On-line biomass monitoring in bench-scale stirred bioreactors using parts of a liquid chromatography system

A cell density monitor, comprising parts of a liquid chromatography system, was adapted for on-line real-time measurement of bacterial cell density up to 75 g l^–1. The device dilutes the sample containing cells and measures the absorbancy at 280 nm. This detector gives an excellent correlation with off-line measurements, good reproducibility, a low limit of detection (3.5 mg l^–1), and becomes a low cost alternative for fermentation monitoring in small stirred bioreactors.     

Development of a new device for on-line measurement of high cell concentrations

A variable-light-path-length flow-through cell to be installed on a spectrophotometer was developed for on-line measurement of high cell concentrations. Cell concentrations of up to about 100 g l^–1 could be measured without dilution. High concentration of protein solution was also measured. A spectrophotometer system combined with the flow-through cell is considered to be cost effective and durable with a minimal maintenance requirement.     

A combination of methods for the on-line determination of alcohols in microbial cultures

Summary A new method for the continuous on-line determination of methanol (range 0.2 to 10 gl^–1) and ethanol (0.2 to 120 gl^–1) is described. The rate limiting step is diffusion of the alcohol through the walls of a silicone tube immersed in the culture broth. A sintered SnO₂ sensor was used instead of a Flame Ionization Detector for alcohol determination. Measurement is not affected by bioreactor aeration or agitation rates, dissolved oxygen, carbon dioxide, ammonia or the concentration of cells in the medium. The assay system was tested in extended batch cultivation of Methylomonas sp. with methanol as the sole carbon source (final biomass concentration, 35 gl^–1). Sensor readings agreed well with simultaneous off-line gas chromatographic methanol determination.     

Evaluation and optimization of factors affecting the performance of a flow-through system based on immobilized acetylcholineesterase as a biosensor

A flow-through system based on acetylcholineesterase (AChE) was studied. The system was prepared by mixing AChE and a multiwall carbon nanotube (MWCNT). Two important parameters, the ratios of AChE:MWCNT (X₁) and AChE-MWCNT:sol-gel (X₂) were optimized using response surface methodology. The results revealed that an enzyme immobilized within the MWCNT-sol-gel was more effective compared to one conducted with sol-gel. The optimum feed flow rate was 0.4 mL/min and ATChI concentration was found to be 1 mM. The optimum ratios of X₁ and X₂ for immobilization on ceramic packing were 1.07 and 0.43, respectively. The sensitivity of this flow-through system was 1.82 × 10⁻⁵/μM and long-term stability analyzed after 120 days was 74% of initial absorbance. With respect to an incubation time of 14 min, the detection limit for paraoxon was 7.3 × 10⁻¹² mol.     

On-line monitoring of pullulanase production during continuous culture of Clostridium thermosulfurogenes

Summary An automated turbidimetric immunoassay for pullulanase from Clostridium thermosulfurogenes was developed for on-line bioprocess monitoring. Measurements are based on the turbidity caused by specific aggregation between pullulanase molecules present in the cultivation medium and antibodies against these enzymes. The time for one assay cycle is 3.0 min. Pullulanase concentrations between 10 units (U)/l and 1000 U/l could be measured. Standard deviations of less than 2% were found. The analysis system could be successfully employed for on-line product monitoring during a 240-h cultivation of C. thermosulfurogenes. An average correlation factor of 0.975 was obtained for the pullulanase data from on-line turbidimetric and the off-line reference assay, demonstrating the accuracy of this on-line method. Offprint requests to: Th. Scheper     

Development of a sequential injection analysis system for monitoring of trehalose concentrations

A sequential injection analysis (SIA) system was developed to on-line monitor the concentrations of trehalose in biological processes. The system is based on the enzymatic reaction of trehalose to glucose in an immobilized trehalase reactor and the subsequent reaction of glucose in the presence of glucose oxidase. The peroxide produced is detected using horseradish peroxidase with a redox indicator (ABTS, 2,2′-azino-di(3-ethylbezthiazoli-sulfonic acid-6)) and a spectrophotometer set at 435 nm. The SIA was fully automated using software written in the LabVIEW^TM development environment. A number of system variablese.g. flow rate of the carrier buffer solution and volume ratio of sample to reagents, were evaluated to increase the sensitivity and performance of the SIA system. The performance of the trehalose-SIA system was linear under partially optimized operating conditions in the concentration range of 0.3 to 2.0 g/L (R²=0.999) with a sample frequency of 6 1/h. The SIA system was successfully employed to on-line monitor the concentrations of trehalose in a continuously stirred tank reactor. The on-line monitored data were in good agreement with the off-line data measured by a HPLC with a refractive index detector (n=14, R²=0.9865).     

Improved methods for investigating the external redox potential in hybridoma cell culture

Because of the interest in understanding and optimizing secretion of proteins from mammalian cells, reliable and more reproducible methods are needed to monitor the external redox potential of animal cells in suspension culture. An improved off-line method was established that greatly reduces the typically long response time of redox electrodes in cell culture media and improves the standardization of redox probes. In addition, the dependence of medium redox potential on dissolved oxygen concentrations and pH was investigated using cell-free medium. Off-line as well as on-line redox potential measurements were then applied to spinner or bioreactor cultures of murine hybridoma cells. Serum containing or protein-free medium were used. The time dependence of the experimentally determined external redox potential was found to be affected not only by oxygen, pH, and medium composition. but to a significant extent by the rate of generation of reductants by hybridoma cells. The observed specific rate of medium reduction by generation of reductants (mV h^–1 viable cell^–1) decreased during exponential growth while cell number increased from 2×10⁵ viable cells ml^–1 to 3.5×10⁶ viable cells ml^–1. This rate, however, was essentially constant at –7.3 mV h^–1±3.7 mV h^–1 per 10¹⁰ viable cells during growth under conditions of constant dissolved oxygen tension and constant pH. Using these observations, the quantity of reductants synthesized and secreted into the medium by viable hybridoma cells was estimated to be approximately 1.3 mole h^–1 per 10¹⁰ viable hybridoma cells. The time course of specific monoclonal antibody secretion rate did not correlate with changes in the external oxidation/reduction potential in either serum containing or protein-free medium.     

Impact of microcarrier coverage on using permittivity for on-line monitoring high adherent Vero cell densities in perfusion bioreactors

The paper presents the interest of on-line permittivity monitoring to estimate the density of Vero cells growing on microcarriers (MCs), even when high cell densities were reached in perfusion bioreactors (4.5 × 10⁶ cells ml⁻¹). Cultures were performed with various MCs concentrations in a reactor equipped with a settling tube. A linear correlation between on-line permittivity and off-line volumetric cell concentration was observed provided that MCs are not fully covered by cells. High permittivities such as 250 pF cm⁻¹ could be measured without signal saturation of the Fogale Biomass system^®. The correlation was no longer linear when cell density per carrier exceeded 100% cell confluency corresponding to 150 cells MC⁻¹ (0.15 × 10⁶ cells cm⁻²). This behaviour was attributed to the decrease of cell volume when cells saturated MCs surface. It mainly happened when low MCs concentration and continuous medium renewing were used. Therefore, permittivity sensor can be considered as a reliable tool to monitor on-line adherent cell densities not exceeding total cell confluency. Moreover, it could be useful to detect when cell confluency occurs.     

Chemiluminescent assays for choline and phospholipase-D using a flow injection system

A flow injection chemiluminescent method is described for the determination of choline. The method is based on the production of hydrogen peroxide from choline using on-line covalently bound immobilized choline oxidase column. The product is mixed downstream and detected via the cobalt catalyzed chemiluminescent oxidation of luminol. The detection limit is 1×10⁻⁷ mol/L, with rsd 1.8 to 2.8% in the range 2–10×10⁻⁵ mol/L. The sample throughput is 30 per hour. The method was applied to the determination of choline produced off-line from phosphatidylcholine using phospholipase-D isolated from cabbage. © 1997 John Wiley & Sons, Ltd.     

Inexpensive Laboratory-Constructed Nephelometer

An inexpensive, simple-to-construct nephelometer which was used to monitor the lysis of spheroplasts is described. The nephelometer is a flow-through device with a linear response to cell concentration from the lower detection limit to 8 × 10⁸ cells per ml.     

On-line determination of glucose and lactate concentrations in animal cell culture based on fibre optic detection of oxygen in flow-injection analysis.

A flow-injection analysis (FIA) system based on fibre optic detection of oxygen consumption using immobilized glucose oxidase (GOD) and lactate oxidase (LOD) is described for the on-line monitoring of glucose and lactate concentrations in animal cell cultures. The consumption of oxygen was determined via dynamic quenching by molecular oxygen of the fluorescence of an indicator. GOD and LOD were immobilized on controlled pore glass (CPG) in enzyme reactors which were directly linked to a specially designed fibre optic flow-through cell covering the oxygen optrode. The system is linear for 0-30 mM glucose, with an r.s.d. of 5% at 30 mM (five measurements) and for 0-30 mM lactate, with an r.s.d. of 5% at 30 mM (five measurements). The enzyme reactors used were stable for more than 4 weeks in continuous operation, and it was possible to analyse up to 20 samples per hour. The system has been successfully applied to the on-line monitoring of glucose and lactate concentrations of an animal cell culture designed for the production of recombinant human antithrombine III (AT-III). Results of the on-line measurement obtained by the FIA system were compared with the off-line results obtained by a glucose and lactate analyser from Yellow Springs Instrument Company (YSI).     

Flow injection analysis of intracellular β-galactosidase in Escherichia coli cultivations, using an on-line system including cell disruption, debris separation and immunochemical quantification

A continuous integrated process for on-line quantification of intracellular components has been developed. By applying the concept of expanded micro-beds in a flow injection system it was possible to first perform on-line cell disintegration followed by an on-line binding assay for quantification of a reporter protein (-galactosidase) from the cell interior. The disintegration process involved the use of an expanded bed with immobilised lysozyme followed by ultrasonic treatment in a flow-through cell. The cell debris does not interfere in the binding assay as it is carried out in an expanded bed. The time for an assay cycle is at present approx. 35 min. This integrated system can be used for quantification of proteins down to at least 10^-7 mol/L.     

Process control during the suspension culture of a human melanoma cell line in a mechanically stirred loop bioreactor

A human melanoma cell line was cultivated for more than 5 months in a serum-free medium without macromolecular growth factors. A mechanically stirred loop bioreactor was used for the culture of the melanoma cells. The tip speed of the marine impellers was 1.5 m s⁻¹. This cell line was able to endure tip speeds of up to 3.5 m s⁻¹ for a few hours without significant cell damage. By using process control it was possible to obtain growth rates and cell numbers close to those found in medium with serum. The pO₂ was controlled at 125 mbar and the pH at 7.15. The signal of an on-line fluorometer, although not caused by the cells, correlates with cell number. The partial pressure of CO₂ in the culture medium and the redox potential of the medium were monitored by on-line sensors.     

The induction of bacterial bioluminescence system on solid medium

Induction of the luminescence system of the marine luminous bacteriumVibrio harveyi growing on a solid medium was studied. The induction occurred when the total number of cells per square centimeter of solid medium approached 10⁴ cells (i.e., either 100 colonies consisting of 100 cells per each colony or 1 colony with about 10⁴ cells). The preinduction period and number of cells per colony at the time of induction decreased as the number of colonies per cm² increased. The ecological significance of the induction of the luminescence system on solid medium is discussed.     

Novel integrated microdialysis-amperometric system for in vitro detection of dopamine secreted from PC12 cells: design, construction, and validation

A novel dual channel in vitro apparatus, derived from a previously described design, has been coupled with dopamine (DA) microsensors for the flow-through detection of DA secreted from PC12 cells. The device, including two independent microdialysis capillaries, was loaded with a solution containing PC12 cells while a constant phosphate-buffered saline (PBS) medium perfusion was carried out using a dual channel miniaturized peristaltic pump. One capillary was perfused with normal PBS, whereas extracellular calcium was removed from extracellular fluid of the second capillary. After a first period of stabilization and DA baseline recording, KCl (75 mM) was added to the perfusion fluid of both capillaries. In this manner, a simultaneous “treatment–control” experimental design was performed to detect K⁺-evoked calcium-dependent DA secretion. For this purpose, self-referencing DA microsensors were developed, and procedures for making, testing, and calibrating them are described in detail. The electronic circuitry was derived from previously published schematics and optimized for dual sensor constant potential amperometry applications. The microdialysis system was tested and validated in vitro under different experimental conditions, and DA secretion was confirmed by high-performance liquid chromatography with electrochemical detection (HPLC–EC). PC12 cell viability was quantified before and after each experiment. The proposed apparatus serves as a reliable model for studying the effects of different drugs on DA secretion through the direct comparison of extracellular DA increase in treatment–control experiments performed on the same initial PC12 cell population.     

Fluorimetric quantification of intracellular lactate dehydrogenase during fermentation using flow injection analysis

A flow injection system for the on-line detection of the intracellular enzyme lactate dehydrogenase (LDH) during fermentation has been developed. The system is comprised of an on-line cell disintegration part, an immobilised dye based expanded bed column for the affinity capture of LDH and a fluorimetric detection unit. The system with a linearity of 0.1–5.4 U LDH ml^–1 was applied for the detection of intracellular accumulation of LDH during Lactococcus lactis subsp.lactis cultivation.     

A Biofeedback System of Baroreceptor Cardiac Reflex Sensitivity

The evidence presently available suggests that the parasympathetic nervous system and sympathetic-parasympathetic interactions could play a role in the pathophysiology of cardiovascular disorders and, specifically, in hypertension. A loss of sensitivity of the baroreceptor reflex is one of the fundamental mechanisms underlying the deficits found in parasympathetic cardiac control. The baroreceptor reflex is a basic mechanism for the regulation of blood pressure, a powerful source of vagal afferent input to the central nervous system, and one of the most important physiological mechanisms affecting efferent cardiac vagal activity. This paper describes a computerized system for the on-line analysis of the baroreceptor cardiac reflex function using the noninvasive spontaneous sequence method in the time domain. The system provides feedback of the baroreceptor reflex sensitivity (the change in heart period per unit change in systolic blood pressure) differentially both when the systolic blood pressure is increasing and when it is decreasing. The accuracy of the described system has been tested against the conventional off-line procedure. None of the parameters supplied by the analysis show a significant difference between the on-line and off-line methods. These results confirm the accuracy of the on-line system to analyze baroreceptor cardiac reflex function.     

Elimination of diastereomer interference to determine Telcagepant (MK-0974) in human plasma using on-line turbulent-flow technology and off-line solid-phase extraction coupled with liquid chromatography/tandem mass spectrometry

To eliminate the diastereomer interference on Telcagepant (MK-0974) determination during clinical study support, on-line high turbulent-flow liquid chromatography (HTLC) methods, HTLC-A and HTLC-B that covered dynamic range of 0.5–500 nM and 5–5000 nM, respectively, were developed. To meet the requirement of rapid assay transfer among multiple laboratories and analysts, a solid-phase extraction (SPE) assay was derived from the existing HTLC-B assay under the same dynamic range. The on-line HTLC assays were achieved through direct injection of plasma samples, extraction of analyte with a Cohesive C₁₈ column (50 mm × 0.5 mm, 50 μm), followed by HPLC separation on a FluoPhase RP column (100 mm × 2.1 mm, 5 μm) and MS/MS detection. The off-line SPE assay used Waters Oasis^®HLB μElution plate to extract the analytes from plasma matrix before injecting on a FluoPhase RP column (150 mm × 2.1 mm, 5 μm) for LC–MS/MS analysis. Under both on-line and off-line assay conditions, the diastereomer 1c was chromatographically separated from MK-0974. Cross-validation with the pooled samples demonstrated that both on-line and off-line assays provided comparable data with a difference of <2.6%. The assays were proved to be specific, accurate and reliable, and have been used to support multiple clinical studies. The pros and cons of on-line and off-line assays with regard to man power involved in sample preparation, total analysis time, carryover, cost efficiency, and the requirement for assay transfer are discussed.     

An on-line approach to monitor ethanol fermentation using FTIR spectroscopy

Fermentation process control is currently limited by its inability to measure parameters such as substrate, product, and biomass concentrations rapidly for consistent on-line feedback. Physical and chemical parameters, such as temperature and pH, currently can be obtained on-line using appropriate sensors. However, to obtain information on the concentration of the substrate, product, and biomass, samples must be taken off-line for measurement. With the use of spectroscopic techniques, real-time monitoring of process constituents such as product and substrate is possible. Spectroscopic techniques are rapid and nondestructive, require minimal or no sample preparation, and can be used to simultaneously assess several constituents in complex matrices. The production of ethanol is the largest fermentation process in terms of production volume and economic value as a result of its prominence in the food, agricultural, and fuel industries. This study attempts to develop an on-line ethanol fermentation monitoring technique using Fourier transform infrared (FTIR) spectroscopy with a flow-through ATR capability. Models developed using multivariate statistics, employed to obtain on-line FTIR measurements, were successfully validated by off-line HPLC analysis and spectrophotometry data. Standard errors of prediction (SEP) values of 0.985 g/L (R2 = 0.996), 1.386 g/L (R2 = 0.998), and 0.546 (R2 = 0.972) were obtained for ethanol, glucose, and OD, respectively. This work demonstrates that FTIR spectroscopy could be used for rapid on-line monitoring of fermentation.