 2,000‐L bioreactor scale) and an enhanced CCR (predicted by the numerical simulations) were found for the same rotation rate range, indicating that there is an optimal operational space with practical consequences on RCF performance. Experimental data of a large‐scale perfusion run employing the simulated RCF showed high cell viabilities for over 100 days, which is probably related to the fact that the computed shear stress level in the system was shown to be relatively low (below 20 Pa under all tested conditions). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1093–1102, 2014     

CFD-aided design of a dynamic filter for mammalian cell separation

In the present work, a rotating disk filter was designed for mammalian cell separation with the aim of avoiding both cell damage and membrane fouling. Different geometric and operational variables of the rotating disk filter were studied using computational fluid dynamics (CFD) by varying rotor radius, rotor angle, membrane-rotor distance, and angular velocity. The combinations of these variables followed a statistical design, so that an analysis of the CFD results provided correlations describing the average shear stress on the membrane surface and the maximum shear stress in the whole module as a function of the variables studied. Based on these correlations, and on the shear resistance levels of Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cell lines, which were investigated using a cone-and-plate viscosimeter, it was possible to determine the geometry and angular velocity that would minimize both cell damage and membrane fouling. After construction, the filter was tested in filtration experiments at increasing permeate fluxes. Cell viability remained >90% for the duration of the experiments (2.5 h), and no indication of fouling was observed. It was shown that the designed dynamic filter is able to effectively avoid both cell damage and membrane fouling, and thus can be used for mammalian cell harvesting and perfusion.     

New technical concept for alternating tangential flow filtration in biotechnological cell separation processes

Robust cell retention devices are key to successful cell culture perfusion. Currently, tangential flow filtration (TFF) and alternating tangential flow filtration (ATF) are most commonly used for this purpose. TFF, however, suffers from poor fouling mitigation, which leads to high filtration resistance and product retention, and ATF suffers from long residence times and cell accumulation. In this work, we propose a filtration system for alternating tangential flow filtration, which takes full advantage of the fouling mitigation effects of alternating flow and reduces cell accumulation. We have tested this novel setup in direct comparison with the XCell ATF® as well as TFF with a model feed comprising yeast cells and bovine serum albumin as protein at harsh permeate to feed flow conditions. We found that by avoiding the dead-end design of a diaphragm pump, the proposed filtration system exhibited a reduced filtration resistance by approximately 20% to 30% (depending on feed rate and permeate flow rate). A further improvement of the novel setup was reached by optimization of phase durations and flow control, which resulted in a fourfold extension of process duration until hollow fiber flow channel blockage occurred. Thus, the proposed concept appears to be superior to current cell retention devices in perfusion technology.     

Hydrodynamic characteristics and microalgae cultivation in a novel flat‐plate photobioreactor

Flat‐plate photobioreactors (FPPBRs) are widely reported for cultivation of microalgae. In this work, a novel FPPBR mounted with inclined baffles was developed, which can make the fluid produce a “spirality” flow. The flow field and cell trajectory in the photobioreactor were investigated by using computational fluid dynamics. In addition, the cell trajectory was analyzed using a Fast Fourier transformation. The influence of height of the baffles, the angle α between the inclined baffle and fluid inlet flow direction (z), and the fluid inlet velocity on the frequency of flashing light effect and pressure drop were examined to optimize the structure parameters of the inclined baffles and operating conditions of the photobioreactor. The results showed that with inclined baffles built‐in, significant swirl flow could be generated in the FPPBR. In this way, the flashing light effect for microalgal cell could also be achieved and the photosynthesis efficiency of microalgae could be promoted. In outdoor cultivation of freshwater Chlorella sp., the maximum biomass productivity of Chlorella sp. cultivated in the photobioreactor with inclined baffles was 29.94% higher than that of the photobioreactor without inclined baffles. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Understanding and modeling alternating tangential flow filtration for perfusion cell culture

Alternating tangential flow (ATF) filtration has been used with success in the Biopharmaceutical industry as a lower shear technology for cell retention with perfusion cultures. The ATF system is different than tangential flow filtration; however, in that reverse flow is used once per cycle as a means to minimize fouling. Few studies have been reported in the literature that evaluates ATF and how key system variables affect the rate at which ATF filters foul. In this study, an experimental setup was devised that allowed for determination of the time it took for fouling to occur for given mammalian (PER.C6) cell culture cell densities and viabilities as permeate flow rate and antifoam concentration was varied. The experimental results indicate, in accordance with D'Arcy's law, that the average resistance to permeate flow (across a cycle of operation) increases as biological material deposits on the membrane. Scanning electron microscope images of the post‐run filtration surface indicated that both cells and antifoam micelles deposit on the membrane. A unique mathematical model, based on the assumption that fouling was due to pore blockage from the cells and micelles in combination, was devised that allowed for estimation of sticking factors for the cells and the micelles on the membrane. This model was then used to accurately predict the increase in transmembane pressure during constant flux operation for an ATF cartridge used for perfusion cell culture. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1291–1300, 2014     

Development of a novel disposable filter bag for separation of biomolecules with functionalized magnetic particles

The integration of disposable magnetic filters in combination with functionalized magnetic particles represents a fast and cost‐effective alternative for enzyme purification in comparison to solid/liquid separation by means of centrifugation followed by chromatographic purification. The main advantage of the particle‐based process is the solid/solid/liquid separation in one step combined with disposable equipment. Furthermore this combination provides the possibility to also process biocatalytic reactions in cell‐containing media into disposable equipment with preimmobilized enzymes onto the magnetic particles. The focus of the presented study is on the design and performance of a disposable filtration unit consisting of a plastic bag with an inlet and outlet and a stainless steel filter matrix. During magnetic separation, the magnetic particles selectively retard at the filter matrix due to the magnetic force, which counteracts the drag force. It was found that the length of a lengthwise aligned filter matrix should be longer than the magnetic pole surfaces in fluid flow direction. Hereby, a filtration capacity of 5.6 g magnetic particles was measured with a loss of below 0.5%. Introducing a two‐phase flow optimizes the cleaning of the bag after a magnetic filtration. The procedure offered a high cleaning efficiency. Herewith, the cleaned filter unit could be discarded with minimum losses of product and magnet particles.     

Acoustic cell filter: a proven cell retention technology for perfusion of animal cell cultures

This article is a review highlighting the application of the acoustic filter as a reliable cell retention device during the long-term perfusion of animal cell cultures. Critical operating parameters such as duty cycle, perfusion and re-circulation flow rates, acoustic power and backflush frequency are discussed with regard to influence on the separation efficiency and optimal operating ranges have been identified. Perfusion data gathered from the literature have been complemented with original data from a series of perfusion experiments carried out in the context of industrial projects for industrially relevant cell lines including NS0, HEK-293, SP2-derived hybridoma and insect cells in different serum-supplemented and serum-free media at different perfusion rates and acoustic chamber volumes. Finally, scale-up potential of the acoustic filter for large-scale industrial applications is discussed.     

Effects of bubble–liquid two‐phase turbulent hydrodynamics on cell damage in sparged bioreactor

According to recent experimental studies on sparged bioreactors, significant cell damage may occur at the gas inlet region near the sparger. Although shear stress was proposed to be one of the potential causes for cell damage, detailed hydrodynamic studies at the gas inlet region of gas–liquid bioreactors have not been performed to date. In this work, a second‐order moment (SOM) bubble–liquid two‐phase turbulent model based on the two‐fluid continuum approach is used to investigate the gas–liquid hydrodynamics in the bubble column reactor and their potential impacts on cell viability, especially at the gas inlet region. By establishing fluctuation velocity and bubble–liquid two‐phase fluctuation velocities correlation transport equations, the anisotropy of two‐phase stresses and the bubble–liquid interactions are fully considered. Simulation results from the SOM model indicate that shear and normal stresses, turbulent energy dissipation rate, and the turbulent kinetic energy are generally smaller at the gas inlet region when compared with those in the fully developed region. In comparison, a newly proposed correlation expression, stress‐induced turbulent energy production (STEP), is found to correlate well with the unusually high cell death rate at the gas inlet region. Therefore, STEP, which represents turbulent energy transfer to a controlled volume induced by a combination of shear and normal stresses, has the potential to provide better explanation for increased cell death at the sparger region. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:48–58, 2014     

Alternating flow filtration as an alternative to internal spin filter based perfusion process: Impact on productivity and product quality

This article reports the results obtained from comparison of internal spin filter (ISF) and alternating flow filtration (ATF) as cell retention systems, regarding cell growth, volumetric perfusion rate, cell specific perfusion rate and cell productivity in the fermentation process. As expected we were able to reach higher cell densities and to achieve longer runs since ATF systems are known to be less affected by fouling. Volumetric production of the reactor using the ATF system was 50‐70% higher than the production achieved using the ISF due to higher cell density and a two‐fold increase in the perfusion rate. On the other hand, downstream processing performances were evaluated regarding chromatographic steps yields and productivity and quality attributes of the purified materials. Similar results were obtained for all evaluated systems. The fact that we were able to achieve a 2 working volumes (WV)/day perfusion rate using an ATF system as cell retention device allowed us to virtually double the WV of a 25 L reactor. These results constitute valuable data for the optimization of recombinant protein production in perfusion processes since a two‐fold increase in the average production of a manufacturing facility could be easily achieved as long as downstream scale up is possible. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1010–1014, 2017     

Glomerular permselection: shape and flow

The selective permeability of the glomerular basement membrane to macromolecules is a function of the size and charge of the macromolecule. Evidence suggests that shape may also be a factor. The orientation of macromolecules in solution is dependent on their size, shape, and frictional interactions with moving solvent molecules. The spaces between the glomerular visceral epithelial cells (slit pores) may produce a non-uniform distribution of fluid flow within the basement membrane, and this non-uniformity may increase during disease. This report is of a model that relates the filtration of rigid prolate ellipsoidal (cigar) shaped macromolecules to the size and shape of the filter and to the velocity of solvent flow. The calculations, using published macromolecular and glomerular parameters correspond well to published data. The glomerular visceral epithelial cell, by altering the number, size and distribution of the intercellular spaces, may regulate the passage of ellipsoidal shaped macromolecules, such as albumin and IgG, into and through glomerular structures.     

Very high density of CHO cells in perfusion by ATF or TFF in WAVE bioreactor™. Part I. Effect of the cell density on the process

High cell density perfusion process of antibody producing CHO cells was developed in disposable WAVE Bioreactor? using external hollow fiber filter as cell separation device. Both “classical” tangential flow filtration (TFF) and alternating tangential flow system (ATF) equipment were used and compared. Consistency of both TFF‐ and ATF‐based cultures was shown at 20–35 × 10⁶ cells/mL density stabilized by cell bleeds. To minimize the nutrients deprivation and by‐product accumulation, a perfusion rate correlated to the cell density was applied. The cells were maintained by cell bleeds at density 0.9–1.3 × 10⁸ cells/mL in growing state and at high viability for more than 2 weeks. Finally, with the present settings, maximal cell densities of 2.14 × 10⁸ cells/mL, achieved for the first time in a wave‐induced bioreactor, and 1.32 × 10⁸ cells/mL were reached using TFF and ATF systems, respectively. Using TFF, the cell density was limited by the membrane capacity for the encountered high viscosity and by the pCO₂ level. Using ATF, the cell density was limited by the vacuum capacity failing to pull the highly viscous fluid. Thus, the TFF system allowed reaching higher cell densities. The TFF inlet pressure was highly correlated to the viscosity leading to the development of a model of this pressure, which is a useful tool for hollow fiber design of TFF and ATF. At very high cell density, the viscosity introduced physical limitations. This led us to recommend cell densities under 1.46 × 10⁸ cell/mL based on the analysis of the theoretical distance between the cells for the present cell line. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:754–767, 2013     

In silico multi‐scale model of transport and dynamic seeding in a bone tissue engineering perfusion bioreactor

Computer simulations can potentially be used to design, predict, and inform properties for tissue engineering perfusion bioreactors. In this work, we investigate the flow properties that result from a particular poly‐L ‐lactide porous scaffold and a particular choice of perfusion bioreactor vessel design used in bone tissue engineering. We also propose a model to investigate the dynamic seeding properties such as the homogeneity (or lack of) of the cellular distribution within the scaffold of the perfusion bioreactor: a pre‐requisite for the subsequent successful uniform growth of a viable bone tissue engineered construct. Flows inside geometrically complex scaffolds have been investigated previously and results shown at these pore scales. Here, it is our aim to show accurately that through the use of modern high performance computers that the bioreactor device scale that encloses a scaffold can affect the flows and stresses within the pores throughout the scaffold which has implications for bioreactor design, control, and use. Central to this work is that the boundary conditions are derived from micro computed tomography scans of both a device chamber and scaffold in order to avoid generalizations and uncertainties. Dynamic seeding methods have also been shown to provide certain advantages over static seeding methods. We propose here a novel coupled model for dynamic seeding accounting for flow, species mass transport and cell advection‐diffusion‐attachment tuned for bone tissue engineering. The model highlights the timescale differences between different species suggesting that traditional homogeneous porous flow models of transport must be applied with caution to perfusion bioreactors. Our in silico data illustrate the extent to which these experiments have the potential to contribute to future design and development of large‐scale bioreactors. Biotechnol. Bioeng. 2013; 110: 1221–1230. © 2012 Wiley Periodicals, Inc.     

Enhancement of cell growth in tissue-engineering constructs under direct perfusion: Modeling and simulation

Perfusion bioreactors improve mass transfer in cell-scaffold constructs. We developed a mathematical model to simulate nutrient flow through cellular constructs. Interactions among cell proliferation, nutrient consumption, and culture medium circulation were investigated. The model incorporated modified Contois cell-growth kinetics that includes effects of nutrient saturation and limited cell growth. Nutrient uptake was depicted through the Michaelis-Menton kinetics. To describe the culture medium convection, the fluid flow outside the cell-scaffold construct was described by the Navier-Stokes equations, while the fluid dynamics within the construct was modeled by Brinkman's equation for porous media flow. Effects of the media perfusion were examined by including time-dependant porosity and permeability changes due to cell growth. The overall cell volume was considered to consist of cells and extracellular matrices (ECM) as a whole without treating ECM separately. Numerical simulations show when cells were cultured subjected to direct perfusion, they penetrated to a greater extent into the scaffold and resulted in a more uniform spatial distribution. The cell amount was increased by perfusion and ultimately approached an asymptotic value as the perfusion rates increased in terms of the dimensionless Peclet number that accounts for the ratio of nutrient perfusion to diffusion. In addition to enhancing the nutrient delivery, perfusion simultaneously imposes flow-mediated shear stress to the engineered cells. Shear stresses were found to increase with cell growth as the scaffold void space was occupied by the cell and ECM volumes. The macro average stresses increased from 0.2 mPa to 1 mPa at a perfusion rate of 20 microm/s with the overall cell volume fraction growing from 0.4 to 0.7, which made the overall permeability value decrease from 1.35 x 10(-2)cm(2) to 5.51 x 10(-4)cm(2). Relating the simulation results with perfusion experiments in literature, the average shear stresses were below the critical value that would induce the chondrocyte necrosis.     

An integrated process for mammalian cell perfusion cultivation and product purification using a dynamic filter

In the present work, a dynamic filter was employed to develop an integrated perfusion/purification process. A recombinant CHO cell line producing a human anti-HIV IgG was employed in the experiments. In the first part of this work, the dynamic filter was fitted with conventional microfiltration membranes and tested as a new external cell retention device for perfusion cultivations. The filter was connected to a running perfusion bioreactor and operated for approximately 400 h at an average cell concentration of 10 million cells mL(-)(1), whereby cell viability remained above 90% and no problems of sterility were experienced. In the second part of this work, the dynamic filter was employed to simultaneously carry out cell separation and product purification, using membrane adsorbers containing Protein A affinity ligands. An automated system was built, which integrated the features of an automated perfusion bioreactor and of a liquid chromatography system. The IgG was continuously adsorbed onto the affinity membranes and was periodically recovered through elution cycles. After connection of the filter, the system was operated for approximately 300 h, whereby three elution cycles were carried out. No progressive increase in transmembrane pressure was observed, indicating no membrane fouling problems, and the IgG was recovered practically free of contaminants in a 14-fold concentrated form, indicating that the integrated, one-step perfusion/purification process developed during this work is a promising alternative for the production of biologicals derived from mammalian cells.     

Flowing biofilms as a transport mechanism for biomass through porous media under laminar and turbulent conditions in a laboratory reactor system

Fluid flow has been shown to be important in influencing biofilm morphology and causing biofilms to flow over surfaces in flow cell experiments. However, it is not known whether similar effects may occur in porous media. Generally, it is assumed that the primary transport mechanism for biomass in porous media is through convection, as suspended particulates (cells and flocs) carried by fluid flowing through the interstices. However, the flow of biofilms over the surfaces of soils and sediment particles, may represent an important flux of biomass, and subsequently affect both biological activity and permeability. Mixed species bacterial biofilms were grown in glass flow cells packed with 1 mm diameter glass beads, under laminar or turbulent flow (porous media Reynolds number = 20 and 200 respectively). The morphology and dynamic behavior reflected those of biofilms grown in the open flow cells. The laminar biofilm was relatively uniform and after 23 d had inundated the majority of the pore spaces. Under turbulent flow the biofilm accumulated primarily in protected regions at contact points between the beads and formed streamers that trailed from the leeward face. Both biofilms caused a 2 to 3-fold increase in friction factor and in both cases there were sudden reductions in friction factor followed by rapid recovery, suggesting periodic sloughing and regrowth events. Time-lapse microscopy revealed that under both laminar and turbulent conditions biofilms flowed over the surface of the porous media. In some instances ripple structures formed. The velocity of biofilm flow was on the order of 10 mum h(-1) in the turbulent flow cell and 1.0 mum h(-1) in the laminar flow cell.     

A new large‐scale manufacturing platform for complex biopharmaceuticals

Complex biopharmaceuticals, such as recombinant blood coagulation factors, are addressing critical medical needs and represent a growing multibillion‐dollar market. For commercial manufacturing of such, sometimes inherently unstable, molecules it is important to minimize product residence time in non‐ideal milieu in order to obtain acceptable yields and consistently high product quality. Continuous perfusion cell culture allows minimization of residence time in the bioreactor, but also brings unique challenges in product recovery, which requires innovative solutions. In order to maximize yield, process efficiency, facility and equipment utilization, we have developed, scaled‐up and successfully implemented a new integrated manufacturing platform in commercial scale. This platform consists of a (semi‐)continuous cell separation process based on a disposable flow path and integrated with the upstream perfusion operation, followed by membrane chromatography on large‐scale adsorber capsules in rapid cycling mode. Implementation of the platform at commercial scale for a new product candidate led to a yield improvement of 40% compared to the conventional process technology, while product quality has been shown to be more consistently high. Over 1,000,000 L of cell culture harvest have been processed with 100% success rate to date, demonstrating the robustness of the new platform process in GMP manufacturing. While membrane chromatography is well established for polishing in flow‐through mode, this is its first commercial‐scale application for bind/elute chromatography in the biopharmaceutical industry and demonstrates its potential in particular for manufacturing of potent, low‐dose biopharmaceuticals. Biotechnol. Bioeng. 2012; 109: 3049–3058. © 2012 Wiley Periodicals, Inc.     

Flowing biofilms as a transport mechanism for biomass through porous media under laminar and turbulent conditions in a laboratory reactor system

Abstract

Fluid flow has been shown to be important in influencing biofilm morphology and causing biofilms to flow over surfaces in flow cell experiments. However, it is not known whether similar effects may occur in porous media. Generally, it is assumed that the primary transport mechanism for biomass in porous media is through convection, as suspended particulates (cells and flocs) carried by fluid flowing through the interstices. However, the flow of biofilms over the surfaces of soils and sediment particles, may represent an important flux of biomass, and subsequently affect both biological activity and permeability. Mixed species bacterial biofilms were grown in glass flow cells packed with 1 mm diameter glass beads, under laminar or turbulent flow (porous media Reynolds number = 20 and 200 respectively). The morphology and dynamic behavior reflected those of biofilms grown in the open flow cells. The laminar biofilm was relatively uniform and after 23 d had inundated the majority of the pore spaces. Under turbulent flow the biofilm accumulated primarily in protected regions at contact points between the beads and formed streamers that trailed from the leeward face. Both biofilms caused a 2 to 3-fold increase in friction factor and in both cases there were sudden reductions in friction factor followed by rapid recovery, suggesting periodic sloughing and regrowth events. Time-lapse microscopy revealed that under both laminar and turbulent conditions biofilms flowed over the surface of the porous media. In some instances ripple structures formed. The velocity of biofilm flow was on the order of 10 μm h^?1 in the turbulent flow cell and 1.0 μm h^?1 in the laminar flow cell.     

External mass transfer in a biological film reactor

A method for calculating the mass transfer coefficient in a biological film system, under turbulent flow conditions, is presented. It is experimentally found that fluid velocity has a positive effect on the rate of substrate utilization when the system is operated in other than the kinetic regime. A correlation is developed which indicates a dependence of the mass transfer coefficient on the fluid velocity raised to the 0.7 power.     

A pumpless perfusion cell culture cap with two parallel channel layers keeping the flow rate constant

This article presents a novel pumpless perfusion cell culture cap, the gravity‐driven flow rate of which is kept constant by the height difference of two parallel channel layers. Previous pumpless perfusion cell culture systems create a gravity‐driven flow by means of the hydraulic head difference (Δh) between the source reservoir and the drain reservoir. As more media passes from the source reservoir to the drain reservoir, the source media level decreases and the drain media level increases. Thus, previous works based on a gravity‐driven flow were unable to supply a constant flow rate for the perfusion cell culture. However, the proposed perfusion cell culture cap can supply a constant flow rate, because the media level remains unchanged as the media moves laterally through each channel having same media level. In experiments, using the different fluidic resistances, the perfusion cap generated constant flow rates of 871 ± 27 μL h^?1 and 446 ± 11 μL h^?1. The 871 and 446 μL h^?1 flow rates replace the whole 20 mL medium in the petridish with a fresh medium for days 1 and 2, respectively. In the perfusion cell (A549 cell line) culture with the 871 μL h^?1 flow rate, the proposed cap can maintain a lactate concentration of about 2200 nmol mL^?1 and an ammonia concentration of about 3200 nmol mL^?1. Moreover, although the static cell culture maintains cell viability for 5 days, the perfusion cell culture with the 871 μL h^?1 flow rate can maintain cell viability for 9 days. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Very high density of Chinese hamster ovary cells in perfusion by alternating tangential flow or tangential flow filtration in WAVE bioreactor™—part II: Applications for antibody production and cryopreservation

A high cell density perfusion process of monoclonal antibody (MAb) producing Chinese hamster ovary (CHO) cells was developed in disposable WAVE Bioreactor? using external hollow fiber (HF) filter as cell separation device. Tangential flow filtration (TFF) and alternating tangential flow (ATF) systems were compared and process applications of high cell density perfusion were studied here: MAb production and cryopreservation. Operations by perfusion using microfiltration (MF) or ultrafiltration (UF) with ATF or TFF and by fed‐batch were compared. Cell densities higher than 10⁸ cells/mL were obtained using UF TFF or UF ATF. The cells produced comparable amounts of MAb in perfusion by ATF or TFF, MF or UF. MAbs were partially retained by the MF using ATF or TFF but more severely using TFF. Consequently, MAbs were lost when cell broth was discarded from the bioreactor in the daily bleeds. The MAb cell‐specific productivity was comparable at cell densities up to 1.3 × 10⁸ cells/mL in perfusion and was comparable or lower in fed‐batch. After 12 days, six times more MAbs were harvested using perfusion by ATF or TFF with MF or UF, compared to fed‐batch and 28× more in a 1‐month perfusion at 10⁸ cells/mL density. Pumping at a recirculation rate up to 2.75 L/min did not damage the cells with the present TFF settings with HF short circuited. Cell cryopreservation at 0.5 × 10⁸ and 10⁸ cells/mL was performed using cells from a perfusion run at 10⁸ cells/mL density. Cell resuscitation was very successful, showing that this system was a reliable process for cell bank manufacturing. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:768–777, 2013