On-line gas analysis in animal cell cultivation: I. Control of dissolved oxygen and pH

To monitor gas reaction rates in animal cell culture at constant dissolved oxygen concentration (DO) and constant pH it was necessary to develop improved control methods. Decoupling of both controllrs was obtained by manipulation of molar fractions of oxygen and carbon dioxide in the gas phase. Two pairs of DO and pH controllers were designed and tested both in simulation and exprimental runs. The first controller pair was developed for headspace aeration only, whereas the second controller pair was designed for bubble aeration using a microsparger and flushing the headspace with helium. pH was controlled by a conventional discrete PID controller in its velocity form. For DO control two linear state space feedback controllers with parameter adaptation were established. In these controllers the oxygen uptake rate (OUR) was considered as a disturbance and was not included in the mathematical model. The feedback gain adaptation was based on the difference between the actual molar fraction of oxygen at time step n and the initial molar fraction. This difference is related to OUR and was used to increase or decrease the state feedback controller gain (k and k(1), respectively) in a slow manner. With these controllers it was possible to get an excellent online estimate of OUR. In the case of bubble aeration a simple gas phase mass balance was sufficient, whereas during the headspace aeration a liquid phase balance was required. It has been shown that determination of OUR using gas balance requires a significantly better controller performance compared to just keeping DO and pH within reasonable limits. (c) 1995 John Wiley & Sons, Inc.     

Efficient and reproducible mammalian cell bioprocesses without probes and controllers?

Bioprocesses for recombinant protein production with mammalian cells are typically controlled for several physicochemical parameters including the pH and dissolved oxygen concentration (DO) of the culture medium. Here we studied whether these controls are necessary for efficient and reproducible bioprocesses in an orbitally shaken bioreactor (OSR). Mixing, gas transfer, and volumetric power consumption (P(V)) were determined in both a 5-L OSR and a 3-L stirred-tank bioreactor (STR). The two cultivation systems had a similar mixing intensity, but the STR had a lower volumetric mass transfer coefficient of oxygen (k(L)a) and a higher P(V) than the OSR. Recombinant CHO cell lines expressing either tumor necrosis factor receptor as an Fc fusion protein (TNFR:Fc) or an anti-RhesusD monoclonal antibody were cultivated in the two systems. The 5-L OSR was operated in an incubator shaker with 5% CO(2) in the gas environment but without pH and DO control whereas the STR was operated with or without pH and DO control. Higher cell densities and recombinant protein titers were obtained in the OSR as compared to both the controlled and the non-controlled STRs. To test the reproducibility of a bioprocess in a non-controlled OSR, the two CHO cell lines were each cultivated in parallel in six 5-L OSRs. Similar cell densities, cell viabilities, and recombinant protein titers along with similar pH and DO profiles were achieved in each group of replicates. Our study demonstrated that bioprocesses can be performed in OSRs without pH or DO control in a highly reproducible manner, at least at the scale of operation studied here.     

Control of dissolved oxygen concentration in mammalian cell culture using a computer-coupled mass flow controller

Summary A simple proportional control system for dissolved oxygen (DO) concentration in cell culture medium was developed by using a computer-coupled mass flow controller. The DO levels were very stable during the cultivation of Vero-6, while flow rates of air and/or oxygen enriched air were gradually changed depending on the DO concentration and the preset DO level. Vero-6 cells could grow normally to the confluence in the range of 30% and 50% of DO. Growth of Vero-6 at 10% of DO was markedly retarded.     

Evaluation of oxygen transfer rates in stirred-tank bioreactors for clinical manufacturing

Several methods are available for determining the volumetric oxygen transfer coefficient in bioreactors, though their application in industrial bioprocess has been limited. To be practically useful, mass transfer measurements made in nonfermenting systems must be consistent with observed microbial respiration rates. This report details a procedure for quantifying the relationship between agitation frequency and oxygen transfer rate that was applied in stirred-tank bioreactors used for clinical biologics manufacturing. The intrinsic delay in dissolved oxygen (DO) measurement was evaluated by shifting the bioreactor pressure and fitting a first-order mathematical model to the DO response. The dynamic method was coupled with the DO lag results to determine the oxygen transfer rate in Water for Injection (WFI) and a complete culture medium. A range of agitation frequencies was investigated at a fixed air sparge flow rate, replicating operating conditions used in Pichia pastoris fermentation. Oxygen transfer rates determined by this method were in excellent agreement with off-gas calculations from cultivation of the organism (P = 0.1). Fermentation of Escherichia coli at different operating parameters also produced respiration rates that agreed with the corresponding dynamic method results in WFI (P = 0.02). The consistency of the dynamic method results with the off-gas data suggests that compensation for the delay in DO measurement can be combined with dynamic gassing to provide a practical, viable model of bioreactor oxygen transfer under conditions of microbial fermentation.     

Control of carbon source supply and dissolved oxygen by use of carbon dioxide concentration of exhaust gas in fed-batch culture

Accurate and automatic control strategies for a feedback-control system of volatile carbon source feeding and dissolved oxygen (DO) level were investigated. To maintain the optimal ethanol concentration for microbial growth, carbon dioxide concentration in exhaust gas was used as a stepwise control parameter of ethanol feeding. A proportional-differential (PD) control program was used to correct the errors. The coefficient of stepwise control was calculated stoichiometrically, and parameters of PD were experimentally preset and were not changed during cultivation. DO was also controlled by the PD control and the stepwise program based on carbon dioxide concentration of the exhaust gas. Agitation speed and partial pressure of oxygen of the inlet gas were changed stepwise in accordance with the oxygen consumption rate. The stepwise coefficients were estimated from stoichiometry and material balance of molecular oxygen. The PD control program was only used for the agitation speed control to correct the fluctuations of DO level. The parameters did not need to be changed during cultivation. By use of these sophisticated control programs for fed-batch culture of Candida brassicae, ethanol concentration and DO level were accurately controlled at 3.4–3.7 g/l and 2.2–2.8 ppm, respectively, while cell mass concentration reached about 80 g/l. No manual operation was needed.     

k(L)a as a predictor for successful probe-independent mammalian cell bioprocesses in orbitally shaken bioreactors

The aim of this study was to gain a better understanding of orbitally shaken bioreactors (OSRs) operated without controllers for pH and dissolved oxygen (DO) concentration. We used cylindrical OSRs with working volumes ranging from 250mL to 200L to determine that the volumetric mass transfer coefficient of oxygen (k(L)a) is a good predictor of the performance of OSRs at different scales. We showed that k(L)a values of 7-10hour(-1) were required to avoid DO limitations and to prevent conditions of low pH during the cultivation of CHO cells. Overall, cell cultures in probe-independent OSRs of different nominal volumes ranging from 250mL to 200L achieved similar cell densities, recombinant protein concentrations, and pH and DO profiles when having the same k(L)a. We conclude that k(L)a is a key parameter for probe-independent bioprocesses in OSRs and can be used as a scale-up factor for their operation.     

Microplates with integrated oxygen sensing for medium optimization in animal cell culture

A new approach using microtiter plate cultivation with on-line measurement of dissolved oxygen (DO) was applied for medium optimization of mammalian cell culture. Applying dynamic liquid phase balance, oxygen uptake rates were calculated from the DO level and used as an indicator for culture viability. The developed method was successfully applied to optimization of the concentration of glucose, glutamine and inorganic salts for cultivation of a Chinese Hamster Ovary (CHO) cell line. Using 2³ full factorial central composite design, the optimum medium composition could be identified in one single run. The concentration of inorganic salts had a significant influence on cultivation. The developed method exhibits high potential to improve procedures of medium optimization for animal cell cultivation by allowing the investigation of large sets of potentially important variables in short time and with reduced effort.     

Adaptive dissolved oxygen control through the glycerol feeding in a recombinant Pichia pastoris cultivation in conditions of oxygen transfer limitation

In high cell density cultivation processes the productivity is frequently constrained by the bioreactor maximum oxygen transfer capacity. The productivity can often be increased by operating the process at low dissolved oxygen concentrations close to the limitation level. This may be accomplished with a closed-loop controller that regulates the dissolved oxygen concentration by manipulating the dominant carbon source feeding rate. In this work we study this control problem in a pilot 50l bioreactor with a high cell density recombinant P. pastoris cultivation in complex media. The study focuses on the design of accurate stable adaptive controllers, with guaranteed exponential convergence and its relation with the calibration of controller parameters. Two adaptive control strategies were tested in the pilot bioreactor: a model reference adaptive controller with a linear reference model and an integral feedback controller with adaptive gain. The latter alternative proved to be more robust to errors in the measurements of the off-gas composition. Concerning the instrumentation, algorithms were derived assuming that both the dissolved oxygen tension and off-gas composition are measured on-line, but also the case of only dissolved oxygen being measured is addressed. It was verified that the measurement of off-gas composition might not improve the controller performance due to measurement and process time delays.     

Multi-input multi-output control of a continuous fermenter using nonlinear model based controllers

Internal Model Control (IMC) and Model Predictive Control (MPC), the two most important members of model based controllers, are favourable alternatives for control of nonlinear processes. However, the performance of these controllers deteriorates drastically in the presence of substantial process-model mismatch. Hu and Rangaiah (1998) proposed feedback augmentation for nonlinear IMC (hence named Augmented IMC, AuIMC) for improving control in the presence of modelling errors, and demonstrated its success on a neutralization process. In the present study, IMC, MPC and AuIMC strategies are tested in a more difficult case of multi-input multi-output (MIMO) operation of a highly nonlinear continuous fermenter. A new control configuration is introduced as the conventional configuration is not applicable. Simulation results for different modelling errors show that IMC is better than MPC for fermenter control. The advantage of augmentation as in AuIMC manifests in the significantly improved regulatory control of the fermenter.     

Characterisation of operation conditions and online monitoring of physiological culture parameters in shaken 24-well microtiter plates

A new online monitoring technique to measure the physiological parameters, dissolved oxygen (DO) and pH of microbial cultures in continuously shaken 24-well microtiter plates (MTP) is introduced. The new technology is based on immobilised fluorophores at the bottom of standard 24-well MTPs. The sensor MTP is installed in a sensor dish reader, which can be fixed on an orbital shaker. This approach allows real online measurements of physiological parameters during continuous shaking of cultures without interrupting mixing and mass transfer like currently available technologies do. The oxygen transfer conditions at one constant shaking frequency (250 1/min) and diameter (25 mm) was examined with the chemical sulphite oxidation method. Varied filling volumes (600–1,200 μL) of Escherichia coli cultures demonstrated the importance of sufficient oxygen transfer to the culture. Cultures with higher filling volumes were subjected to an oxygen limitation, which influenced the cell metabolism and prolongated the cultivation time. The effects could be clearly monitored by online DO and pH measurements. A further study of different media in an E. coli fermentation elucidated the different growth behaviour in response to the medium composition. The MTP fermentations correlated very well with parallel fermentations in shake flasks. The new technique gives valuable new insights into biological processes at a very small scale, thus enabling parallel experimentation and shorter development times in bioprocessing.     

Hyperproduction of cordycepin by two-stage dissolved oxygen control in submerged cultivation of medicinal mushroom Cordyceps militaris in bioreactors

Effect of oxygen supply on cordycepin production was investigated in submerged cultivation of Cordyceps militaris, a famous traditional Chinese medicinal mushroom, in a 5-L turbine-agitated bioreactor (TAB). Initial volumetric oxygen transfer coefficient (kLa) within the range of 11.5-113.8 h(-1) had significant influence on cordycepin production. The highest cordycepin concentration of 167.5 mg/L was obtained at an initial kLa value of 54.5 h(-1), where a moderate dissolved oxygen (DO) pattern was observed throughout cultivation. The possible correlation between cordycepin production and DO level was explored by DO control experiments, and the results showed that DO within the range of 10-80% of air saturation greatly affected the cultivation process. To obtain a high specific cordycepin formation rate (rho) throughout cultivation, a two-stage DO control strategy was developed based on the analysis of the relationship of rho and DO. That is, DO was controlled at 60% from the beginning of cultivation and then shifted to a lower control level of 30% when rho started to decrease. As a result, a high cordycepin production of 201.1 mg/L and a high productivity of 15.5 mg/(L.d) were achieved, which was enhanced by about 15% and 30% compared to the highest titers obtained in conventional DO control experiments, respectively. The proposed DO control strategy was also applied to a recently developed 5-L centrifugal impeller bioreactor (CIB) with cordycepin production and productivity titers of 188.3 mg/L and 14.5 mg/(L.d). Furthermore, the scale-up of the two-stage DO control process from 5-L CIB to 30-L CIB was successfully demonstrated. The work is useful for the efficient large-scale production of bioactive metabolites by mushroom cultures.     

Microfluidic reactor for continuous cultivation of Saccharomyces cerevisiae

A diffusion-based microreactor system operated with a reaction volume of 8 μL is presented and characterized to intensify the process understanding in microscale cultivations. Its potential as screening tool for biological processes is evaluated. The advantage of the designed microbioreactor is the use for the continuous cultivation mode by integrating online measurement technique for dissolved oxygen (DO) and optical density (OD). A further advantage is the broaden application for biological systems. The bioreactor geometry was chosen to achieve homogeneous flow during continuous process operation. The device consisted of a microstructured top layer made of poly(dimethylsiloxane) (PDMS), which was designed and fabricated using UV-depth and soft lithography assembled with a glass bottom. CFD simulation data used for geometry design were verified via microparticle-image-velocimetry (μPIV). In the used microreactor geometry no concentration gradients occurred along the entire reaction volume because of rapid diffusive mixing, the homogeneous medium flow inside the growth chamber of the microreactor could be realized. Undesirable bubble formation before and during operation was reduced by using degassed medium as well as moistened and moderate incident air flow above the gas permeable PDMS membrane. Because of this a passive oxygen supply of the culture medium in the device is ensured by diffusion through the PDMS membrane. The oxygen supply itself was monitored online via integrated DO sensors based on a fluorescent dye complex. An adequate overall volumetric oxygen transfer coefficient K(L)a as well as mechanical stability of the device were accomplished for a membrane thickness of 300 μm. Experimental investigations considering measurements of OD (online) and several metabolite concentrations (offline) in a modified Verduyn medium. The used model organism Saccharomyces cerevisiae DSM 2155 tended to strong reactor wall growth resembling a biofilm.     

Determination of external and internal mass transfer limitation in nitrifying microbial aggregates

In this article we present a study of the effects of external and internal mass transfer limitation of oxygen in a nitrifying system. The oxygen uptake rates (OUR) were measured on both a macro-scale with a respirometric reactor using off-gas analysis (Titrimetric and Off-Gas Analysis (TOGA) sensor) and on a micro-scale with microsensors. These two methods provide independent, accurate measurements of the reaction rates and concentration profiles around and in the granules. The TOGA sensor and microsensor measurements showed a significant external mass transfer effect at low dissolved oxygen (DO) concentrations in the bulk liquid while it was insignificant at higher DO concentrations. The oxygen distribution with anaerobic or anoxic conditions in the center clearly shows major mass transfer limitation in the aggregate interior. The large drop in DO concentration of 22-80% between the bulk liquid and aggregate surface demonstrates that the external mass transfer resistance is also highly important. The maximum OUR even for floccular biomass was only attained at much higher DO concentrations (approximately 8 mg/L) than typically used in such systems. For granules, the DO required for maximal activity was estimated to be >20 mg/L, clearly indicating the effects of the major external and internal mass transfer limitations on the overall biomass activity. Smaller aggregates had a larger volumetric OUR indicating that the granules may have a lower activity in the interior part of the aggregate.     

Development of a strategy to control the dissolved concentrations of oxygen and carbon dioxide at constant shear in a plant cell bioreactor

To examine the effects of volatile components on plant cell growth, a bioreactor control system was developed to simultaneously control the dissolved concentrations of both oxygen and carbon dioxide. The first step in this work was to develop a mathematical model to account for gas-liquid mass transfer; biological utilization and production of O(2) and CO(2); and the series of chemical reactions of CO(2) in water. Using this model and dynamic measurements for dissolved O(2) and CO(2), it was observed that (1) both absorption and desorption of a volatile component could be described by a single mass transfer coefficient, K(l)a, and (2) K(l)a values for oxygen and carbon dioxide transfer were directly proportional. The second step of this work was to employ the mathematical model in an adaptive feed-forward strategy to control the dissolved O(2) and CO(2) concentrations by manipulating the inlet gas composition to the bioreactor. This strategy allowed dissolved concentrations to be controlled without the need for changing either the total gas flow rate or agitator speed. Adaptive control was required because the volumetric rates of O(2) and CO(2) consumption and production vary with time during long term operation and therefore these rates must be continually updated. As the final step, we demonstrated that this control strategy was capable of controlling the dissolved gas concentrations in both short- and long-term studies involving the cultivation of Catharanthus roseus plant cells.     

Adaptive control of dissolved oxygen concentration in a bioreactor

A new adaptive DO (dissolved oxygen) concentration control algorithm considering DO electrode dynamics with response time delay has been developed. A system model with two time-varying parameters was used to relate the DO concentration with two control variables: air flow rate and agitation speed. Parameters of this model were estimated on-line using a regularized constant trace recursive least-squares method. An extended Kalman filter was used to remove the effect of noises from the DO concentration measurements and thus to improve control performance. A discrete one-step ahead control scheme was adopted to determine control actions based on the parameter estimation results. Experimental results showed that the new adaptive DO concentration control algorithm performed better than other algorithms tested, a PID controller and adaptive algorithms without the DO electrode dynamics.     

Effects of dissolved oxygen on the morphology of an arachidonic acid production by Mortierella alpina 1S-4

Arachidonic acid (AA) production by Mortierella alpina 1S-4 was investigated using a 50-L fermentor. In order to optimize the dissolved oxygen (DO) concentration and to investigate the effect of DO on morphology, cultivation was carried out under constant DO at various levels in the range of 3-50 ppm. To maintain a DO concentration above 7 ppm, two methods, i.e., the oxygen-enrichment (OE) method (experimental range, 25-90% oxygen gas supplied) and the pressurization (PR) method (experimental range, 180-380 kPa headspace pressure), were used. As a result, the optimum DO concentration range was found to be 10-15 ppm. In this optimum DO concentration range, the AA yield was enhanced about 1.6-fold compared to that obtained at 7 ppm DO, and there was no difference in the AA productivity between the OE and PR methods. When the DO concentration was maintained at 20-50 ppm using the OE method, the morphology changed from filaments to pellets, and the AA yield decreased drastically because of stress due to the limited mass transfer through the pellet wall. When the DO concentration was maintained at 15-20 ppm using the PR method, the morphology did not change, and the AA yield decreased gradually.     

Simulation of L/A control in fed-batch culture of baker's yeast

L/A controllers have extended their use from continuous to fed-batch fermentation where the control is applied from the start of an initial batch phase. As opposed to proportional integral derivative (PID) controllers where even a startup procedure is recommended prior to fed-batch, the L/A controller is not upset by an early connection. It is easily retuned continuously by means of ethanol measurements and can cope with a large range of output conditions. The performance of an L/A algorithm, which uses biomass concentration as the controlled variable, is assessed through simulation. The self-contained algorithm is relatively simple with no greater intrinsic complexity than modern PID stand alone controllers.     

Impact of oxygen mass transfer on nitrification reactions in suspended carrier reactor biofilms

Biofilm-internal and external mass transfer resistance was investigated in laboratory-scale nitrifying suspended carrier reactors (SCR), demonstrating the importance of these factors for these increasingly popular reactor systems. Controlled respirometric experiments revealed that oxygen mass transfer resistance regulated the process performance up to a DO concentration of 20 mg L^?1. External mass transfer exerts significant control over the overall reaction rate, thus biofilm models must adequately account for this resistance. Whilst carrier type and characteristics have some influence, biofilm structure seems primarily responsible for differences in mass transfer and nitrification performance. Heterogeneous biofilms grown under high ammonium loadings had much greater area-specific rates than the gel-like biofilms sourced from low loaded systems.Being a mass-transfer controlled process, the overall reaction rate of these SCR systems could be immediately increased by elevating the DO above normal operating levels (up to 20 mg L^?1). Long-term oxygen deficiency in the lower biofilm sections does not negatively affect the biomass activity.     

Oxygen transfer in three-phase airlift and stirred tank reactors using silicone oil as transfer vector

The use of organic liquids as vectors to enhance mass transfer has been applied since the 1970s. However, mass transfer in three-phase reactors is only partially understood. This paper aimed to characterize oxygen transfer in three-phase reactors containing air as gas, silicone oil as vector and water as aqueous phase. A mass transfer model that considers separately air/vector, vector/water and air/water oxygen transfers was developed. The model was used to describe oxygen transfer in airlift and stirred tank reactors containing from 0 to 50% of silicone oil. Under the experimental conditions, silicone oil had a positive effect on the overall oxygen transfer. In both reactor designs, the maximum overall oxygen transfer was observed with 10% silicone oil which was increased by 65 and 84% in the airlift and stirred reactor, respectively, compared to reactors operated without silicone oil. The overall transfer increase was mainly due to an enhanced air/water transfer. With 10% silicone oil, the air/water contribution to the overall oxygen transfer was 94.7 and 93.0% for the airlift and stirred reactor, respectively.     

Intra-particle oxygen diffusion limitation in solid-state fermentation

Oxygen limitation in solid-state fermentation (SSF) has been the topic of modeling studies, but thus far, there has been no experimental elucidation on oxygen-transfer limitation at the particle level. Therefore, intra-particle oxygen transfer was experimentally studied in cultures of Rhizopus oligosporus grown on the surface of solid, nutritionally defined, glucose and starch media. The fungal mat consisted of two layers--an upper layer with sparse aerial hyphae and gas-filled interstitial pores, and a dense bottom layer with liquid-filled pores. During the course of cultivation ethanol was detected in the medium indicating that oxygen was depleted in part of the fungal mat. Direct measurement of the oxygen concentrations in the fungal mat during cultivation, using oxygen microelectrodes, showed no oxygen depletion in the upper aerial layer, but revealed development of steep oxygen concentration gradients in the wet bottom layer. Initially, the fungal mat was fully oxygenated, but after 36.5 hours oxygen was undetectable at 100 microm below the gas-liquid interface. This was consistent with the calculated oxygen penetration depth using a reaction-diffusion model. Comparison of the overall oxygen consumption rate from the gas phase to the oxygen flux at the gas-liquid interface showed that oxygen consumption of the microorganisms occurred mainly in the wet part of the fungal mat. The contribution of the aerial hyphae to overall oxygen consumption was negligible. It can be concluded that optimal oxygen transfer in SSF depends on the available interfacial gas-liquid surface area and the thickness of the wet fungal layer. It is suggested that the moisture content of the matrix affects both parameters and, therefore, plays an important role in optimizing oxygen transfer in SSF cultures.