An automated laboratory‐scale methodology for the generation of sheared mammalian cell culture samples

Continuous disk‐stack centrifugation is typically used for the removal of cells and cellular debris from mammalian cell culture broths at manufacturing‐scale. The use of scale‐down methods to characterise disk‐stack centrifugation performance enables substantial reductions in material requirements and allows a much wider design space to be tested than is currently possible at pilot‐scale. The process of scaling down centrifugation has historically been challenging due to the difficulties in mimicking the Energy Dissipation Rates (EDRs) in typical machines. This paper describes an alternative and easy‐to‐assemble automated capillary‐based methodology to generate levels of EDRs consistent with those found in a continuous disk‐stack centrifuge. Variations in EDR were achieved through changes in capillary internal diameter and the flow rate of operation through the capillary. The EDRs found to match the levels of shear in the feed zone of a pilot‐scale centrifuge using the experimental method developed in this paper (2.4×10⁵ W/Kg) are consistent with those obtained through previously published computational fluid dynamic (CFD) studies (2.0×10⁵ W/Kg). Furthermore, this methodology can be incorporated into existing scale‐down methods to model the process performance of continuous disk‐stack centrifuges. This was demonstrated through the characterisation of culture hold time, culture temperature and EDRs on centrate quality.     

A scale‐down mimic for mapping the process performance of centrifugation,depth and sterile filtration

In the production of biopharmaceuticals disk‐stack centrifugation is widely used as a harvest step for the removal of cells and cellular debris. Depth filters followed by sterile filters are often then employed to remove residual solids remaining in the centrate. Process development of centrifugation is usually conducted at pilot‐scale so as to mimic the commercial scale equipment but this method requires large quantities of cell culture and significant levels of effort for successful characterization. A scale‐down approach based upon the use of a shear device and a bench‐top centrifuge has been extended in this work towards a preparative methodology that successfully predicts the performance of the continuous centrifuge and polishing filters. The use of this methodology allows the effects of cell culture conditions and large‐scale centrifugal process parameters on subsequent filtration performance to be assessed at an early stage of process development where material availability is limited. Biotechnol. Bioeng. 2016;113: 1934–1941. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

CFD of mixing of multi‐phase flow in a bioreactor using population balance model

Mixing in bioreactors is known to be crucial for achieving efficient mass and heat transfer, both of which thereby impact not only growth of cells but also product quality. In a typical bioreactor, the rate of transport of oxygen from air is the limiting factor. While higher impeller speeds can enhance mixing, they can also cause severe cell damage. Hence, it is crucial to understand the hydrodynamics in a bioreactor to achieve optimal performance. This article presents a novel approach involving use of computational fluid dynamics (CFD) to model the hydrodynamics of an aerated stirred bioreactor for production of a monoclonal antibody therapeutic via mammalian cell culture. This is achieved by estimating the volume averaged mass transfer coefficient (k_La) under varying conditions of the process parameters. The process parameters that have been examined include the impeller rotational speed and the flow rate of the incoming gas through the sparger inlet. To undermine the two‐phase flow and turbulence, an Eulerian‐Eulerian multiphase model and k‐ε turbulence model have been used, respectively. These have further been coupled with population balance model to incorporate the various interphase interactions that lead to coalescence and breakage of bubbles. We have successfully demonstrated the utility of CFD as a tool to predict size distribution of bubbles as a function of process parameters and an efficient approach for obtaining optimized mixing conditions in the reactor. The proposed approach is significantly time and resource efficient when compared to the hit and trial, all experimental approach that is presently used. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:613–628, 2016     

Simulation of the dynamic packing behavior of preparative chromatography columns via discrete particle modeling

Preparative packed‐bed chromatography using polymer‐based, compressible, porous resins is a powerful method for purification of macromolecular bioproducts. During operation, a complex, hysteretic, thus, history‐dependent packed bed behavior is often observed but theoretical understanding of the causes is limited. Therefore, a rigorous modeling approach of the chromatography column on the particle scale has been made which takes into account interparticle micromechanics and fluid–particle interactions for the first time. A three‐dimensional deterministic model was created by applying Computational Fluid Dynamics (CFD) coupled with the Discrete Element Method (DEM). The column packing behavior during either flow or mechanical compression was investigated in‐silico and in laboratory experiments. A pronounced axial compression–relaxation profile was identified that differed for both compression strategies. Void spaces were clearly visible in the packed bed after compression. It was assumed that the observed bed inhomogeneity was because of a force‐chain network at the particle scale. The simulation satisfactorily reproduced the measured behavior regarding packing compression as well as pressure‐flow dependency. Furthermore, the particle Young's modulus and particle–wall friction as well as interparticle friction were identified as crucial parameters affecting packing dynamics. It was concluded that compaction of the chromatographic bed is rather because of particle rearrangement than particle deformation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:363–371, 2016     

Effects of adsorbent properties on zone spreading in expanded bed chromatography

The mixing performance as well as the adsorption performance in expanded bed chromatography (EBC) was investigated by using various types of adsorption media (average particle size = 100–700 m, density = 1100–1700 kg/m³, base matrix = hydroxyapatite, styrene-divinylbenzene, cross-linked agarose). The scale down study with 0.8 cm diameter columns was also attempted. Pulse response curves were measured with vitamin B₁₂ as a tracer [Residence time distribution RTD experiments], and the HETP (height equivalent to a theoretical plate or plate height) values were calculated from the peak variance and the peak retention time. The HETP values for different types of packing media tested showed very similar values (0.5–1.0 cm), which did not depend on the flow-rate or the column diameter (0.8–2.6 cm). Dynamic binding capacity (DBC) values of lactic acid on a Dowex anion-exchange resin were determined from breakthrough curve (BTC) measurements for both EBC and fixed bed chromatography (FBC). The DBC values for EBC were similar to those for FBC. When the liquid feed contained insoluble particles (yeast cells) the degree of mixing increased. However, the contribution of the mixing to the total spreading of BTCs for EBC was usually small so that this increase in the mixing did not affect the adsorption performance or the DBC values significantly.     

Ultra scale‐down device to predict dewatering levels of solids recovered in a continuous scroll decanter centrifuge

During centrifugation operation, the major challenge in the recovery of extracellular proteins is the removal of the maximum liquid entrapped within the spaces between the settled solids–dewatering level. The ability of the scroll decanter centrifuge (SDC) to process continuously large amounts of feed material with high concentration of solids without the need for resuspension of feeds, and also to achieve relatively high dewatering, could be of great benefit for future use in the biopharmaceutical industry. However, for reliable prediction of dewatering in such a centrifuge, tests using the same kind of equipment at pilot‐scale are required, which are time consuming and costly. To alleviate the need of pilot‐scale trials, a novel USD device, with reduced amounts of feed (2 mL) and to be used in the laboratory, was developed to predict the dewatering levels of a SDC. To verify USD device, dewatering levels achieved were plotted against equivalent compression (Gt_comp) and decanting (Gt_dec) times, obtained from scroll rates and feed flow rates operated at pilot‐scale, respectively. The USD device was able to successfully match dewatering trends of the pilot‐scale as a function of both Gt_comp and Gt_dec, particularly for high cell density feeds, hence accounting for all key variables that influenced dewatering in a SDC. In addition, it accurately mimicked the maximum dewatering performance of the pilot‐scale equipment. Therefore the USD device has the potential to be a useful tool at early stages of process development to gather performance data in the laboratory thus minimizing lengthy and costly runs with pilot‐scale SDC. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1494–1502, 2013     

Packing of large‐scale chromatography columns with irregularly shaped glass based resins using a stop‐flow method

Rigid chromatography resins, such as controlled pore glass based adsorbents, offer the advantage of high permeability and a linear pressure‐flow relationship irrespective of column diameter which improves process time and maximizes productivity. However, the rigidity and irregularly shaped nature of these resins often present challenges in achieving consistent and uniform packed beds as formation of bridges between resin particles can hinder bed consolidation. The standard flow‐pack method when applied to irregularly shaped particles does not yield well‐consolidated packed beds, resulting in formation of a head space and increased band broadening during operation. Vibration packing methods requiring the use of pneumatically driven vibrators are recommended to achieve full packed bed consolidation but limitations in manufacturing facilities and equipment may prevent the implementation of such devices. The stop‐flow packing method was developed as an improvement over the flow‐pack method to overcome these limitations and to improve bed consolidation without the use of vibrating devices. Transition analysis of large‐scale columns packed using the stop‐flow method over multiple cycles has shown a two‐ to three‐fold reduction of change in bed integrity values as compared to a flow‐packed bed demonstrating an improvement in packed bed stability in terms of the height equivalent to a theoretical plate (HETP) and peak asymmetry (A_s). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1319–1325, 2014     

On-line monitoring of glucose and/or lactate in a fermentation process using an expanded micro-bed flow injection analyser

A novel flow injection biosensor system for monitoring fermentation processes has been developed using an expanded micro bed as the enzyme reactor. An expanded bed reactor is capable of handling a mobile phase containing suspended matter like cells and cell debris. Thus, while the analyte is free to interact with the adsorbent, the suspended particulate matter passes through unhindered. With the use of a scaled down expanded bed in the flow injection analysis (FIA) system, it was possible to analyse samples directly from a fermentor without the pretreatment otherwise required to extract the analyte or remove the suspended cells. This technique, therefore, provides a means to determine the true concentrations of the metabolites in a fermentor, with more ease than possible with other techniques.Glucose oxidase immobilised on STREAMLINE was used to measure glucose concentration in a suspension of dead yeast cells. There was no interference from the cell particles even at high cell densities such as 15 gm dry weight per litre. The assay time was about 6 min. Accuracy and reproducibility of the system was found to be good. In another scheme, lactate oxidase was covalently coupled to STREAMLINE for expanded bed operation. With the on-line expanded micro bed FIA it was possible to follow the fermentation with Lactobacillus casei.     

CFD simulation of non-Newtonian fluid flow in anaerobic digesters

A general mathematical model that predicts the flow fields in a mixed-flow anaerobic digester was developed. In this model, the liquid manure was assumed to be a non-Newtonian fluid, and the flow governed by the continuity, momentum, and k-epsilon standard turbulence equations, and non-Newtonian power law model. The commercial computational fluid dynamics (CFD) software, Fluent, was applied to simulate the flow fields of lab-scale, scale-up, and pilot-scale anaerobic digesters. The simulation results were validated against the experimental data from literature. The flow patterns were qualitatively compared for Newtonian and non-Newtonian fluids flow in a lab-scale digester. Numerical simulations were performed to predict the flow fields in scale-up and pilot-scale anaerobic digesters with different water pump power inputs and different total solid concentration (TS) in the liquid manure. The optimal power inputs were determined for the pilot-scale anaerobic digester. Some measures for reducing dead and low velocity zones were proposed based upon the CFD simulation results.     

Direct comparison of four methods to construct xylem vulnerability curves: Differences among techniques are linked to vessel network characteristics

During periods of dehydration, water transport through xylem conduits can become blocked by embolism formation. Xylem embolism compromises water supply to leaves and may lead to losses in productivity or plant death. Vulnerability curves (VCs) characterize plant losses in conductivity as xylem pressures decrease. VCs are widely used to characterize and predict plant water use at different levels of water availability. Several methodologies for constructing VCs exist and sometimes produce different results for the same plant material. We directly compared four VC construction methods on stems of black cottonwood (Populus trichocarpa), a model tree species: dehydration, centrifuge, X‐ray–computed microtomography (microCT), and optical. MicroCT VC was the most resistant, dehydration and centrifuge VCs were intermediate, and optical VC was the most vulnerable. Differences among VCs were not associated with how cavitation was induced but were related to how losses in conductivity were evaluated: measured hydraulically (dehydration and centrifuge) versus evaluated from visual information (microCT and optical). Understanding how and why methods differ in estimating vulnerability to xylem embolism is important for advancing knowledge in plant ecophysiology, interpreting literature data, and using accurate VCs in water flux models for predicting plant responses to drought.     

The effect of antiapoptosis genes on clarification performance

Optimal bioreactor harvest time is typically determined based on maximizing product titer without compromising product quality. We suggest that ease of downstream purification should also be considered during harvest. In this view, we studied the effect of antiapoptosis genes on downstream performance. Our hypothesis was that more robust cells would exhibit less cell lysis and thus generate lower levels of cell debris and host‐cell contaminants. We focused on the clarification unit operation, measuring postclarification turbidity and host‐cell protein (HCP) concentration as a function of bioreactor harvest time/cell viability. In order to mimic primary clarification using disk‐stack centrifugation, a scale‐down model consisting of a rotating disk (to simulate shear in the inlet feed zone of the centrifuge) and a swinging‐bucket lab centrifuge was used. Our data suggest that in the absence of shear during primary clarification (typical of depth filters), a 20–50% reduction in HCP levels and 50–65% lower postcentrifugation turbidity was observed for cells with antiapoptosis genes compared to control cells. However, on exposing the cells to shear levels typical in a disk‐stack centrifuge, the reduction in HCP was 10–15% while no difference in postcentrifugation turbidity was observed. The maximum benefit of antiapoptosis genes is, therefore, realized using clarification options that involve low shear, <1 × 10⁶ W/m³ and minimal damage to the cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:100–107, 2014     

Laccase production from oil palm industry solid waste: Statistical optimization of selected process parameters

Oil palm frond parenchyma tissue was used as a solid substrate for the production of laccase via solid‐state fermentation using the white rot fungus Pycnoporus sanguineus. With a rectangular aluminium tray as solid‐state fermentation bioreactor, process parameters such as bed height, moisture and supplemented nitrogen (as urea solution) levels were studied and optimized using a statistical design of experiment. The moisture level exerted a significant effect on the process. The interaction effect observed between bed height and supplemented nitrogen level suggested that uniform distribution of supplemented nitrogen into the substrate bed was important. The proposed regression model sufficiently predicted the process response over the experimental range tested. The optimum parameter combination for laccase production was a 3‐cm bed height, 72% w/w moisture and 0.21% w/v supplemented nitrogen. Laccase productivity remained constant when the tray size was increased from 1.4 to 3.4‐fold.     

Ultra scale‐down prediction using microwell technology of the industrial scale clarification characteristics by centrifugation of mammalian cell broths

This article describes how a combination of an ultra scale‐down (USD) shear device feeding a microwell centrifugation plate may be used to provide a prediction of how mammalian cell broth will clarify at scale. In particular a method is described that is inherently adaptable to a robotic platform and may be used to predict how the flow rate and capacity (equivalent settling area) of a centrifuge and the choice of feed zone configuration may affect the solids carry over in the supernatant. This is an important consideration as the extent of solids carry over will determine the required size and lifetime of a subsequent filtration stage or the passage of fine particulates and colloidal material affecting the performance and lifetime of chromatography stages. The extent of solids removal observed in individual wells of a microwell plate during centrifugation is shown to correlate with the vertical and horizontal location of the well on the plate. Geometric adjustments to the evaluation of the equivalent settling area of individual wells (Σ_M) results in an improved prediction of solids removal as a function of centrifuge capacity. The USD centrifuge settling characteristics need to be as for a range of equivalent flow rates as may be experienced at an industrial scale for a machine of different shear characteristics in the entry feed zone. This was shown to be achievable with two microwell‐plate based measurements and the use of varying fill volumes in the microwells to allow the rapid study of a fivefold range of equivalent flow rates (i.e., at full scale for a particular industrial centrifuge) and the effect of a range of feed configurations. The microwell based USD method was used to examine the recovery of CHO‐S cells, prepared in a 5 L reactor, at different points of growth and for different levels of exposure to shear post reactor. The combination of particle size distribution measurements of the cells before and after shear and the effect of shear on the solids remaining after centrifugation rate provide insight into the state of the cells throughout the fermentation and the ease with which they and accumulated debris may be removed by continuous centrifugation. Hence bioprocess data are more readily available to help better integrate cell culture and cell removal stages and resolve key bioprocess design issues such as choice of time of harvesting and the impact on product yield and contaminant carry over. Operation at microwell scale allows data acquisition and bioprocess understanding over a wide range of operating conditions that might not normally be achieved during bioprocess development. Biotechnol. Bioeng. 2009; 104: 321–331 © 2009 Wiley Periodicals, Inc.     

Mathematical model for a three-phase fluidized bed biofilm reactor in wastewater treatment

A mathematical model for a three phase fluidized bed bioreactor (TFBBR) was proposed to describe oxygen utilization rate, biomass concentration and the removal efficiency of Chemical Oxygen Demand (COD) in wastewater treatment. The model consisted of the biofilm model to describe the oxygen uptake rate and the hydraulic model to describe flow characteristics to cause the oxygen distribution in the reactor. The biofilm model represented the oxygen uptake rate by individual bioparticle and the hydrodynamics of fluids presented an axial dispersion flow with back mixing in the liquid phase and a plug flow in the gas phase. The difference of settling velocity along the column height due to the distributions of size and number of bioparticle was considered. The proposed model was able to predict the biomass concentration and the dissolved oxygen concentration along the column height. The removal efficiency of COD was calculated based on the oxygen consumption amounts that were obtained from the dissolved oxygen concentration. The predicted oxygen concentration by the proposed model agreed reasonably well with experimental measurement in a TFBBR. The effects of various operating parameters on the oxygen concentration were simulated based on the proposed model. The media size and media density affected the performance of a TFBBR. The dissolved oxygen concentration was significantly affected by the superficial liquid velocity but the removal efficiency of COD was significantly affected by the superficial gas velocity. An erratum to this article can be found online at .     

A new method to predict flowability using a microscale fluid bed

The purpose of this research was to develop a new method to predict the flow behavior of pharmaceutical powders using a multichamber microscale fluid bed. Different amounts of poorly flowing paracetamol were added to various grades of microcrystalline celluloses and silicified microcrystalline cellulose powders. Magnesium stearate was used as a lubricant. Experimental minimum fluidization velocities (u _mf) were defined using 2 to 4 g (equal to 10 mL) of material (Video 1). The reference flowability of the powders was determined using a specific flow meter. Also, the weight variation of the compressed powders, using a single-punch press, was measured. When the amount of paracetamol in the excipients was increased, the experimentalu _mf increased and the fluidization behavior grew worse (Video 2). Principal component analysis (PCA) established that the pressure difference over the bed as a function of fluidization velocity could be used to characterize the behavior of powders. The increase in poor fluidization behavior of the powders was in accordance with the increasing amount of paracetamol and with the increasing weight variation of the tablets. Furthermore, the angle of repose and the flow rate of silicified microcrystalline cellulose powders were predicted using a partial least squares (PLS) model. The developed method to predict flowability is a promising approach for use in the preformulation and formulation stages of new drug candidates, for example.     

Integrative analyses reveal signaling pathways underlying familial breast cancer susceptibility

The signaling events that drive familial breast cancer (FBC) risk remain poorly understood. While the majority of genomic studies have focused on genetic risk variants, known risk variants account for at most 30% of FBC cases. Considering that multiple genes may influence FBC risk, we hypothesized that a pathway‐based strategy examining different data types from multiple tissues could elucidate the biological basis for FBC. In this study, we performed integrated analyses of gene expression and exome‐sequencing data from peripheral blood mononuclear cells and showed that cell adhesion pathways are significantly and consistently dysregulated in women who develop FBC. The dysregulation of cell adhesion pathways in high‐risk women was also identified by pathway‐based profiling applied to normal breast tissue data from two independent cohorts. The results of our genomic analyses were validated in normal primary mammary epithelial cells from high‐risk and control women, using cell‐based functional assays, drug‐response assays, fluorescence microscopy, and Western blotting assays. Both genomic and cell‐based experiments indicate that cell–cell and cell–extracellular matrix adhesion processes seem to be disrupted in non‐malignant cells of women at high risk for FBC and suggest a potential role for these processes in FBC development.     

Influences of Woody Debris on Flow Patterns and Channel Morphology in a Low Energy,Sand‐Bed Stream Reach

In lowland areas, such as the glacial landscapes of eastern Germany, sand‐bed streams are the most common stream type. They have low gradients and their hydrological regime is often subdued due to the frequent interruption by lakes. Very few is known about the influence of woody debris in these streams, since nearly all previous studies are from high‐gradient conditions, where streams have coarse bed sediments and harsh hydrological regimes. The research objectives of this study were first to assess the quasi‐natural quantity and quality of wood in a lowland sand‐bed stream and second to understand the influence of wood on the channel morphology and the flow patterns at base‐flow. The three‐dimensional stream bed relief was surveyed by electronic distance measurement. The position and the size of large woody debris was assessed by close‐up photography. An acoustic Doppler velocimeter was used to record the patterns of flow velocity and turbulence. Overlay and analysis of the spatial data was done using a Geographic Information System. The standing stock of wood was 1.9 m³ and 39 woody elements per 100 m² of stream bed. The flow pattern was clearly controlled by the wood. Woody elements elevated above the stream bed deflected flow and locally caused strong secondary current, high turbulence, and scour of the stream bed at baseflow. Wood resting directly on the stream bed, which contributed the majority of the wood inside the bank‐full channel, determined the roughness of the stream bed. Near‐bed flow patterns observed were isolated roughness flow and wake interference flow, which was registered inside the accumulations of wood. 68% of the stream bed had shear stress above critical. Hence, the secondary morphological structures of the sand‐bed were controlled at base‐flow by the flow which was determined by the woody debris distribution.     

Flow pattern visualization in a mimic anaerobic digester using CFD

Three-dimensional steady-state computational fluid dynamics (CFD) simulations were performed in mimic anaerobic digesters to visualize their flow pattern and obtain hydrodynamic parameters. The mixing in the digester was provided by sparging gas at three different flow rates. The gas phase was simulated with air and the liquid phase with water. The CFD results were first evaluated using experimental data obtained by computer automated radioactive particle tracking (CARPT). The simulation results in terms of overall flow pattern, location of circulation cells and stagnant regions, trends of liquid velocity profiles, and volume of dead zones agree reasonably well with the experimental data. CFD simulations were also performed on different digester configurations. The effects of changing draft tube size, clearance, and shape of the tank bottoms were calculated to evaluate the effect of digester design on its flow pattern. Changing the draft tube clearance and height had no influence on the flow pattern or dead regions volume. However, increasing the draft tube diameter or incorporating a conical bottom design helped in reducing the volume of the dead zones as compared to a flat-bottom digester. The simulations showed that the gas flow rate sparged by a single point (0.5 cm diameter) sparger does not have an appreciable effect on the flow pattern of the digesters at the range of gas flow rates used.     

Modeling of transient flow through a viscoelastic preparative chromatography packing

The common method for purification of macromolecular bioproducts is preparative packed‐bed chromatography using polymer‐based, compressible, viscoelastic resins. Because of a downstream processing bottleneck, the chromatography equipment is often operated at its hydrodynamic limit. In this case, the resins may exhibit a complex behavior which results in compression–relaxation hystereses. Up to now, no modeling approach of transient flow through a chromatography packing has been made considering the viscoelasticity of the resins. The aim of the present work was to develop a novel model and compare model calculations with experimental data of two agarose‐based resins. Fluid flow and bed permeability were modeled by Darcy's law and the Kozeny–Carman equation, respectively. Fluid flow was coupled to solid matrix stress via an axial force balance and a continuity equation of a deformable packing. Viscoelasticity was considered according to a Kelvin–Voigt material. The coupled equations were solved with a finite difference scheme using a deformable mesh. The model boundary conditions were preset transient pressure drop functions which resemble simulated load/elution/equilibration cycles. Calculations using a homogeneous model (assuming constant variables along the column height) gave a fair agreement with experimental data with regard to predicted flow rate, bed height, and compression–relaxation hysteresis for symmetric as well as asymmetric pressure drop functions. Calculations using an inhomogeneous model gave profiles of the bed porosity as a function of the bed height. In addition, the influence of medium wall support and intraparticle porosity was illustrated. The inhomogeneous model provides insights that so far are not easily experimentally accessible. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:958–967, 2013     

Biodiesel production using Aspergillus niger as a whole‐cell biocatalyst in a packed‐bed reactor

Enzymatic lipase transesterification of palm oil to biodiesel in a packed‐bed reactor (PBR) using a novel strain of the fungus Aspergillus niger, immobilized within polyurethane biomass support particles (BSPs), was investigated. A three‐step addition of methanol was used to reduce lipase inhibition by immiscible methanol. The influence of water content and PBR flow rate was investigated. FAME yield was enhanced with an increase of PBR flow rate in the range of 0.15–30 L h^?1, where inefficient mixing of the reaction mixture at lower flow rates resulted in low conversion rates i.e. 69% after 72‐h reaction. Adding the third mole equivalent of methanol resulted in lipase inhibition due to methanol migration into the accumulated glycerol layer. Glutaraldehyde (GA) solution (0.5 vol.%) was used to stabilize lipase activity, which led to a high FAME yield (>90%) in the PBR after 72‐h of reaction time at a flow rate of 15 L h^?1, and a water content of 15%. Moreover, a high conversion rate (>85%) was maintained after four palm oil batch conversion cycles in the PBR. In contrast, lipase activity of non‐GA‐treated cells decreased with each PBR batch cycle, where only 70% FAME was produced after the forth PBR cycle. Transesterification of palm oil in a PBR system using BSPs‐immobilized A. niger as a whole‐cell biocatalyst is a viable process for enzymatic biodiesel production.