Evaluation of intrinsic immobilized kinetics in hollow fiber reactor systems.

Immobilized cell and enzyme hollow fiber reactors have been developed for a variety of biochemical and biomedical applications. Reported mathematical models for predicting substrate conversion in these reactors have been limited in accuracy because of the use of free-solution kinetic parameters. This paper describes a method for determining the intrinsic kinetics of enzymes immobilized in hollow fiber reactor systems using a mathematical model for diffusion and reaction in porous media and an optimization procedure to fit intrinsic kinetic parameters to experimental data. Two enzymes, a thermophilic beta-galactosidase that exhibits product inhibition and L-lysine alpha-oxidase, were used in the analysis. The intrinsic kinetic parameters show that immobilization enhanced the activity of the beta-galactosidase while decreasing the activity of L-lysine alpha-oxidase. Both immobilized enzymes had higher Km values than did the soluble enzyme, indicating less affinity for the substrate. These results are used to illustrate the significant improvement in the ability to predict substrate conversion in hollow fiber reactors.     

Mammalian cell and protein distributions in ultrafiltration hollow fiber bioreactors

The heterogeneous nature of hollow fiber reactors for cell cultivation requires special considerations for proper design and operation. Downstream concentration of high-molecular-weight proteins has been measured in the shell side of ultrafiltration hollow fiber bioreactors. This distribution resulted from shell-side convective fluxes which caused a concentration polarization of proteins retained by the ultrafiltration membranes (nominal 3 x 10(4) D cutoff). Measurements of the axial hybridoma cell distribution also revealed a downstream concentration of viable cells during the first month of perfusion operation. This is believed to result from the shell-side convective flow and its influence on the inoculum and high-molecular-weight growth factor distributions. The heterogeneous distribution of cells leads to reduced cell numbers and reactor productivities. The mechanisms responsible for these phenomena have been investigated and their implications in process design and operation are considered. The heterogeneous protein and cell distributions on the shell side of hollow fiber bioreactors have been reduced significantly by periodic alternation of the direction of recycle flow and the reactor antibody productivities have been doubled.     

Hollow fiber bioreactors with internal aeration circuits

New hollow fiber bioreactors for aerobic culture were introduced and Aspergillus niger for citric acid production was cultivated as a model system. These reactors consisted of a bundle mixed of hydrophilic membranes for liquid nutrient transport and hydrophobic membranes for gaseous nutrient transport. The cells were successfully cultivated. However, the polymeric hollow fiber membranes were compressed and blocked by excessive fungal cell growth. Citric acid was produced with a high volumetric productivity compared with that of shake-flask fermentation, but the long-term operation was not successful due to a rapid decrease of the production rate.     

Development of a hollow-fiber system for large-scale culture of mammalian cells

A flat-bed hollow-fiber cell culture system has been developed which maximizes the utilization of the large fiber surface while diminishing significantly the problems inherent in a cartridge-type reactor. The reactor core consists of a shallow bed of hollow fibers sandwiched between two stainless-steel microporous filter plates through which the media flow is directed normal to the plane of the fiber bed. Reactors with both 930 and 9300 cm² of fiber surface have been successfully constructed and operated. A variety of cells has been grown in these reactors including SV3T3 cells, baby hamster kidney cells, Vero cells, and rhesus money kidney cells, and cell products such as plasminogen activator and migration inhibition factor (MIF) were produced. This system offers an excellent prototype for scaleup design.     

Continuous Asymmetric Reduction Of 4-Oxoisophorone By Thermophilic Bacteria Using A Hollow Fiber Reactor

Continuous asymmetric reduction of 4-oxoisophorone by the thermophilic bacterium Thermomonospora curvata JTS321 was examined using three reactor systems: packed bed, fluidized bed and hollow fiber. T. curvata was immobilized in polyacrylamide-hydrazide gels when used in the packed and fluidized bed reactors. Of the three reactor systems, the highest productivity (964 mg.1^-1.h^-1) was observed in the fluidized bed reactor. However, many cells grew outside of the gel matrix, causing product contamination. The productivity of the hollow fiber reactor was 504 mg.1^-1.h^-1; the problem of cell contamination of the product was avoided, as the molecular cut-off of the hollow fibers (400 000) was of an appropriate size to prevent cell leakage to the product stream. We therefore consider that the hollow fiber reactor is most suitable for continuous microbial conversions.     

Membrane bioreactors: Engineering aspects

Membrane bioreactors have in-situ separation capability lacking in other types of immobilized cell reactors. This makes them very useful for certain systems. Enzyme reactions utilizing cofactors and hydrolysis of macromolecules are advantageous in membrane reactors. Anaerobic cell culture may be efficiently carried out in membrane cell recycle systems, while aerobic cultures work well in dual hollow fiber reactors. Animal and plant cells have much a better chance of success in membrane reactors because of the protective environment of the reactor and the small oxygen uptake rate of these cells.     

Bioartificial kidney. I. Theoretical analysis of convective flow in hollow fiber modules: application to a bioartificial hemofilter

Analytical expressions describing convective flow in a continuous arteriovenous hollow fiber hemofilter were developed. In the lumen of the hollow fiber membrane, existing analytical expressions were applied to describe velocity profiles and pressure. For flow in the shell (the extracapillary space separating the fibers), analytical expressions for the radial and axial velocity profiles and pressure distribution were derived by first finding the stream function. The expressions are based on a similarity solution. Previous analyses of ultrafiltration have either ignored osmotic pressure or assumed constant shell pressure. In this paper, the axial variation in lumen pressure, shell pressure, and osmotic pressure were accounted for. The predicted filtration rates agree well with the experimental results. This flow model is general enough to describe flow in hollow fiber membrane systems employed as bioreactors (e.g., for cell cultures and as bioartificial organs) and as separators (e.g., ultrafiltration and microfiltration) operating in the open-shell mode. The results were applied to determine the design of an optimally functioning bioartificial hemofilter for use ex vivo or in vivo.     

Continuous Asymmetric Reduction Of 4-Oxoisophorone By Thermophilic Bacteria Using A Hollow Fiber Reactor

Continuous asymmetric reduction of 4-oxoisophorone by the thermophilic bacterium Thermomonospora curvata JTS321 was examined using three reactor systems: packed bed, fluidized bed and hollow fiber. T. curvata was immobilized in polyacrylamide-hydrazide gels when used in the packed and fluidized bed reactors. Of the three reactor systems, the highest productivity (964 mg.1^-1.h^-1) was observed in the fluidized bed reactor. However, many cells grew outside of the gel matrix, causing product contamination. The productivity of the hollow fiber reactor was 504 mg.1^-1.h^-1; the problem of cell contamination of the product was avoided, as the molecular cut-off of the hollow fibers (400 000) was of an appropriate size to prevent cell leakage to the product stream. We therefore consider that the hollow fiber reactor is most suitable for continuous microbial conversions.     

Operation and pressure distribution of immobilized cell hollow fiber bioreactors

It has been cited in the literature on hollow fiber systems that pressure gradients persist, and the transmembrane flux of the hollow fiber system is dependent on the pattern of the pressure gradients. The pattern can be used to its advantage in immobilized enzyme systems. However, with immobilized living cell systems, the pressure gradients lead to a nonuniform environment within the hollow fiber cartridge and not necessarily favorable results. This article provides pertinent pressure-drop data on hollow fiber cartridges which are in flow configurations typical of immobilized cell culture work. The results illuminate operational problems that may arise in the culture of either anchorage dependent or independent cells. Possible solutions with crossflow systems are suggested.     

A radial flow hollow fiber bioreactor for the large-scale culture of mammalian cells

A radial flow hollow fiber bioreactor has been developed that maximizes the utilization of fiber surface for cell growth while eliminating nutrient and metabolic gradients inherent in conventional hollow fiber cartridges. The reactor consists of a central flow distributor tube surrounded by an annular bed of hollow fibers. The central flow distributor tube ensures an axially uniform radial convective flow of nutrients across the fiber bed. Cells attach and proliferate on the outer surface of the fibers. The fibers are pretreated with polylysine to facilitate cell attachment and long-term maintenance of tissuelike densities of cell mass. A mixture of air and CO(2) is fed through the tube side of the hollow fibers, ensuring direct oxygenation of the cells and maintenance of pH. Spent medium diffuses across the cell layer into the tube side of the fibers and is convected away along with the spent gas stream. The bioreactor was run as a recycle reactor to permit maximum utilization of nutrient medium. A bioreactor with a membrane surface area of 1150 cm(2) was developed and H1 cells were grown to a density of 7.3 x 10(6) cells/cm(2).     

Theoretical analysis of the effect of convective flow on solute transport and insulin release in a hollow fiber bioartificial pancreas

The bioartificial pancreas, in which transplanted pancreatic tissue or isolated cells are cultured on a hollow fiber membrane, is an attractive approach to restore physiologic insulin delivery in the treatment of diabetes. Insulin response in prototype devices has been unacceptable due to the large mass transport limitations associated with the membrane and the surrounding shell region. Although available theoretical analyses provide some insight into the combined effects of transport and reaction in the bioartificial pancreas, they cannot quantitatively account for the effects of convective recirculation flow, complex intrinsic insulin secretory kinetics, and non-uniform distribution of pancreatic cells. We have developed a detailed model for glucose and insulin transport and insulin secretion in the hollow fiber bioartificial pancreas based on the solution of the mass and momentum conservation equations describing flow and transport in the lumen, matrix, and shell. Model predictions are in good agreement with literature data obtained in a hollow fiber device with minimal radial convective flow. Although no quantitative data are available for a device with significant radial convection, model simulations demonstrate that convective recirculation flow can dramatically improve insulin response, allowing the device to accurately capture the bi-phasic insulin secretion characteristic of the normal physiologic response. Results provide fundamental insights into the coupling between kinetics and transport in the hollow fiber system and a rational basis for the design of clinical devices.     

Protein fractionation using fast flow immobilized metal chelate affinity membranes

A new group-specific affinity membrane using metal chelates as ligands and inorganic glass hollow fiber microfiltration membranes as support matrices is developed and tested. The study focused on developing the optimum activation and coupling procedures to bind the chelating agent (iminodiacetic acid, IDA) to the surface of the microporous glass hollow fiber membrane and testing the resultant affinity membrane. Starting with three different glass surfaces, five modification reactions were evaluated. All the modified "active surfaces" were first tested for their protein adsorptive properties in batch mode with suspended microporous glass grains using model proteins with known binding characteristics with Cu-IDA systems. The metal loading capacities of the surfaces exhibiting favorable fractionation were then measured by atomic absorption spectroscopy.The results were compared with the results obtained with a commercial material used in immobilized metal affinity column chromatography. The protein binding characteristics of the hollow fiber affinity membranes were also evaluated under conditions of convective flow. This was performed by flowing single solute protein solutions through the microporous membrane at different flow rates. These results were then used to estimate the optimum loading and elution times for the process. A mathematical model incorporating radial diffusion was solved using a finite difference discretization method. Comparison between model predictions and experimental results was performed for four different proteins at one flow rate. These results suggested that the kinetics of adsorption was concentration dependent. Finally, the hollow fiber affinity membranes were challenged with two component mixtures to test their ability to fractionate mixed protein solutions. Efficient separation and good purity were obtained.The results presented here represent the development of a new fast flow affinity membrane process-immobilized metal affinity membranes (IMAM). (c) 1994 John Wiley & Sons, Inc.     

Hybridoma perfusion system using a sedimentation device

Summary A novel sedimentation method with a spiral decanter was utilized with a bioreactor for propagation of hybridoma cells at high densities. The live cell concentration was increased and cell lysis was greatly reduced in this system compared to a tangential flow hollow fiber perfusion system. The specific monoclonal antibody productivity was higher than that obtained using a hollow fiber perfusion system or in a batch culture. Cell specific productivity usually declined over time in long term experiments. The use of the sedimentation device eliminated progressive deterioration of reactor performance usually associated with a perfusion device.     

Enhancing Cell Viability with Pulsating Flow in a Hollow Fiber Bioartificial Liver

A pulsating flow of medium was used to alleviate diffusion and transport limitations in a hollow fiber bioreactor containing a human hepatoblastoma cell line. The strategy is easy to implement but effective. The pulsating flow is introduced by a solenoid pinch valve at the outlet of the bioreactor and regulated by a timing circuit. In a permeability test, the system with pulsating flow had much less membrane fouling as compared to the control, a conventional hollow fiber unit. In hepatocyte culture test runs, the pulsating-flow bioreactor demonstrated the ability to maintain a higher cell viability. Histological sections indicated significantly smaller necrotic regions in the pulsating-flow bioreactor as compared to the conventional unit.     

Comparison of 2 ultrafiltration systems for the concentration of seeded viruses from environmental waters

The use of ultrafiltration as a concentration method to recover viruses from environmental waters was investigated. Two ultrafiltration systems (hollow fiber and tangential flow) in a large- (100 L) and small-scale (2 L) configuration were able to recover greater than 50% of multiple viruses (bacteriophage PP7 and T1 and poliovirus type 2) from varying water turbidities (10-157 nephelometric turbidity units (NTU)) simultaneously. Mean recoveries (n = 3) in ground and surface water by the large-scale hollow fiber ultrafiltration system (100 L) were comparable to recoveries observed in the small-scale system (2 L). Recovery of seeded viruses in highly turbid waters from small-scale tangential flow (2 L) (screen and open channel) and hollow fiber ultrafilters (2 L) (small pilot) were greater than 70%. Clogging occurred in the hollow fiber pencil module and when particulate concentrations exceeded 1.6 g/L and 5.5 g/L (dry mass) in the screen and open channel filters, respectively. The small pilot module was able to filter all concentrates without clogging. The small pilot hollow fiber ultrafilter was used to test recovery of seeded viruses from surface waters from different geographical regions in 10-L volumes. Recoveries >70% were observed from all locations.     

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.     

pH-induced flocculation,indirect electrocoagulation,and hollow fiber filtration techniques for harvesting the saltwater microalga Dunaliella

This research assessed the efficacy of three harvesting methods on a strain of Dunaliella viridis. While there is strong potential to use lipids from microalgae as a feedstock for biofuels to replace petroleum-based fuel, at present microalgal harvesting for biofuel production is not yet economically feasible or energy efficient. pH-induced flocculation (by adjusting the pH of exponentially growing cells), indirect electrocoagulation (applying aluminum hydroxide coagulant to culture), and hollow fiber filtration (separating biomass from medium using tangential flow) were compared as potential harvesting mechanisms for small-scale (3–10 L) and large-scale (30–150 L) volumes of D. viridis. Both pH-induced flocculation and electrocoagulation yielded significant biomass recovery (>95 %), but both methods required removal of added chemicals and/or coagulant before the medium could be reused. In contrast, hollow fiber filtration did not require added chemicals or coagulant, and as another advantage, the filtrate was successfully reused as culture medium without apparent detrimental effects on cell size, cell shape, or cell production. When high salinity stress was imposed on the concentrate produced from the filtration method, total fatty acids (FAs) did not increase. However, total FAs did significantly increase after hollow fiber filtration (49 %) in comparison to FA content before filtration (36 %). This research indicates that hollow fiber filtration as a commercial harvesting mechanism offers attractive advantages as a harvesting mechanism for microalgae such as Dunaliella, relative to pH-induced flocculation and indirect electrocoagulation.     

Pulsatile flow and oxygen transport past cylindrical fiber arrays for an artificial lung: computational and experimental studies

The influence of time-dependent flows on oxygen transport from hollow fibers was computationally and experimentally investigated. The fluid average pressure drop, a measure of resistance, and the work required by the heart to drive fluid past the hollow fibers were also computationally explored. This study has particular relevance to the development of an artificial lung, which is perfused by blood leaving the right ventricle and in some cases passing through a compliance chamber before entering the device. Computational studies modeled the fiber bundle using cylindrical fiber arrays arranged in in-line and staggered rectangular configurations. The flow leaving the compliance chamber was modeled as dampened pulsatile and consisted of a sinusoidal perturbation superimposed on a steady flow. The right ventricular flow was modeled to depict the period of rapid flow acceleration and then deceleration during systole followed by zero flow during diastole. Experimental studies examined oxygen transfer across a fiber bundle with either steady, dampened pulsatile, or right ventricular flow. It was observed that the dampened pulsatile flow yielded similar oxygen transport efficiency to the steady flow, while the right ventricular flow resulted in smaller oxygen transport efficiency, with the decrease increasing with Re. Both computations and experiments yielded qualitatively similar results. In the computational modeling, the average pressure drop was similar for steady and dampened pulsatile flows and larger for right ventricular flow while the pump work required of the heart was greatest for right ventricular flow followed by dampened pulsatile flow and then steady flow. In conclusion, dampening the artificial lung inlet flow would be expected to maximize oxygen transport, minimize work, and thus improve performance.     

Model of oxygen transport limitations in hollow fiber bioreactors

Axial and radial oxygen depletion are believed to be critical scale-limiting factors in the design of cell culture hollow fiber bioreactors. A mathematical analysis of oxygen depletion has been performed in order to develop effectiveness factor plots to aid in the scaling of hollow fiber bioreactors with cells immobilized in the shell-side. Considerations of the lumen mass transport resistances and the axial gradients were added to previous analyses of this immobilization geometry. An order of magnitude analysis was used to evaluate the impact of the shell-side convective fluxes on the oxygen transport. A modified Thiele modulus and a lumen and membrane resistance factor have been derived from the model. Use of these terms in the effectiveness factor plots results in a considerable simplification of the presentation and use of the model. Design criteria such as fiber dimensions and spacing, reactor lengths, and recycle flow rates can be selected using these plots. Model predictions of the oxygen limitations were compared to experimental measurements of the axial cell distributions in a severely oxygen limited hollow fiber bioreactor. Despite considerable uncertainty in our parameters and nonidealities in hollow fiber geometry, the cell distribution correlated well with the modeling results.     

Optimal NS0 cell growth in a hollow fiber bioreactor requires increased serum concentration or a cholesterol supplement on the cell side of the fiber

An NSO/GS cell line secreting a humanized antibody was routinely propagated in a T-flask using 2% serum. For scale-up of antibody production, this cell line was inoculated into a hollow fiber system using the same serum concentration. The metabolic activity increased for a few days in the hollow fiber system, but invariably the activity dropped dramatically as the cells died by day 7. A hollow fiber micro-bioreactor was used as a screening tool to examine possible reasons for cell death in the large-scale system. As seen in the hollow fiber system, cells died when 2% serum was used either on the cell side only or on both sides of the fiber in the micro-bioreactor. In contrast, the use of 20% serum on the cell side of the fiber and basal medium on the non-cell side resulted in good cell expansion at high viability. Regardless of the cell side serum concentration, no further growth enhancements were seen when up to 20% serum was placed on the non-cell side of the fiber. These results suggest that a serum component that does not readily cross the fiber is limiting cell growth in the hollow fiber bioreactors. The addition of a cholesterol-rich lipid supplement resulted in better cell growth in the micro-bioreactor, while the addition of other non-cholesterol lipid supplements resulted in no growth enhancement. The growth-enhancing properties of the cholesterol supplement were more pronounced at lower serum concentrations, suggesting that poor growth at low serum concentration was due to suboptimal cholesterol levels. When the cell side serum concentration was increased to 20% in the hollow fiber system, cells grew and filled the bioreactor, allowing a 39-day production run. These results demonstrate that this NSO cell line requires an increased cell side serum concentration for optimal growth and that this requirement is likely due to the inherent cholesterol dependency of this cell line.