Determination of ammonium and L-Glutamine in hybridoma cell cultures by sequential flow injection analysis

A flow injection anlytical system based on a gas diffusion membrane module for ammonia and an ammonium flow-through potentiometric detector has been set up for measurement of L-glutamine and ammonium ions in hybridoma cell cultures. The main feature of the system is that the same basic analytical concept and equipment is used in both measurements, the only difference being for the determination of L-glutamine, in which the sample flows through an immobilized glutaminase cartridge. The conditions to enable the performance of both analysis consecutively, avoiding potential interferences by unwanted deamination of other compounds in the samples, have been determined. Finally, the proposed system has been compared with reference analytical methods for batch hybridoma cell culture experiments.     

The effect of glucose and glutamine on the intracellular nucleotide pool and oxygen uptake rate of a murine hybridoma

The effects of media concentrations of glucose andglutamine on the intracellular nucleotide pools andoxygen uptake rates of a murine antibody-secretinghybridoma cell line were investigated. Cells takenfrom mid-exponential phase of growth were incubated inmedium containing varying concentrations of glucose(0–25 mM) and glutamine (0–9 mM). The intracellularconcentrations of ATP, GTP, UTP and CTP, and theadenylate energy charge increased concomitantly withthe medium glucose concentration. The total adenylatenucleotide concentration did not change over a glucose concentration range of 1–25 mM but therelative levels of AMP, ADP and ATP changed as theenergy charge increased from 0.36 to 0.96. Themaximum oxygen uptake rate (OUR) was obtained in thepresence of 0.1–1 mM glucose. However at glucoseconcentrations >1 mM the OUR decreased suggestinga lower level of aerobic metabolism as a result of theCrabtree effect.A low concentration of glutamine (0.5 mM) caused asignificant increase (45–128%) in the ATP, GTP,CTP, UTP, UDP-GNac, and NAD pools and a doubling ofthe OUR compared to glutamine-free cultures. Theminimal concentration of glutamine also caused anincrease in the total adenylate pool indicating thatthe amino acid may stimulate thede novosynthesis of nucleotides. However, all nucleotidepools and the OUR remained unchanged within the rangeof 0.5–9 mM glutamine.Glucose was shown to be the major substrate forenergy metabolism. It was estimated that in thepresence of high concentrations of glucose (10–25 mM),glutamine provided the energy for the maintenance ofup to 28% of the intracellular ATP pool, whereas theremainder was provided by glucose metabolism.(Author for correspondence; E-mail:     

Growth characteristics in fed-batch culture of hybridoma cells with control of glucose and glutamine concentrations

An online system using HPLC was developed for the measurement of glucose, glutamine, and lactate in a culture broth. Using the system, the glucose and glutamine concentrations were controlled simultaneously by an adaptive-control algorithm within the ranges of 0.2 to 2.0 and 0.1 to 0.6 g/L, respectively. When the glucose concentration was controlled at the low level of 0.2 g/L, the intracellular lactate dehydrogenase activity decreased by one-half and the lactate concentration by one-third, whereas the uptake rates of serine and glycine were about twice as high, compared with the amounts when the glucose concentration was controlled at 1.0 g/L. On the other hand, ammonia production increased when the glucose concentration was kept low. To reduce the production of inhibitory metabolites such as ammonia and lactate and improve the antibody production rate in a hybridoma cell culture, the concentrations of glucose and glutamine were controlled at 0.2 and 0.1 g/L, respectively. With these low concentrations of glucose and glutamine, the cell concentration (4.1 x 10(6) cells/mL) and antibody production (172 mg/L) both increased about twofold compared with the amounts when the glucose was controlled at higher levels. From these results, simultaneous control of the glucose and glutamine concentrations was shown to be useful in the production of antibody by hybridoma cell cultivation. (c) 1994 John Wiley & Sons, Inc.     

In situ growth of gold nanoparticles by enzymatic glucose oxidation within alginate gel matrix

In situ growth of gold nanoparticles (Au NPs) was performed in an alginate gel matrix co‐encapsulating Au NP seeds and glucose oxidase (GOx) for the development of a glucose‐sensing platform. We observed a simultaneous growth of Au NPs in the alginate matrix through the reduction of AuCl by H₂O₂ on Au NP seeds. The detection of the glucose level was carried out successfully via the coupling of Au NP enlargement with the oxidation of glucose catalyzed by the immobilized GOx. The enlargement of Au NPs in the alginate matrix exhibited only a red‐shift in absorbance maxima, while the generation of small Au NPs in a free solution caused a blue‐shift in higher glucose concentrations. This study shows that the co‐encapsulation of metal NPs and bioreceptor in a gel matrix may provide a simple route for the fabrication of an optical biosensor. Biotechnol. Bioeng. 2010;105: 210–214. © 2009 Wiley Periodicals, Inc.     

The use of differential measurements with a glucose biosensor for interference compensation during glucose determinations by flow injection analysis

A novel detection system for the determination of glucose in the presence of clinically important interferents, based on the use of dual sensors and flow-injection analysis (FIA), is described. The normalisation methodology involves measurement of the interference signal at a reference sensor; this signal can then be subtracted from the glucose sensor signal (post-run) to give a corrected measurement of the glucose concentration. The detection system consists of a thin layer cell with dual glassy carbon working electrodes. One electrode was surface modified to act asglucose biosensor by immobilisation of glucose oxidase (GOx) (from Aspergillus niger) with 1% glutaraldehyde and bovine serum albumin. The second electrode (glucose oxidase omitted) was utilised to measure the interference signal responding only to electroactive species present in the injected sample. A computer controlled multichannel potentiostat was used for potential application and current monitoring duties. The sensor responses were saved in ASCII format to facilitate post-run analysis in Microsoft Excel. Cyclic voltammetry (CV) was utilised to investigate the manner in which the interference signal contributed to the total signal obtained at the biosensor in the presence of glucose. The kinetic parameters I_max and the apparent Michaelis-Menten constant, K′_m, were calculated for the sensor operating under flow-injection conditions.     

葡萄糖测定方法的比较研究

比较了传统斐林定糖,葡萄糖氧化酶－过氧化物酶比色法,葡萄糖氧化酶电极自动分析仪法测定葡萄糖。比较测定了的结果显示,三法的平均标准误差（ＳＤ）,变异系数（ＣＶ）均十分接近。通过对此三种方法的回归相关性分析显示：斐林法－酶终点比色法的回归方程为ｙ＝０．９８４３ｘ＋６．３２３９,相关系数Ｒ＾２＝０．９９８９,斐林法－自动分析仪法的回归方程为ｙ＝１．００８８ｘ＋２．０４８３,相关系数Ｒ＾２＝０．９９９１,     

A kinetic analysis of hybridoma growth and metabolism in continuous suspension culture on serum-free medium

The steady-state metabolic parameters for a murine hybridoma cell line have been determined in continuous suspension culture over a wide range of dilution rates. Long-term adaption occurred over seven months in culture and resulted in lower glucose consumption rates, reduced lactate production, higher cell viability, and, consequently, growth rates more nearly matching the dilution rate. Antibody production rates decreased over the first two months and then remained stable for at least 75 days. The antibody production rate was not found to be growth associated. Steadystate amino acid uptake rates are presented for a wide range of growth rates.     

Construction of multi-chimeric pyrroloquinoline quinone glucose dehydrogenase with improved enzymatic properties and application in glucose monitoring

A multi-chimeric enzyme was constructed by combining the protein regions responsible for the enzymatic properties of Escherichia coli and Acinetobacter calcoaceticus pyrroloquinoline quinone glucose dehydrogenase (PQQGDH). The constructed multi-chimeric PQQGDH showed increased co-factor binding stability, thermal stability, an alteration in substrate specificity and a 10-fold increase in the K _m value for glucose compared with the wild-type E. coli PQQGDH. The cumulative effect of each introduced protein region on the improvement of enzymatic properties was observed. The application of the multi-chimeric PQQGDH in amperometric glucose sensor construction achieved an expanded dynamic range together with increased operational stability and narrower substrate specificity. The glucose sensor can measure glucose from 5 to 40 mM, suggesting its potential for the direct measurement of high blood-glucose levels in diabetic patients.     

Methods for determination of growth-rate-dependent changes in hybridoma volume,shape and surface structure during continuous recycle

Hybridoma volume and surface membrane structure were found to vary as a function of specific growth rate using a method of cell recycle with continuous medium perfusion to vary growth rate. Mean hybridoma volume determined at constant osmolality by both electronic particle counting and scanning electron microscopic (SEM) methods indicated that rapidly growing cells are significantly larger than very slowly growing cells. We have previously determined that during both rapid and slow growth over a range of L-glutamine provision rates (Gln PR) that specific monoclonal antibody (MoAb) secretion rate was not changed. In this study a constant MoAb secretion rate per unit of membrane area was found which may indicate that changing membrane area is not a rate-determining factor in MoAb secretion. SEM methods were of limited use for accurate determination of cell volume due to cell shrinkage and large coefficients of variations. In spite of this limitation, SEM stereology methods were useful in confirming that cells remained spherical over a wide range of specific growth rates and that hybridoma cells were not circular. Sequential SEM observations also revealed that surface membrane structure of the 9.2.27 murine hybridoma investigated was correlated with growth rate. Under conditions of very slow growth, hybridoma surface microvilli density appeared to be significantly reduced.     

Production of monoclonal antibodies against human chorionic gonadotropin by hybridoma cultures in calcium alginate capsules

A new method of making microcapsules with calcium alginate gel was developed and the cultivation of the encapsulated hybridoma cells producing monoclonal antibodies against human chorionic gonadotropin was investigated. A high cell density of 2.0×10⁸ cells/cm³ in the capsules led to a high dilution rate of 0.68 per hour and resulted in the high volumetric monoclonal antibody productivity of 652.8 mg/l/day, which was 20–30 times higher than those of traditional continuous suspension cultures. However, long-term continuous culture was not achieved with this capsule system probably because of the limitation in nutrient supply and the accumulation of waste products. Also the analysis of oxygen transfer in this system showed that oxygen supply was not enough to support such a high cell density.     

Simultaneous determination of the monoamine neurotransmitters and glucose in rat brain by microdialysis sampling coupled with liquid chromatography-dual electrochemical detector

A new type of chemically modified electrode based ring-disk electrode as the dual electrochemical detector (DECD) for high-performance liquid chromatography (HPLC) to simultaneously determine the monoamine neurotransmitters and glucose is described. The ring electrode was modified with an ion-exchange polymer-overoxidized polypyrrole (OPPy) and the disk electrode was modified with nano Au colloid and glucose oxidase (GOD). The electrochemical behaviors of dopamine (DA) and ascorbic acid (AA) at the OPPy chemically modified electrode (CME) were investigated by differential pulse voltammetry (DPV). It was found that the CME could permeate dopamine cations and repelled the ascorbate anions, which could be used to determine the monoamine neurotransmitters and avoid the interference of AA. The electrochemical behavior of glucose at the Nafion/GOD-Au colloid/GC CME was investigated by amperometry and flow injection analysis (FIA). It was found that the sensitivity of the CME increased apparently in determination of glucose. In order to obtain better separation and current responses of the analytes in HPLC-DECD, several operational parameters have been investigated. Under the optimum conditions, the method showed good stability and reproducibility. The application of this method coupled with microdialysis sampling for in vivo simultaneous determination of monoamine neurotransmitters and glucose in rat brain was satisfactory.     

Offline glucose biomonitoring in yeast culture by polyamidoamine/cysteamine-modified gold electrodes

This article deals with the use of pyranose oxidase (PyOx) and glucose oxidase (GOx) enzymes in amperometric biosensor design and their application in monitoring fermentation processes with the combination of flow injection analysis (FIA). The amperometric studies were carried out at -0.7 V by following the oxygen consumption due to the enzymatic reactions for both batch and FIA modes. Optimization studies (enzyme amounts and pH) and analytical parameters such as linearity, repeatability, effect of interference, storage, and operational stabilities have been studied. Under optimized conditions, for the PyOx-based biosensor, linear graph was obtained from 0.025 to 0.5 mM glucose in phosphate buffer (50 mM) at pH 7.0 with the equation of y = 3.358x + 0.028 and R(2) = 0.998. Linearity was found to be 0.01-1.0 mM in citrate buffer (50 mM and pH 4.0) with the equation of y = 1.539x + 0.181 and R(2) = 0.992 for the GOx biosensor. Finally, these biosensor configurations were further evaluated in a conventional flow injection system. Results from batch experiments provide a guide to design sensitive, stable, and interference-free biosensors for FIA mode. Biosensor stability, dynamic range, and repeatability were also studied in FIA conditions, and the applicability for the determination of glucose in fermentation medium could be successfully demonstrated. The FIA-combined glucose biosensor was used for the offline monitoring of yeast fermentation. The obtained results correlated well with HPLC measurements.     

Simultaneous transdermal extraction of glucose and lactate from human subjects by reverse iontophoresis

This study investigated the possibility of simultaneously extracting glucose and lactate from human subjects, at the same skin location, using transdermal reverse iontophoresis. Transdermal monitoring using iontophoresis is made possible by the skin's permeability to small molecules and the nanoporous and microporous nature of the structure of skin. The study was intended to provide information which could be used to develop a full, biosensor-based, monitoring system for multiple parameters from transdermal extraction. As a precursor to the human study, in vitro reverse iontophoresis experiments were performed in an artificial skin system to establish the optimum current waveforms to be applied during iontophoresis. In the human study, a bipolar DC current waveform (with reversal of the electrode current direction every 15 minutes) was applied to ten healthy volunteers via skin electrodes and utilized for simultaneous glucose and lactate transdermal extraction at an applied current density of 300 microA/cm2. Glucose and lactate were successfully extracted through each subject's skin into the conducting gel that formed part of each iontophoresis electrode. The results suggest that it will be possible to noninvasively and simultaneously monitor glucose and lactate levels in patients using this approach and this could have future applications in diagnostic monitoring for a variety of medical conditions.     

Synthesis and characterization of drug–saccharide conjugates by enzymatic strategy in organic media

A facile and efficient strategy to prepare drug derivatives with saccharides was developed, and 12 drug–saccharide conjugates were obtained by selective enzymatic synthesis methods. When the transesterification of chlorphenesin vinyl esters with glucose was chosen as a model reaction, the influence of reaction conditions was optimized. Then, we successfully prepared a series of chlorphenesin–saccharide derivatives of three monosaccharides (glucose, mannose and galactose) and three disaccharides (maltose, lactose and sucrose). In order to study the relationship between the solubility of drug–saccharide conjugates and the structure of parent drugs, five hydrophobic drugs (chlorphenesin, mephenesin, guaifenesin, propranolol and clorprenaline) were chosen as substrates to synthesize drug–saccharide conjugates. The results indicated that the aqueous solublity of drug–saccharide derivatives was greatly improved, which was changed by different saccharides and the structure of parent drugs.     

Influence of glucose solubility and dissolution rate on the kinetics of lipase catalyzed synthesis of glucose laurate in 2-methyl 2-butanol

The lipase catalyzed acylation of glucose by dodecanoic acid in 2-methyl 2-butanol was studied. The initial reaction rate was strongly dependent on the dissolved glucose concentration in the medium. Several methods were shown to increase dissolved glucose concentrations and initial reaction rates, namely, the use of solid beta-glucose, amorphous solid glucose, and supersaturated glucose solution. Supersaturated glucose solutions in 2-methyl 2-butanol showed a high stability even in the presence of solid crystalline glucose. During the reaction, the dissolved glucose concentration falls as the reaction proceeds, before recovering later as more of the excess solid dissolves. However, the ester synthesis rate continues to fall even after glucose concentration reaches its minimum, so glucose dissolution rate limitation is not responsible for the synthesis rate decline. Experiments with added molecular sieves show that the main reason is the accumulation of product water. In the presence of molecular sieves, 70% of glucose was converted to ester, independent of the initial soluble glucose in the medium.     

Role of vitamins in determining apoptosis and extent of suppression by bcl-2 during hybridoma cell culture

The identification of cell culture media components that may instigate apoptosis in cell lines used for the production of commercial antibodies and recombinant proteins, is crucial to aid the development of improved media for reduced cell death and to understand the role of nutrient components in cell survival and maintenance. Here we determine the impact of depriving all or individual B-group media vitamins either, D-CaPantothenate (DCaP), choline chloride (CC), riboflavin (Rb), i-inositol, nicotinamide (NAM), pyridoxal hydrochloride (PyrHCl), folic acid (FA), or thiamine hydrochloride (ThHCl) on hybridoma cell growth and viability using fluorescence microscopy techniques. Cultivation in media deprived of all these vitamins prevented cell proliferation from reaching maximum capacity while increasing cell death rate, predominantly via apoptosis. Deletion of either DCaP, CC, or Rb showed that these components were most likely responsible for the development of apoptosis. Exclusion of either i-inositol, NAM or PyrHCl failed to inhibit cell growth and viability, while marginal improvements in viability were noted by ThHCl deprivation and more so by FA exclusion. Over-expression of the anti-apoptotic gene bcl-2 suppressed cell death initiated by all or single vitamin (either DCaP, CC or Rb) deprivation. The involvement of bcl-2 activity, established a close association between small vitamin molecules particularly DCaP, CC or Rb and the biochemical activation of apoptosis.     

Immobilization of glucose oxidase in thin polypyrrole films: influence of polymerization conditions and film thickness on the activity and stability of the immobilized enzyme

Using monomers that polymerize to form electrically conducting polymers, one can control the thickness of the polymer film and the amount of enzyme that can be immobilized in the films. First, an investigation of the major variables that influence the immobilization of glucose oxidase by entrapment in polypyrrole films, prepared by electropolymerization from aqueous solutions containing the enzyme and monomer, was carried out. Then the optimized conditions were used to assess the effects of film thickness on the activity and stability of immobilized enzyme. For the films ranged in thickness from 0.1 mum to 1.6 mum, the resulting apparent activity and stability of the immobilized enzyme were found to be a strong function of the polymer film thickness. Above a thickness of 1.0 mum, the apparent activity of the immobilized enzyme increases linearly with increasing film thickness. The nonlinearity observed for films of thickness less than 1.0 mum can be attributed to the changes observed in the morphology of the resulting polypyrrole films. Furthermore, it was noted that when the glucose oxidase/polypyrrole films are stored in phosphate buffer, at 4 degrees C, the observed rate of loss in apparent activity of the immobilized enzyme is highest for the first few days, also being higher for the thinner films. However, after the loosely entrapped enzyme is leached from the polymer film, the rate of loss in activity is very low indicating that the well-entrapped enzyme, as well as the polypyrrole films, exhibit good stability. Finally, the reproducibility of the immobilization technique is excellent. (c) 1993 John Wiley & Sons, Inc.     

A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells

A goal in recombinant protein production using Chinese hamster ovary (CHO) cells is to achieve both high specific productivity and high cell density. Addition of glucose to the culture media is necessary to maintain both cell growth and viability. We varied the glucose concentration in the media from 5 to 16 g/L and found that although specific productivity of CHO‐DG44 cells increased with the glucose level, the integrated viable cell density decreased. To examine the biological basis of these results, we conducted a discovery proteomic study of CHO‐DG44 cells grown under batch conditions in normal (5 g/L) or high (15 g/L) glucose over 3, 6, and 9 days. Approximately 5,000 proteins were confidently identified against an mRNA‐based CHO‐DG44 specific proteome database, with 2,800 proteins quantified with at least two peptides. A self‐organizing map algorithm was used to deconvolute temporal expression profiles of quantitated proteins. Functional analysis of altered proteins suggested that differences in growth between the two glucose levels resulted from changes in crosstalk between glucose metabolism, recombinant protein expression, and cell death, providing an overall picture of the responses to high glucose environment. The high glucose environment may enhance recombinant dihydrofolate reductase in CHO cells by up‐regulating NCK1 and down‐regulating PRKRA, and may lower integrated viable cell density by activating mitochondrial‐ and endoplasmic reticulum‐mediated cell death pathways by up‐regulating HtrA2 and calpains. These proteins are suggested as potential targets for bioengineering to enhance recombinant protein production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1026–1038, 2015     

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.     

葡萄糖和木糖双底物生物转化生产2,3-丁二醇和氢气的代谢计量分析

以Klebsiella pneumoniae利用葡萄糖和木糖双底物生物转化生产2,3-丁二醇和氢气过程为研究对象,对其进行代谢计量分析。分析结果显示:2,3-丁二醇和氢气相对于底物葡萄糖和木糖的质量收率依赖于还原能力NADH2氧化磷酸化的分率(δ)。当δ=27时,即在总还原能力NADH2中有27mol NADH2被氧化磷酸化,剩余部分用来产生氢气,呼吸商为14时,2,3-丁二醇和氢气的最优质量收率分别为50%和0.8%;而当δ=1,即还原能力NADH2全部被氧化磷酸化、不产生氢气,呼吸商为4时,2,3-丁二醇的质量收率为37.5%;2,3-丁二醇和氢气的质量收率与底物中葡萄糖和木糖的比值无关。而氢气的摩尔收率与底物中葡萄糖和木糖的比值相关,当底物全部是葡萄糖或木糖时,其最优摩尔收率分别为71%和60%。该分析结果为葡萄糖和木糖双底物生物转化生产2,3-丁二醇过程的实验研究奠定了理论基础。