Lysis of Micrococcus lysodeikticus cells by lysozyme covalently immobilized on the lumen of hollow fibers

Summary A lytic enzyme reactor for microbial cell lysis is described in which lysozyme is immobilized on the lumen of hemodialyzer hollow fibers using epichlorohydrin as a coupling agent. The cell suspension flows through the lumen without any hindrance where the cells are lyzed by the immobilized lysozyme efficiently. Micrococcus lysodeikticus cells at concentrations of 0.25 g/L and 5 g/L were successfully lyzed without clogging the hollow fiber. In comparison with lysozyme immobilized on submicron particles, the activity retention was at least 8 times higher.     

Structure and mechanical properties of wet-spun fibers made from natural cellulose nanofibers

Cellulose nanofibers were prepared by TEMPO-mediated oxidation of wood pulp and tunicate cellulose. The cellulose nanofiber suspension in water was spun into an acetone coagulation bath. The spinning rate was varied from 0.1 to 100 m/min to align the nanofibers to the spun fibers. The fibers spun from the wood nanofibers had a hollow structure at spinning rates of >10 m/min, whereas the fibers spun from tunicate nanofibers were porous. Wide-angle X-ray diffraction analysis revealed that the wood and tunicate nanofibers were aligned to the fiber direction of the spun fibers at higher spinning rates. The wood spun fibers at 100 m/min had a Young's modulus of 23.6 GPa, tensile strength of 321 MPa, and elongation at break of 2.2%. The Young's modulus of the wood spun fibers increased with an increase in the spinning rate because of the nanofiber orientation effect.     

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).     

Modulation of transmembrane pressure in manufacturing scale tangential flow filtration N-1 perfusion seed culture

Mammalian cells were grown to high density in a 3,000 L culture using perfusion with hollow fibers operated in a tangential flow filtration mode. The high-density culture was used to inoculate the production stage of a biomanufacturing process. At constant permeate flux operation, increased transmembrane pressures (TMPs) were observed on the final day of the manufacturing batches. Small scale studies suggested that the filters were not irreversibly fouled, but rather exposed to membrane concentration polarization that could be relieved by tangential sweeping of the hollow fibers. Studies were undertaken to analyze parameters that influence the hydrodynamic profile within hollow fibers; including filter area, cell density, recirculation flow rate, and permeate flow rate. Results indicated that permeate flow rate had the greatest influence on modulating TMP. Further evaluation showed a significant decrease in TMP when permeate flow was reduced, and this occurred without any negative effect on cell growth or viability. Hence, a 30% reduction of permeate flow rate was implemented at manufacturing scale. A stable operation was achieved as TMP was successfully reduced by 75% while preserving all critical factors for performance in the perfusion bioreactor.     

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.     

High density culture of hybridoma cells in a dual hollow fiber bioreactor

Summary Hybridoma cells were cultured for two months in the dual hollow fiber bioreactor (DHFBR) which had been successfully used for high cell density cultures of various microbial cells. In batch suspension culture the concentration of monoclonal antibody (Mab) against human Chorionic Gonadotropin (hCG) and the cell density of Alps 25-3 hybridoma cells were obtained in 30 μg/mL and 2.35×10⁶ cells/mL, respectively. The continuous culture with DHFBR produced Mab of 100–130 μg/mL for 30 days and the estimated cell density in the extracapillary space of DHFBR was 1.87×10⁸ cells/mL based on the antibody production rate. The productivity of Mab was 205 mg/day per litre of the total reactor volume while that of the batch suspension culture was only 10 mg/L day.     

Rapid deoxygenation of red cells and hemoglobin solution using hollow capillary fibers

A newly designed capillary deoxygenator has been constructed by using microporous polypropylene hollow fibers sealed into an airtight plexiglass housing. Oxygenated red cell suspensions and hemoglobin solutions flowing through the hollow fibers were subjected to deoxygenation with a gas mixture composed of 95 percent N2 and 5 percent CO2 passed through the housing. At a given flow rate of the oxygenated fluid, the outgoing fluid pO2 varied directly with hematocrit and inversely with the residence time. With a deoxygenator composed of 144 parallel 100-micrometers fibers with an active length of 10 cm, 2 ml of blood at 10 percent hematocrit can be converted from arterial to venous pO2 in approximately 1 min. The design of this deoxygenator provides a method for rapid deoxygenation of blood without red cell membrane damage or hemolysis.     

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

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

Characterization of an aerated submerged hollow fiber ultrafiltration device for efficient microalgae harvesting

The present work characterizes a submerged aerated hollow fiber polyvinylidene fluorid (PVDF) membrane (0.03 μm) device (Harvester) designed for the ultrafiltration (UF) of microalgae suspensions. Commercial baker''s yeast served as model suspension to investigate the influence of the aeration rate of the hollow fibers on the critical flux (CF, J _c) for different cell concentrations. An optimal aeration rate of 1.25 vvm was determined. Moreover, the CF was evaluated using two different Chlorella cultures (axenic and non‐axenic) of various biomass densities (0.8–17.5 g DW/L). Comparably high CFs of 15.57 and 10.08 L/m/²/h were measured for microalgae concentrations of 4.8 and 10.0 g DW/L, respectively, applying very strict CF criteria. Furthermore, the J _c‐values correlated (negative) linearly with the biomass concentration (0.8–10.0 g DW/L). Concentration factors between 2.8 and 12.4 and volumetric reduction factors varying from 3.5 to 11.5 could be achieved in short‐term filtration, whereat a stable filtration handling biomass concentrations up to 40.0 g DW/L was feasible. Measures for fouling control (aeration of membrane fibers, periodic backflushing) have thus been proven to be successful. Estimations on energy consumption revealed very low energy demand of 17.97 kJ/m³ treated microalgae feed suspension (4.99 × 10⁻³ kWh/m³) and 37.83 kJ/kg treated biomass (1.05 × 10⁻² kWh/kg), respectively, for an up‐concentration from 2 to 40 g DW/L of a microalgae suspension.     

Performance of a membrane reactor for cellobiose hydrolysis

A continuous enzymatic hollow fiber reactor (HFR), obtained by immobilizing cellobiose active cells into the shell side of hollow-fiber modules, was studied. The HFR yield was monitored by glucose analysis resulting from hydrolysis of cellobiose. The residence time of substrate in the bioreactor to obtain convenient hydrolysis yields was calculated from tests carried out by varying the reactor dilution rate in the range 0.001-0.004 L/min. The glucose yield was measured for 300 h (continuous substrate flux). The yield decreased from 40 to 15%. This decrease was due to the loss of specific activity in the operating conditions and to the pressure drop increase from 0.2 to 1.7 atm. The pressure drop increase is in turn dependent on the cell loading (0.2-2.1 g dry cell) and the substrate flux.     

Crossflow microfiltration of animal cells

Laminar shear is the primary mechanism of cell damage, limiting flow rate (and hence flux) in crossflow microfiltration of animal cells. Sensitivity to hydrodynamic and interfacial stress is reduced by the addition of 0.1% Pluronic polyol. A critical average wall shear rate of 3000 s(-1) (above which damage occurs) is found for several cell types, including mammalian and insect cells. Hydrodynamic stress also limits the maximum tip speed in a rotary lobe pump to less than 350 cm/s. Turbulent flow in the recirculation loop piping at Reynolds numbers of up to 71,000 does not cause cell damage. Maximum sustainable flux decreases with cell concentration and increases with cell size (in qualitative agreement with the hydrodynamic lift model). A flux of 30 to 75 L/m(2) h (depending on cell size) can be sustained during 20-fold concentration from 2.5 x 10(6) cells/ml, while maintaining high cell viability.     

Design and performance study of a novel immobilized hollow fiber membrane bioreactor

A dual-layer coaxial hollow fiber (DLHF) bioreactor for cell immobilization developed to overcome nutrients transport limitation is presented. Cells were contained in the annular space between two coaxial hollow fibers, and nutrients were supplied by a forced convective transport from the shell side through the annular space to the lumen side. With judicious selection of the membrane materials, a low operating transmembrane pressure of 50 kPa, and using E. coli as the model organism, a high cell density of 10(11) cells/mL annular space volume and a high cell viability of (up to 80%) were obtained.     

Crossflow filtration of baker's yeast with periodical stopping of permeation flow and bubbling

The periodical stopping of permeation flow was applied to increase the permeation flux in crossflow filtration of commercially available baker's yeast cell suspension. The permeation flux after 3 h filtration in the crossflow filtration increased to 8 x 10(-5) m(3) /m(2) s (290 L/m(2) h) from 2 x 10(-5) m(3)/m(2) s (72 L/m(2) h) by applying the periodical stopping of permeation. Introduction of air bubbles during the stopping period of permeation further increased the flux.(c) John Wiley & Sons, Inc.     

CH4 production from H2 and CO2 byMethanobacterium thermoautotrophicum cells fixed on hollow fibers

Summary Methane was produced from H₂ and CO₂ byMethanobacterium thermoautotrophicum cells fixed on the surface of hollow fibers. The mineral solution permeated through the inside of fibers was consumed by the cells, while the gaseous substrate flowing outside the fibers was directly metabolized to methane. Methane production was proportional to hollow fiber length i.e., contact area between cell layer and gas phase. In repeated batch cultures, the production rates of methane and cell mass were 33.1 L/L reactor/day and 1.75 g cells/L reactor/day, respectively with 90% conversion rate.     

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     

A theoretical method to improve and optimize the design of bioartificial livers

Bioartificial livers (BALs) are a potentially effective countermeasure against liver failure, particularly in cases of acute or fulminant liver failure. It is hoped these devices can sustain a patient's liver function until recovery or transplant. However, no large‐scale clinical trial has yet proven that BALs are particularly effective and evidently design issues remain to be addressed. One aspect of BAL design that must be considered is the mass transfer of adequate oxygen to the hepatocytes within the device. We present here a mathematical modeling approach to oxygen mass transport in a BAL. A mathematical model based upon Krogh cylinders is outlined to describe a diffusion‐limited hollow fiber bioreactor. In addition, operating constraints are defined on the system—cells should not experience hypoxia and the cell population should be of adequate size. By combining modeling results with these operating constraints and presenting the results graphically, “operating region” charts can be constructed for the hollow fiber BAL (HF‐BAL). The effects of varying various operating parameters on the BAL are then established. It is found that smaller radii and short, thin walled fibers are generally advantageous while cell populations in excess of 10 billion could be supported in the BAL with a plasma flow rate of 200 mL/min. For fibers of intermediate length and lumen radius, the minimum number of fibers required to produce a viable design ranges approximately from 7,000–10,000. In theory, this may be enough to support patients with failing livers. Biotechnol. Bioeng. 2010;106: 980–988. © 2010 Wiley Periodicals, Inc.     

Artificial capillary perfusion cell culture: metabolic studies.

Glucose, lactic-acid, and oxygen metabolism of BHK and L929 cells on artificial capillary perfusion units have been studied using several different modes of perfusion. After 7 to 10 days, cells planted in the extracapillary compartment of culture units containing 80 to 150 fibers reached populations that used 0.073 +/- 0.025 mumol per min glucose and 0.76 +/- 0.26 microliter per min oxygen and excreted 0.078 +/- 0.038 mumol per min lactic acid. From these data it is estimated that these units contain approximately 2 x 10(7) cells. The metabolic rate of cultures perfused through the capillaries or through the extracapillary compartment was not affected significantly by change in flow rate except at perfusion flow rates less than or equal to 0.05 ml per min. The cell population, as measured by metabolic activity, did not increase significantly when the serum content of the medium was less than or equal to 1%. No major differences were found in glucose utilization rates of equal numbers of cells on artificial capillaries, on short-term suspension culture, or as monolayers in plastic flasks. Artificial capillary perfusion may provide a simple system for studying metabolism of mammalian cells in culture.     

Optimum fiber spacing in a hollow fiber bioreactor

A high surface area hollow fiber reactor was developed for mammalian cell culture. The reactor employs an interfiber gel matrix of agar or collagen for cell support. A model was developed to predict cell density as a function of fiber spacing. Optimum spacings are calculated for two sizes of Celgard hollow fibers. Ehrlich Ascites Tumor (EAT) cells were grown to an estimated density of 1.1 x 10(8) viable cells/mL in the extracapillary space-corresponding to an overall reactor density of 7 x 10(7) cells/mL. On the basis of available kinetic and diffusivity data, the model predicts that lactate accumulation may limit cell growth in the early stage of medium utilization, while oxygen delivery becomes limiting at later stages.     

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)     

Ultrasonic cell separator as a cell retaining device for high density cultures of plant cell

A system of ultrasonic filter device consisted of an ultrasonic generator, ultrasonic cell separation chamber (resonator) and a guide column, which was developed for suspension cultures of a plant cell. The key operation parameters affecting the efficiency of separation of cells from medium fluid were found to be the voltage of ultrasonic generator, the convective flow rate, and the distance between transducer and reflector. In the high density cultures ofAloe saponaria (>17 g DCW/L), the ultrasonic filter was so efficient that the cell holding time in the separation chamber was 10-fold higher than the case without ultrasonic wave at a convective flow rate of 0.24 cm/min. Furthermore, in perfusion type of high cell density cultures, cell aggregates were observed to be densely held in the ultrasonic chamber by ultrasonic force overcoming both gravitational and drag forces by pump. The accumulated cells were finally overflowed after the holding capacity of the chamber was reached. Back pressure was applied periodically to the resonator to flush cells back to bioreactor. The ultrasonic cell separator could operate over 75 min at a convective flow rate of 0.1 cm/min and at a cell concentration of 17 g DCW/L.