Toward continuous glucose monitoring with planar modified biosensors and microdialysis. Study of temperature, oxygen dependence and in vivo experiment

Glucose biosensors based on the use of planar screen-printed electrodes modified with an electrochemical mediator and with glucose oxidase have been optimised for their application in the continuous glucose monitoring in diabetic patients. A full study of their operative stability and temperature dependence has been accomplished, thus giving useful information for in vivo applications. The effect of dissolved oxygen concentration in the working solution was also studied in order to evaluate its effect on the linearity of the sensors. Glucose monitoring performed with serum samples was performed to evaluate the effect of matrix components on operative stability and demonstrated an efficient behaviour for 72 h of continuous monitoring. Finally, these studies led to a sensor capable of detecting glucose at concentrations as low as 0.04 mM and with a good linearity up to 2.0 mM (at 37 degrees C) with an operative stability of ca. 72 h, thus demonstrating the possible application of these sensors for continuous glucose monitoring in conjunction with a microdialysis probe. Moreover, preliminary in vivo experiments for ca. 20 h have demonstrated the feasibility of this system.     

The Protective Role of <Emphasis Type="SmallCaps">d</Emphasis>-Glucose Against 1-Methyl-4-Phenylpyridinium Ion (MPP<Superscript>+</Superscript>): Induced Mitochondrial Dysfunction in C6 Astroglial Cells

Impaired mitochondrial function in glial and neuronal cells in the substantia nigra is one of the most likely causes of Parkinson’s disease. In this study, we investigated the protective role of glucose on early key events associated with MPP⁺-induced changes in rat C6 astroglial cells. Studies were carried out to examine alterations in mitochondrial respiratory status, membrane potential, glutathione levels, and cell cycle phase inhibition at 48 h in 2 and 10 mM glucose in media. The results obtained suggest that MPP⁺ caused significant cell death in 2 mM glucose with LC₅₀ 0.14 ± 0.005 mM, while 10 mM glucose showed highly significant protection against MPP⁺ toxicity with LC₅₀ 0.835 ± 0.03 mM. This protection was not observed with cocaine, demonstrating its compound specificity. MPP⁺ in 2 mM glucose decreased significantly mitochondrial respiration, membrane potential and glutathione levels in a dose dependent manner, while 10 mM glucose significantly restored them. MPP⁺ in 2 mM glucose arrested the cells at G0/G1 and G2/M phases, demonstrating its dual inhibitory effects. However, in 10 mM glucose, MPP⁺ caused G0/G1 arrest only. In summary, the results suggest that loss of cell viability in 2 mM glucose group with MPP⁺ treatments was due to mitochondrial dysfunction caused by multilevel mechanism, involving significant decrease in mitochondrial respiration, membrane potential, glutathione levels, and dual arrest of cell phases, while 10 mM glucose rescued astroglial cells from MPP⁺ toxicity by significant maintenance of these factors.     

Fermentation process for continuous production of succinic acid in a fibrous bed bioreactor

Succinic acid (SA) was produced from Actinobacillus succinogenes with high cell density by continuous fermentation using fibrous bed bioreactor (FBB). The effects of feeding glucose concentration, dilution rate, and pH on continuous production of SA were examined to achieve an efficient and economical bioprocess. The optimum feeding glucose concentration, dilution rate, and pH were 80 g/L, 0.05 1/h, and 6.0–6.5, respectively. A SA concentration of 55.3 ± 0.8 g/L, productivity of 2.77 ± 0.04 g/L/h, and yield of 0.8 ± 0.02 g/g were obtained, and the continuous fermentation exhibited long-term stability for as long as 18 days (440 h) with no obvious fluctuations in both SA and biomass levels. The Jerusalimsky equation for the specific rate of SA production presented the inhibition phenomenon of the product, demonstrating that 60 g/L SA might be a critical concentration in this continuous FBB system. The results obtained could be beneficial for future fermentor designs and improvements in SA production.     

Continuous glucose monitoring and control in a rotating wall perfused bioreactor

A glucose control system consisting of a single in-line glucose sensor, concentrated glucose solution, and computer hardware and software were developed. The system was applied to continuously control glucose concentrations of a perfusion medium in a rotating wall perfused vessel (RWPV) bioreactor culturing BHK-21 cells. The custom-made glucose sensor was based on a hydrogen peroxide electrode. The sensor continuously and accurately measured the glucose concentration of GTSF-2 medium in the RWPV bioreactor during cell culture. Three sets of two-point calibrations were applied to the glucose sensor during the 55-day cell culture. The system first controlled the glucose concentration in perfusing medium between 4.2 and 5.6 mM for 36 days and then at different glucose levels for 19 days. A stock solution with a high glucose concentration (266 mM) was used as the glucose injection solution. The standard error of prediction (SEP) for glucose measurement by the sensor, compared to measurement by the Beckman glucose analyzer, was +/-0.4 mM for 55 days.     

On-line glucose monitoring by near infrared spectroscopy during the scale up steps of mammalian cell cultivation process development

NIR spectroscopy is a non-destructive tool for in-situ, on-line bioprocess monitoring. One of its most frequent applications is the determination of metabolites during cultivation, especially glucose. Previous studies have usually investigated the applicability of Near Infrared (NIR) spectroscopy at one bioreactor scale but the effect of scale up was not explored. In this study, the complete scale up from shake flask (1 L) through 20 L, 100 L and 1000 L up to 5000 L bioreactor volume level was monitored with on-line NIR spectroscopy. The differences between runs and scales were examined using principal component analysis. The bioreactor runs were relatively similar regardless of scales but the shake flasks differed strongly from bioreactor runs. The glucose concentration throughout five 5000 L scale bioreactor runs were predicted by partial least squares regression models that were based on pre-processed spectra of bioreactor runs and combinations of them. The model that produced the lowest error of prediction (4.18 mM on a 29 mM concentration range) for all five runs in the prediction set was based on the combination of 20 L and 100 L data. This result demonstrated the capabilities and the limitations of an NIR system for glucose monitoring in mammalian cell cultivations.

     

Taurine prevents high-glucose-induced human vascular endothelial cell apoptosis

Elevated blood glucose in uncontrolled diabetes is causallycorrelated with diabetic microangiopathy. Hyperglycemia-triggered accelerated endothelial cell apoptosis is a critical event in theprocess of diabetes-associated microvascular disease. The conditionallysemiessential amino acid taurine has been previously shown to protectagainst human endothelial cell apoptosis. Therefore, this study wasdesigned to investigate the role of taurine in the prevention ofhigh-glucose-mediated cell apoptosis in human umbilical veinendothelial cells (HUVEC) and the mechanisms involved. Exposure ofHUVEC to 30 mM glucose for 48 h (short-term) and 14 days (long-term)resulted in a significant increase in apoptosis, compared with normalglucose (5.5 mM; P < 0.05).High-glucose-induced DNA fragmentation preferentially occurred in the Sphase cells. Mannitol (as osmotic control) at 30 mM failed to induceHUVEC apoptosis. Taurine prevented high-glucose-induced HUVECapoptosis, which correlates with taurine attenuation ofhigh-glucose-mediated increased intracellular reactive oxygen species(ROS) formation and elevated intracellularCa²⁺ concentration([Ca²⁺]_i)level. Antioxidants, DMSO, N-acetylcysteine, and glutathione, only partly attenuated high-glucose-inducedHUVEC apoptosis. Glucose at 30 mM did not cause HUVEC necrosis.However, both glucose and mannitol at 60 mM caused HUVEC necrosis asrepresented by increased lactate dehydrogenase release and cell lysis.Taurine failed to prevent hyperosmolarity-induced cell necrosis. Theseresults demonstrate that taurine attenuates hyperglycemia-induced HUVECapoptosis through ROS inhibition and[Ca²⁺]_istabilization and suggest that taurine may exert a beneficial effect inpreventing diabetes-associated microangiopathy.

     

Increased hexosamine pathway flux and high fat feeding are not additive in inducing insulin resistance: evidence for a shared pathway

Excess fatty acids and carbohydrates have both been implicated in the pathogenesis of type 2 diabetes, and both can reproduce essential features of the disease including insulin resistance and beta cell failure. It has been proposed that both nutrients may regulate metabolism through a common fuel sensing mechanism, namely hexosamine synthesis. We have previously shown that transgenic overexpression of the rate-limiting enzyme for hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), targeted to muscle and fat, leads to insulin resistance mediated by increased O-linked glycosylation of nuclear and cytosolic proteins. We report here that hexosamine-induced insulin resistance is not additive with that induced by high fat feeding. In control mice fed a high fat diet, glucose disposal rates during euglycemic hyperinsulinemia were decreased by 37% (p < 0.02) compared to mice on a low fat diet. Transgenic mice overexpressing GFA and fed a low fat diet exhibited a 51% decrease in glucose disposal compared to controls on a low fat diet (p < 0.001), but no further decrease was evident in the transgenic mice fed a high fat diet. Decreased glucose disposal rates were mirrored by increases in skeletal muscle levels of the principal end product of the hexosamine pathway, UDP-N-acetyl glucosamine. Serum leptin levels, which are modulated both by feeding and hexosamine flux, also show no additivity in their stimulation by GFA overexpression and high fat feeding. These data are consistent with a shared nutrient sensing pathway for high fat and carbohydrate fluxes and a common pathway by which glucose and lipids induce insulin resistance.     

Novel planar glucose biosensors for continuous monitoring use

Novel planar glucose biosensors to be used for continuous monitoring have been developed. The electrodes are produced with the "screen printing" technique, and present a high degree of reproducibility together with a low cost and the possibility of mass production. Prior to enzyme immobilisation, electrodes are chemically modified with ferric hexacyanoferrate (Prussian Blue). This allows the detection of the hydrogen peroxide produced by the enzymatic reaction catalysed by GOD, at low applied potential (ca. 0.0 V versus Ag/AgCl), highly limiting any electrochemical interferences. The layer of Prussian Blue (PB) showed a high stability at the working conditions (pH 7.4) and also after 1 year of storage dry at RT, no loss of activity was observed. The assembled glucose biosensors, showed high sensitivity towards glucose together with a long-term operational and storage stability. In a continuous flow system, with all the analytical parameters optimised, the glucose biosensors detected glucose concentration as low as 0.025 mM with a linear range up to 1.0mM. These probes were also tested over 50-60 h in a continuous flow mode to evaluate their operational stability. A 0.5 mM concentration of glucose was continuously fluxed into a biosensor wall-jet cell and the current due to the hydrogen peroxide reduction was continuously monitored. After 50-60 h, the drift of the signal observed was around 30%. Because of their high stability, these sensors suggest the possibility of using such biosensors, in conjunction with a microdialysis probe, for a continuous monitoring of glucose for clinical purposes.     

Optimization of glucose feeding approaches for enhanced glucosamine and N-acetylglucosamine production by an engineered Escherichia coli

In this work, a recombinant Escherichia coli was constructed by overexpressing glucosamine (GlcN) synthase and GlcN-6-P N-acetyltransferase for highly efficient production of GlcN and N-acetylglucosamine (GlcNAc). For further enhancement of GlcN and GlcNAc production, the effects of different glucose feeding strategies including constant-rate feeding, interval feeding, and exponential feeding on GlcN and GlcNAc production were investigated. The results indicated that exponential feeding resulted in relatively high cell growth rate and low acetate formation rate, while constant feeding contributed to the highest specific GlcN and GlcNAc production rate. Based on this, a multistage glucose supply approach was proposed to enhance GlcN and GlcNAc production. In the first stage (0–2 h), batch culture with initial glucose concentration of 27 g/l was conducted, whereas the second culture stage (2–10 h) was performed with exponential feeding at μ _set = 0.20 h⁻¹, followed by feeding concentrated glucose (300 g/l) at constant rate of 32 ml/h in the third stage (10–16 h). With this time-variant glucose feeding strategy, the total GlcN and GlcNAc yield reached 69.66 g/l, which was enhanced by 1.59-fold in comparison with that of batch culture with the same total glucose concentration. The time-dependent glucose feeding approach developed here may be useful for production of other fine chemicals by recombinant E. coli.     

Multi-stage high cell continuous fermentation for high productivity and titer

We carried out the first simulation on multi-stage continuous high cell density culture (MSC-HCDC) to show that the MSC-HCDC can achieve batch/fed-batch product titer with much higher productivity to the fed-batch productivity using published fermentation kinetics of lactic acid, penicillin and ethanol. The system under consideration consists of n-serially connected continuous stirred-tank reactors (CSTRs) with either hollow fiber cell recycling or cell immobilization for high cell-density culture. In each CSTR substrate supply and product removal are possible. Penicillin production is severely limited by glucose metabolite repression that requires multi-CSTR glucose feeding. An 8-stage C-HCDC lactic acid fermentation resulted in 212.9 g/L of titer and 10.6 g/L/h of productivity, corresponding to 101 and 429% of the comparable lactic acid fed-batch, respectively. The penicillin production model predicted 149% (0.085 g/L/h) of productivity in 8-stage C-HCDC with 40 g/L of cell density and 289% of productivity (0.165 g/L/h) in 7-stage C-HCDC with 60 g/L of cell density compared with referring batch cultivations. A 2-stage C-HCDC ethanol experimental run showed 107% titer and 257% productivity of the batch system having 88.8 g/L of titer and 3.7 g/L/h of productivity. MSC-HCDC can give much higher productivity than batch/fed-batch system, and yield a several percentage higher titer as well. The productivity ratio of MSC-HCDC over batch/fed-batch system is given as a multiplication of system dilution rate of MSC-HCDC and cycle time of batch/fed-batch system. We suggest MSC-HCDC as a new production platform for various fermentation products including monoclonal antibody.     

Feed development for fed‐batch CHO production process by semisteady state analysis

Semisteady state cultures are useful for studying cell physiology and facilitating media development. Two semisteady states with a viable cell density of 5.5 million cells/mL were obtained in CHO cell cultures and compared with a fed‐batch mode control. In the first semisteady state, the culture was maintained at 5 mM glucose and 0.5 mM glutamine. The second condition had threefold higher concentrations of both nutrients, which led to a 10% increase in lactate production, a 78% increase in ammonia production, and a 30% reduction in cell growth rate. The differences between the two semisteady states indicate that maintaining relatively low levels of glucose and glutamine can reduce the production of lactate and ammonia. Specific amino acid production and consumption indicated further metabolic differences between the two semisteady states and fed‐batch mode. The results from this experiment shed light in the feeding strategy for a fed‐batch process and feed medium enhancement. The fed‐batch process utilizes a feeding strategy whereby the feed added was based on glucose levels in the bioreactor. To evaluate if a fixed feed strategy would improve robustness and process consistency, two alternative feeding strategies were implemented. A constant volume feed of 30% or 40% of the initial culture volume fed over the course of cell culture was evaluated. The results indicate that a constant volumetric‐based feed can be more beneficial than a glucose‐based feeding strategy. This study demonstrated the applicability of analyzing CHO cultures in semisteady state for feed enhancement and continuous process improvement. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Effects of aluminium on ultrastructure and antioxidant activity in leaves of tea plant

Seedlings of Camellia sinensis (L.) were grown hydroponically to study the effect of aluminium (Al) on leaf antioxidant defence system and cell ultrastructure. We found that malondialdehyde (MDA) content decreased at 0–0.32 mM Al, but increased significantly at 0.53 mM Al. Like MDA, hydrogen peroxide (H₂O₂) content increased at 0.53 mM Al; however, no differences were observed at 0–0.32 mM Al. Superoxide dismutase (SOD, EC1.15.1.1) activity remained practically constant at 0–0.32 mM Al, but increased sharply at 0.53 mM Al; catalase (CAT, EC1.11.1.6) and guaiacol peroxidase (GPX, EC1.11.1.7) activities decreased following an initial increase, reaching their peaks at 0.32 mM Al. Ascorbate peroxidase (APX, EC 1.11.1.11) activity increased and glutathione (GR, EC 1.6.4.2) level fluctuated with increasing Al concentrations. Transmission electron microscope analysis of Al-treated leaves showed that although cell ultrastructural integrity was maintained at 0–0.32 mM, significant membrane damage was observed at 0.53 mM. Our results suggest that at low Al concentrations, the leaf antioxidant defence system can scavenge reactive oxygen species and sufficiently protect cells from free radical injury. However, at higher Al concentrations (0.53 mM), the balance between formation and detoxification of ROS is lost, resulting in the destruction of cell ultrastructure.     

Targeted functional investigations guided by integrative proteome network analysis revealed significant perturbations of renal tubular cell functions induced by high glucose

Recently, several studies employed various proteomic approaches to define diabetes‐induced changes in renal proteins. However, functional significance of those datasets in diabetic nephropathy remained unclear. We thus performed integrative proteome network analysis of such datasets followed by various targeted functional studies in distal renal tubular cells treated with high glucose (HG) (25 mM) compared to normal glucose (NG) (5.5 mM) and NG + mannitol (M) (5.5 + 19.5 mM). The data showed that at 96 h when cell proliferation/death, tight junction protein and β‐/F‐actin expression and organization, and transepithelial resistance remained unchanged, only HG caused increased levels of HSP90, HSP70, and HSP60, and increased accumulation of intracellular protein aggregates. In addition, HG also induced overproduction of intracellular ROS, decreased catalase level, increased level of oxidatively modified proteins, increased intracellular ATP level, and defective transepithelial Ca²⁺ transport. However, both HG and M increased the levels of ubiquitinated proteins. Taken together, this study demonstrated significant perturbations of distal renal tubular cells induced by HG based on targeted functional studies guided by integrative proteome network analysis. These data may, at least in part, lead to better understanding of the pathogenic mechanisms of diabetic nephropathy.     

Simultaneous measurement of glucose and glutamine in insect cell culture media by near infrared spectroscopy

The purpose of this study was to develop non-invasive techniques to monitor the composition of cell culture media in insect cell bioreactors. Such a monitor could be used in conjunction with a fed-batch feeding scheme to ensure that cells are maintained in an optimal environment for growth and protein production. Glucose and glutamine concentrations in an insect cell culture bioreactor were determined off-line with near-infrared (NIR) absorption spectroscopy. Spectra were collected from 5000 to 4000 cm(-1) with a 1.5-mm optical path length. Partial least squares (PLS) regression was applied to correlate the collected spectra with the concentration of the desired analytes. Under the culture conditions evaluated here, glucose and glutamine concentrations ranged from 38 to 55 mM and from 3 to 13 mM, respectively. Accurate measurements of glucose and glutamine in insect cell culture samples were possible over these entire ranges. The standard error of prediction (SEP) and mean percent error (MPE) for glutamine were 0.52 mM and 5.3%, respectively. Glucose could be measured with an SEP of 1.30 mM and an MPE of 2.3%. These levels of error are quite low considering the changing complexity of the growth media due to the shifting levels of amino acids, carbohydrates, yeastolate, proteins, and cell debris. This study represents an important step in the development of noninvasive on-line monitoring devices for cell culture bioreactors. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 11-15, 1997.     

Bioreactor culture of recombinant <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> S2 cells: characterization of metabolic features related to cell growth and production of the rabies virus glycoprotein

Although several reports have been published on recombinant protein expression using Drosophila cells, information on their metabolism and growth in vitro is relatively scarce. In the present study, we have analyzed the growth and metabolism of transfected S2 cells (S2AcRVGP) in bioreactor cultures with serum-free medium Sf900 II, to evaluate its potential for mass production of a rabies virus glycoprotein (RVGP). Cells were cultured in a 3 l-stirred-tank bioreactor at 28 °C with pH controlled at 6.2 and dissolved oxygen at 50% air saturation. The cells attained a specific growth rate and maximum cell density as high as 0.084 h⁻¹ and 2.3 × 10⁷ cell ml⁻¹, respectively. The main substrates consumed during this rapid growth phase were glucose, glutamine and proline. An atypical accumulation of ammonia and alanine was observed in the culture medium, up to 62 mM and 47 mM, respectively, but lactate was produced in low levels. After exhaustion of glutamine and proline as energy sources, alanine was consumed and production of ammonia increased. The production of recombinant RVGP reached concentrations as high as 178 μg l⁻¹. Premature exhaustion of glutamine, serine and cysteine could be related to degradation of the recombinant glycoprotein. In general, the results demonstrated that S2AcRVGP can be considered an effective vehicle for large-scale recombinant glycoprotein expression and that several critical factors of the bioprocess could be optimized to increase the quality and productivity of the RVGP.     

Controlled release of nutrients to mammalian cells cultured in shake flasks

Though cell culture-based protein production processes are rarely carried out under batch mode of operation, cell line and initial process development operations are usually carried out in batch mode due to simplicity of operation in widely used scale down platforms like shake flasks. Nutrient feeding, if performed, is achieved by bolus addition of concentrated feed solution at different intervals, which leads to large transient increases in nutrient concentrations. One negative consequence is increased waste metabolite production. We have developed a hydrogel-based nutrient delivery system for continuous feeding of nutrients in scale down models like shake flasks without the need for manual feed additions or any additional infrastructure. Continuous delivery also enables maintaining nutrient concentrations at low levels, if desired. The authors demonstrate the use of these systems for continuous feeding of glucose and protein hydrolysate to a suspension Chinese Hamster Ovary (CHO) culture in a shake flask. Glucose feeding achieved using the glucose-loaded hydrogel resulted in a 23% higher integral viable cell density and an 89% lower lactate concentration at the end of the culture when compared with a bolus-feed of glucose.     

Diversity of neurodegenerative processes in the model of brain cortex tissue ischemia

Stroke is known to induce massive cell death in the ischemic brain. Either necrotic or apoptotic types of cell death program were observed in neurons in zone of ischemia. We suggest that spatial heterogeneity of glucose and oxygen distribution plays a crucial role in this phenomenon. In order to elucidate the role of glucose and oxygen in ischemic neurons choice of cell death pathway, conditions corresponding to different areas of insult were reproduced in vitro in the model of surviving brain cortex tissue slices. Three zones were modeled in vitro by varying glucose and oxygen concentration in surviving slices incubation media. Modeled ischemic area I (MIA I) was corresponded to the center of suggested ischemic zone where the levels of glucose and oxygen were considered to be extremely low. MIA II was assigned as intermediate area where oxygen concentration was still very low but glucose was present (this area was also divided into two sub-areas MIA IIa and MIA IIb with physiologically low (5 mM) and normal (10 mM) level of glucose respectively). MIA III was considered as a periphery area where glucose concentration was close to physiological level and high level of ROS production had been induced by reoxygenation after anoxia. Analysis of molecular mechanisms of cell death in MIA I, IIa, IIb and III was carried out. Cell death in MIA I was found to proceed by necrotic manner. Apoptosis characterized by cyt c release, caspase 3 activation and internucleosomal DNA fragmentation was observed in MIA III. Cell death in MIA II was accompanied by several (not all) hallmarks of apoptosis. Mechanisms of cell death in MIA IIa and MIA IIb were found to be different. Internucleosomal DNA fragmentation in MIA IIa but not in MIA IIb was sensitive to glycine (5 mM), inhibitor of NMDA receptor MK-801 (10 μM) and PTP inhibitor cyclosporine A (10 μM). Activation of caspase 3 was detected in MIA IIb but not in MIA IIa. However cytochrome c release was observed neither in MIA IIa nor in MIA IIb. In MIAs II–III apoptosis was accompanied by uncoupling of oxidative phosphorylation, which was induced by rise of intracellular Ca²⁺ and intensive ROS production. Results obtained in present study allow us to propose existence of at least four molecular pathways of cell death development in brain ischemic zone. The choice of cell death pathway is determined by oxygen and glucose concentration in the particular area of the ischemic zone.     

Kinetics of cell growth and heterologous glucoamylase production in recombinant Aspergillus nidulans

In the work, a study of cell growth and the regulation of heterologous glucoamylase synthesis under the control of the positively regulated alcA promoter in a recombinant Aspergillus nidulans is presented. We found that similar growth rates were obtained for both the host and recombinant cells when either glucose or fructose was employed as sole carbon and energy source. Use of the potent inducer cyclopentanone in concentrations greater than 3 mM resulted n maximum glucoamylase concentration and maximum overall specific glucoamylase concentration over 80 h of batch cultivation. However, cyclopentanone concentrations in excess of 3 mM also showed an inhibitory effect on spore germination as well as fungal growth. In contrast, another inducer, threonine, had no negative effect on spore germination even when concentrations of up to 100 mM were used with either glucose or fructose as carbon source. Glucoamylase synthesis in the presence of glucose plus either inducer did not begin until glucose was totally depleted, suggesting strong catabolite repression. Similar results were obtained when fructose was employed, although low levels of glucoamylase were detected before fructose depletion, suggesting partial catabolite repression. The highest enzyme concentration (570 mg/L) and overall specific enzyme concentration (81 mg/g cell) were observed in batch culture when cyclopentanone was the inducer and fructose the primary carbon source. A maximum glucoamylase concentration of 1.1 g/L and an overall specific glucoamylase concentration of 167 mg/g cell were obtained in a bioreactor using cyclopentanone as the inducer and limited-fructose feeding strategy, which nearly doubles the glucoamylase productivity from batch cultures. (c) 1993 John Wiley & Sons, Inc.     

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).