Microbial hollow fiber bioreactors

Hollow fiber membranes have been used for more than a decade to culture mammalian cells and immobilize enzymes. More recently, hollow fiber bioreactors have shown encouraging potential for culturing microbes but many of the practical aspects of their operation have not been explored.     

Immobilized mammalian cell cultivation in hollow fiber bioreactors

Cultivation of animal cells for the production of recombinant proteins is an important method for manufacturing complex proteins requiring posttranslational processing. One of the often considered methods for cultivation is by immobilization of the cells in hollow fiber bioreactors (HFBRs). These systems allow the cells to grow to high densities in a shear protected environment; furthermore the product can be accumulated in high concentration in the case of ultrafiltration HFBRs. Operation and scale-up are constrained by nutrient and product transport with oxygen transfer to growing cells being the most critical parameter. Mathematical models describing HFBRs have proved to be useful in quantitating and understanding the constraints and guiding the scale-up of this approach to animal cell cultivation.     

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.     

Design and operating criteria for hollow fiber bioreactors

We have analyzed the design and operation of hollow fiber bioreactors for cell culture on the basis of cell growth efficiency and non-uniform fiber spacing. Operating diagrams are presented that describe reactor performance as a function of important operating variables like oxygen concentration and the fiber packing density. The diagrams allow one to find the best set of operating conditions for a fixed reactor design, or to rationally adjust the design parameters for fixed operating conditions.     

Comparison of cell growth in T-flasks, in micro hollow fiber bioreactors, and in an industrial scale hollow fiber bioreactor system

In this article, cell growth in a novel micro hollow fiberbioreactor was compared to that in a T-flask and theAcuSyst-Maximizer®, a large scale industrial hollowfiber bioreactor system. In T-flasks, there was relativelylittle difference in the growth rates of one murine hybridomacultured in three different media and for three other murinehybridomas cultured in one medium. However, substantialdifferences were seen in the growth rates of cells in themicro bioreactor under these same conditions. These differencecorrelated well with the corresponding rates of initial cellexpansion in the Maximizer. Quantitative prediction of thesteady-state antibody production rate in the Maximizer was moreproblematic. However, conditions which lead to faster initialcell growth and higher viable cell densities in the microbioreactor correlated with better performance of a cell line inthe Maximizer. These results demonstrate that the microbioreactor is more useful than a T-flask for determining optimalconditions for cell growth in a large scale hollow fiberbioreactor system.     

Protein adsorption in polysulfone hollow fiber bioreactors used for serum-free mammalian cell culture

The recovery of serum-free medium proteins from poly-sulfone hollow fiber bioreactors (HFBRs) was investigated. More than 99% of the initial transferrin was adsorbed to the hydrophobic hollow fibers within 2 h of HFBR operation. A methodology to minimize transferrin adsorption by pre-adsorption of bovine serum albumin (BSA) was developed. BSA adsorption on suspended cut fibers was virtually complete within 1 h. BSA-coated fibers adsorbed only 5% of the transferrin within 10 days, whereas uncoated cut fibers adsorbed more than 99% of the transferrin within 1 h. An improved HFBR startup procedure, using a BSA-coating step before inoculation, resulted in substantially higher transferrin recovery. Additional factors influenced extracapillary space (ECS) transferrin concentrations. Pronounced downstream polarization of transferrin was observed in the ECS. In addition, the 30-kDa nominal molecular weight cutoff ultrafiltration membranes rapidly leaked transferrin from the ECS to the lumen. (c) 1993 John Wiley & Sons, Inc.     

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.     

Mass transfer limitations in diffusion-limited isotropic hollow fiber bioreactors

Diffusional mass transfer limitations in hollow fiber bioreactors – with densely packed whole cells in its extracapillary space to perform biotransformation reactions – have been studied theoretically using a steady-state diffusion and reaction model. Simple analytical expressions have been derived to calculate the radial and axial concentration profiles for zero- and first-order kinetics, as well as to plot effectiveness factor versus Thiele modulus plots for first-order kinetics. The influence of the magnitude of the effective diffusion coefficients, the thickness of the isotropic membrane as well as the size of the annular cell region have been assessed to optimise the reactor performance.     

Large-scale production of murine monoclonal antibodies using hollow fiber bioreactors

We have produced large quantities of murine monoclonal antibodies for in vivo human clinical trials using hollow fiber bioreactors (HFBRs). Thirty-three different hybridoma cell lines have been evaluated in various HFBR systems. Monoclonal antibody (Ab) productivity is highly dependent on the intrinsic secretory rate of each cell line. Other factors that affect Ab production include capillary membrane molecular weight cutoff, and HFBR design. Studies comparing HFBRs to static and suspension culture systems revealed similar Ab productivity. An advantage of the HFBR is that the Ab is concentrated in the extracapillary space, simplifying downstream processing.     

Monoclonal antibody production in hollow fiber bioreactors using serum-free medium

Murine hybridoma cells that produce monoclonal antibody directed against human fibronectin have been cultured in VITAFIBER II and VITAFIBER V hollow fiber bioreactors using defined, serum-free WRC 935 medium. During a two-week growth period, following inoculation of the bioreactors, the cells proliferated to an extent where the bioreactor was filled with cultured cells. Using a 5 sq. ft. VITAFIBER V bioreactor, over 15 grams of antibody were produced during the 40 days of the experiment. This antibody was greater than 95% IgG. During the production period, this packed mass of cells produced 579 +/- 15 mg IgG per day. Because the medium is formulated for air equilibration and high cell densities, WRC 935 medium is especially useful for production of gram quantities of monoclonal antibodies using continuous feed hollow fiber bioreactor cell culture systems.     

Liquid residence time distributions in immobilized cell bioreactors

Previous work has demonstrated that high ethanol productivities can be achieved using yeast or bacterial cells adsorbed onto the surface of ion exchange resin in vertical packed bed bioreactors. The present work quantitatively characterizes the overall degree of backmixing in such reactors at two scales of operation: 2.0 and 8.0 L. Stimulus-response experiments, using two solvents (2,3-butanediol and 2-ethoxyethanol) as tracers, were performed to measure the liquid phase residence time distribution (RTD) during continuous ethanol fermentations using the yeast Saccharomyces cerevisiae and the bacterium Zymomonas mobilis at the 2-L scale, and with S. cerevisiae at the 8-L scale. In order to separately determine the effects of liquid flow rate and gas evolution on the degree of mixing, stimulus-response experiments were also performed in the systems without microbial cells present. The evolution of CO(2) was found to dramatically increase the extent of mixing; however, the tanks-in-series model for non-ideal flow represented the systems adequately. The packed beds were equivalent to over 70 tanks-in-series during abiotic operation while during fermentations, with similar liquid flow rates, they ranged in equivalence from 35 to 15 tanks-in-series. This increased knowledge of the overall degree of mixing in packed bed, immobilized cell bioreactors will allow for more accurate kinetic modelling and efficient scale up of the process.     

A strategy to determine operating parameters in tissue engineering hollow fiber bioreactors

The development of tissue engineering hollow fiber bioreactors (HFB) requires the optimal design of the geometry and operation parameters of the system. This article provides a strategy for specifying operating conditions for the system based on mathematical models of oxygen delivery to the cell population. Analytical and numerical solutions of these models are developed based on Michaelis–Menten kinetics. Depending on the minimum oxygen concentration required to culture a functional cell population, together with the oxygen uptake kinetics, the strategy dictates the model needed to describe mass transport so that the operating conditions can be defined. If c_min ≫ K_m we capture oxygen uptake using zero‐order kinetics and proceed analytically. This enables operating equations to be developed that allow the user to choose the medium flow rate, lumen length, and ECS depth to provide a prescribed value of c_min. When , we use numerical techniques to solve full Michaelis–Menten kinetics and present operating data for the bioreactor. The strategy presented utilizes both analytical and numerical approaches and can be applied to any cell type with known oxygen transport properties and uptake kinetics. Biotechnol. Bioeng. 2011; 108:1450–1461. © 2011 Wiley Periodicals, Inc.     

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.     

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.     

Targeted oxygen delivery within hepatic hollow fiber bioreactors via supplementation of hemoglobin-based oxygen carriers

Hepatic hollow fiber bioreactors are considered a promising class of bioartificial liver assist device (BLAD). Unfortunately, limited oxygen (O(2)) transport to hepatocytes within this device hinders further development. Hepatocytes in vivo (in the liver sinusoid) experience a wide range of oxygen tensions (pO(2) = 25-70 mmHg), which is important for development of proper differentiated function (zonation). Previously, we observed that bovine red blood cell (bRBC) supplementation of the circulating media stream enhanced oxygenation of cultured C3A hepatoma cells compared to a culture with no O(2) carrier (Gordon, J.; Palmer, A. F. Artif. Cells, BloodSubstitutes, Biotechnol. 2006, 33 (3), 297-306). Despite this success, the cells were not exposed to the desired in vivo O(2) spectrum (Sullivan, J.; Gordon, J.; Palmer, A. Biotechnol. Bioeng. 2006, 93 (2) 306-317). We hypothesize that altering the kinetics of O(2) binding/release to/from hemoglobin-based O(2) carriers (HBOCs) could potentially target O(2) delivery to cell cultures. High P(50) (low O(2) affinity) HBOCs preferentially targeted O(2) delivery at high inlet pO(2) values. Conversely, low P(50) (high O(2) affinity) HBOCs targeted O(2) delivery at low inlet pO(2) values. Additionally, inlet pO(2), flow rate, and HBOC concentration were varied to find optimal bioreactor operating conditions. Our results demonstrate that HBOCs can enhance O(2) delivery to cultured hepatocytes, while exposing them to in vivo-like O(2) tensions, which is critical to create a fully functional BLAD.     

Heparin removal from blood using poly(L-lysine) immobilized hollow fiber

Based on the negative charge density characteristics of heparin, an affinity adsorption technique has been developed for the removal of heparin from blood. Poly(L-lysine) . HBr (PLL . HBr), a polycation, was immobilized with the help of cyanogen bromide (BrCN) onto poly(ethylene-vinyl alcohol) (PEVAL) copolymer coated polyethylene (PE) hollow fibers. Heparin bound rapidly onto PLL . HBr imobilized surface in buffer, plasma, and blood. The heparin binding capacity of PLL immobilized surface increased sevenfold as compared to a non-PLL-treated control. When heparinized blood was recirculated through a PLL immobilized PEVAL hollow fiber cartridge, the anticoagulant activity of heparin decreased by 85% from initial activity in 25 min. Moreover, circulation of blood through PLL immobilized hollow fiber did not show any adverse effects; no hemolysis was observed and no significant loss of plasma proteins was noted during the heparin removal process. These results suggest that PLL immobilized surface may be utilized for rapid and effective removal of heparin from blood. (c) 1992 John Wiley & Sons, Inc.     

Urocanic acid production using whole cells immobilized in a hollow fiber reactor

The feasibility of using hollow fiber membrane dialyzers (C-DAK) for immobilization of microbial whole cells was investigated. The cells are located on the shell side of the dialyzer, while substrates and products are free to diffuse across the hollow fiber membranes. The biochemical reaction studied was the conversion of L -histidine to urocanic acid and catalyzed by L -histidine ammonia-lyase. C-DAK dialyzers containing a heat-treated suspension of Pseudomonas fluorescens ATCC 11299b (with L -histidine ammonia–lyase activity) were incorporated into constant volume recycle reactor systems for continuous product formation. A simple model successfully correlated the data and predicted performance. It was found that the reaction was not likely to be diffusion limited, and such a cell immobilization scheme is convenient and workable for continuous production of biochemicals.     

Membrane biofouling behaviors at cold temperatures in pilot-scale hollow fiber membrane bioreactors with quorum quenching

Abstract

In this study, the seasonality of the biofouling behavior of pilot-scale membrane bioreactors (MBRs) run in parallel with vacant sheets and quorum quenching (QQ) sheets using real municipal wastewater was investigated. QQ media delayed fouling, but low temperatures caused severe biofouling. The greater amount of extracellular polymeric substances (EPSs) produced in cold weather was responsible for the faster biofouling of a membrane, even with QQ media. There were significant negative relationships between EPS levels and water temperature. Cold weather was detrimental to the degradation of quorum sensing signal molecules by QQ sheets, whose activity was restored with a higher dose of QQ bacteria. The QQ bacteria in the sheets experienced a slight loss in activity during the early stage of the field test, but survived in the pilot-scale MBR fed with real wastewater. There were no significant discrepancies in treatment efficiency among conventional, vacant, and QQ MBRs.     

Use of ATP to characterize biomass viability in freely suspended and immobilized cell bioreactors

This work describes investigations into the viability of cells growing on 3,4-dichloroaniline (34DCA). Two bioreactors are employed for microbial growth, a continuous stirred tank (CST) bioreactor with a 2-L working volume, and a three-phase air lift (TPAL) bioreactor with a 3-L working volume. Experiments have been performed at several dilution rates between 0.027 and 0.115 h(-1) in the CST bioreactor and between 0.111 and 0.500 h(-1) in the TPAL bioreactor. The specific ATP concentration was calculated at each dilution rate in the suspended biomass in both bioreactors as well as in the immobilized biomass in the TPAL bioreactor. The ATP was extracted from the cells using boiling tris-EDTA buffer (pH 7.75), and the quantity determined using a firefly (bioluminescence) technique. The cultures were inspected under an electron microscope to monitor compositional changes. Results from the CST bioreactor showed that the biomass-specific ATP concentration increases from 0.44 to 1.86 mg ATP g(-1) dry weight (dw) as dilution rate increases from 0.027 to 0.115 h(-1). At this upper dilution rate the cells were washed out. The specific ATP concentration reached a limiting average value of 1.73 mg ATP g(-1) dw, which is assumed to be the quantity of ATP in 100% viable biomass. In the TPAL bioreactor, the ATP level increased with dilution rate in both the immobilized and suspended biomass. The specific ATP concentration in the immobilized biomass increased from approximately 0.051 mg ATP g(-1) dw at dilution rates between 0.111 and 0.200 h(-1) to approximately 0.119 mg ATP g(-1) dw at dilution rates between 0.300 and 0.500 h(-1). This indicates that the immobilized biomass contained a viable cell fraction of around 5%. Based on these results, kinetic data for freely suspended cells should not be applied to the modeling of immobilized cell systems on the assumption that immobilized biomass is 100% viable. (c) 1993 John Wiley & Sons, Inc.