Application of dielectric spectroscopy for monitoring high cell density in monoclonal antibody producing CHO cell cultivations

The application of dielectric spectroscopy was frequently investigated as an on-line cell culture monitoring tool; however, it still requires supportive data and experience in order to become a robust technique. In this study, dielectric spectroscopy was used to predict viable cell density (VCD) at industrially relevant high levels in concentrated fed-batch culture of Chinese hamster ovary cells producing a monoclonal antibody for pharmaceutical purposes. For on-line dielectric spectroscopy measurements, capacitance was scanned within a wide range of frequency values (100–19,490 kHz) in six parallel cell cultivation batches. Prior to detailed mathematical analysis of the collected data, principal component analysis (PCA) was applied to compare dielectric behavior of the cultivations. PCA analysis resulted in detecting measurement disturbances. By using the measured spectroscopic data, partial least squares regression (PLS), Cole–Cole, and linear modeling were applied and compared in order to predict VCD. The Cole–Cole and the PLS model provided reliable prediction over the entire cultivation including both the early and decline phases of cell growth, while the linear model failed to estimate VCD in the later, declining cultivation phase. In regards to the measurement error sensitivity, remarkable differences were shown among PLS, Cole–Cole, and linear modeling. VCD prediction accuracy could be improved in the runs with measurement disturbances by first derivative pre-treatment in PLS and by parameter optimization of the Cole–Cole modeling.     

Multifrequency permittivity measurements enable on‐line monitoring of changes in intracellular conductivity due to nutrient limitations during batch cultivations of CHO cells

Lab and pilot scale batch cultivations of a CHO K1/dhfr^? host cell line were conducted to evaluate on‐line multifrequency permittivity measurements as a process monitoring tool. The β‐dispersion parameters such as the characteristic frequency (f_C) and the permittivity increment (Δε_max) were calculated on‐line from the permittivity spectra. The dual‐frequency permittivity signal correlated well with the off‐line measured biovolume and the viable cell density. A significant drop in permittivity was monitored at the transition from exponential growth to a phase with reduced growth rate. Although not reflected in off‐line biovolume measurements, this decrease coincided with a drop in OUR and was probably caused by the depletion of glutamine and a metabolic shift occurring at the same time. Sudden changes in cell density, cell size, viability, capacitance per membrane area (C_M), and effects caused by medium conductivity (σ_m) could be excluded as reasons for the decrease in permittivity. After analysis of the process data, a drop in f_C as a result of a fall in intracellular conductivity (σ_i) was identified as responsible for the observed changes in the dual‐frequency permittivity signal. It is hypothesized that the β‐dispersion parameter f_C is indicative of changes in nutrient availability that have an impact on intracellular conductivity σ_i. On‐line permittivity measurements consequently not only reflect the biovolume but also the physiological state of mammalian cell cultures. These findings should pave the way for a better understanding of the intracellular state of cells and render permittivity measurements an important tool in process development and control. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Analysis of mammalian viable cell biomass based on cellular ATP

Analysis of cellular ATP as a means of measuring viable biomass loading was investigated in hybridoma cell culture. ATP analysis by the luciferin-luciferase assay was compared with trypan blue-stained hemocytometer counts. The cell-specific ATP content varied between 2 and 6 fmol per viable cell over a batch culture. ATP levels were highest during exponential growth, and decreased during the stationary and decline phases. Electronic counting and volume measurements were performed to assay the viable cell biomass. Cell sorting, using fluorescein diacetate, was used to separate viable and nonviable cells in cultures with between 35% and 90% viable cells. Viable cells contained over 2 orders of magnitude greater cell-specific ATP than nonviable cells. Cell-specific ATP correlated directly with the viable cell volume rather than viable cell numbers. Over the range of batch culture conditions, ATP analysis should provide a more accurate measurement of hybridoma viable biomass than hemocytometer counts.     

A novel approach for using dielectric spectroscopy to predict viable cell volume (VCV) in early process development

Online monitoring of viable cell volume (VCV) is essential to the development, monitoring, and control of bioprocesses. The commercial availability of steam‐sterilizable dielectric‐spectroscopy probes has enabled successful adoption of this technology as a key noninvasive method to measure VCV for cell‐culture processes. Technological challenges still exist, however. For some cell lines, the technique's accuracy in predicting the VCV from probe‐permittivity measurements declines as the viability of the cell culture decreases. To investigate the cause of this decrease in accuracy, divergences in predicted vs. actual VCV measurements were directly related to the shape of dielectric frequency scans collected during a cell culture. The changes in the shape of the beta dispersion, which are associated with changes in cell state, are quantified by applying a novel “area ratio” (AR) metric to frequency‐scanning data from the dielectric‐spectroscopy probes. The AR metric is then used to relate the shape of the beta dispersion to single‐frequency permittivity measurements to accurately predict the offline VCV throughout an entire fed‐batch run, regardless of cell state. This work demonstrates the possible feasibility of quantifying the shape of the beta dispersion, determined from frequency‐scanning data, for enhanced measurement of VCV in mammalian cell cultures by applying a novel shape‐characterization technique. In addition, this work demonstrates the utility of using changes in the shape of the beta dispersion to quantify cell health. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:479–487, 2014     

The use of culture redox potential and oxygen uptake rate for assessing glucose and glutamine depletion in hybridoma cultures

Culture redox potential (CRP) and oxygen uptake rate (OUR) were monitored on-line during glucose- and glutamine-limited batch cultures of a murine hybridoma cell line that secretes a neutralizing monoclonal antibody specific to toxin 2 of the scorpion Centruroides noxius Hoffmann. It was found that OUR and CRP can be used for assessing the viable cell concentration and growth phases of the culture. Before nutrient depletion, OUR increased exponentially with viable cell concentration, whereas CRP decreased monotonically until cell viability started to decrease. During the death phase, CRP gradually increased. A sudden decrease in OUR occurred upon glucose or glutamine depletion. CRP traced the dissolved oxygen profile during a control action or an operational eventuality, however, during nutrient depletion it did not follow the expected behavior of a system composed mainly by the O(2)/H(2)O redox couple. Such a behavior was not due to the accumulated lactate or ammonia, nor to possible intracellular redox potential changes caused by nutrient depletion, as inferred from respiration inhibition by rotenone or uncoupled respiration by 2,4-dinitrophenol. As shown in this study, operational eventualities can be erroneously interpreted as changes in OUR when using algorithms based solely on oxygen balances. However, simultaneous measurements of CRP and OUR may be used to discriminate real metabolic events from operational failures. The results presented here can be used in advanced real-time algorithms for controling glucose and glutamine at low concentrations, avoiding under- or over-feeding them in hybridoma cultures, and consequently reducing the accumulation of metabolic wastes and improving monoclonal antibody production. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 555-563, 1997.     

On-line monitoring of the progress of infection in Sf-9 insect cell cultures using relative permittivity measurements

The use of on-line relative permittivity (epsilon') measurements for monitoring cultures of Sf-9 cells was evaluated in a batch culture and a batch infected with a baculovirus expressing beta-galactosidase. It was found that viable cell density and volume essentially accounted for all the variation in epsilon' in both non-infected and synchronously infected cultures, indicating that the epsilon' of a cell suspension was sensitive only to changes in the viable cell population. Additionally the parameter provided clearly defined signposts of the progress of the infection.     

Cell cycle,cell size and mitochondrial activity of hybridoma cells during batch cultivation

Cell cycle, cell size and rhodamine 123 fluorescence in cell populations of two batch cultures were analysed and quantified with a fluorescence-activated cell sorter (FACS). Two cultures derived from either exponential or stationary phase innocula were investigated in order to demonstrate the dependency of the subsequent cell growth on innoculum condition. The results demonstrated that the level of activity of cells in the innoculum culture could have a significant effect on cellular activity during the initial phase of the inoculated culture, as it advances through its growth cycle. Positive correlation was found between the cell size and mitochondrial activity (as measured by rhodamine 123 uptake) with S and G₂ fractions as the cell progressed through the cell cycle. The enumeration of the fractions of cell cycle phases has helped in prediction of the changes in cell numbers following perturbation of the culture condition.     

On-line monitoring of physiological parameters of insect cell cultures during the growth and infection process

On-line monitoring of insect cell cultures used for the production of recombinant proteins with the baculovirus expression vector system (BEVS) provides valuable tools for the optimization, operation, and control of the production process. The relative permittivity (epsilon') and CO(2) evolution rates (CER) were measured on-line using the biomass monitor and the infrared CO(2) analyzer, respectively. The growth and infection phases of two different cell lines, Spodoptera frugiperda (Sf-9) and Trichoplusia ni(High-5), were monitored using the above measurements. These in turn were correlated to the progress of the culture by using the off-line measurements of protein produced, virus titer, and biovolume, which is the product of viable cell density and mean cell volume. The epsilon', CER, and the biovolume profiles were closely matched during the growth phase of cells when grown in a batch or fed batch culture. The relationship became more complex when the cultures were either in stationary phase or in the postinfection phase. The epsilon' profile was found to be a good indicator of the process of synchronous baculoviral infection, showing a plateau between 18 and 24 h postinfection (hpi), the period during which budded virus is produced, and a peak at approximately 48 hpi correlated to the onset of accelerated cell lysis. The CER profile continues to increase after the growth period with a peak around the 24 hpi period, after which there is a decline in the profile corresponding to release of virus as seen from virus titer determinations. This was examined for Sf-9 cultures under conditions of cell densities from 3 to 50 x 10(6) cells/mL and MOI values ranging from 0.001 to 1000. The profiles were found to be similar also in the case of the High-5 cells. Thus both measurements give reliable information regarding the physiological status of the cells as seen from their correlation to virus and protein production. A further combination of these with the off-line measured parameters such as the biovolume and metabolite concentrations can give a more detailed understanding of the process and help in the better design and automation of these processes.     

On-line monitoring of respiration in recombinant-baculovirus infected and uninfected insect cell bioreactor cultures

Respiration rates in Spodoptera frugiperda (Sf-9) cell bioreactor cultures were successfully measured on-line using two methods: The O(2) uptake rate (OUR) was determined using gas phase pO(2) values imposed by a dissolved oxygen controller and the CO(2) evolution rate (CER) was measured using an infrared detector. The measurement methods were accurate, reliable, and relatively inexpensive. The CER was routinely determined in bioreactor cultures used for the production of several recombinant proteins. Simple linear relationships between viable cell densities and both OUR and CER in exponentially growing cultures were used to predict viable cell density. Respiration measurements were also used to follow the progress of baculoviral infections in Sf-9 cultures. Infection led to increases in volumetric and per-cell respiration rates. The relationships between respiration and several other culture parameters, including viable cell density, cell protein, cell volume, glucose consumption, lactate production, viral titer, and recombinant beta-galactosidase accumulation, were examined. The extent of the increase in CER following infection and the time postinfection at which maximum CER was attained were negatively correlated with the multiplicity of infection (MOI) at multiplicities below the level required to infect all the cells in a culture. Delays in the respiration peak related to the MOI employed were correlated with delays in the peak in recombinant protein accumulation. DO levels in the range 5-100% did not exert any major effects on viable cell densities, CER, or product titer in cultures infected with a baculovirus expressing recombinant beta-galactosidase. (c) 1996 John Wiley & Sons, Inc.     

Optimization of HEK-293S cell cultures for the production of adenoviral vectors in bioreactors using on-line OUR measurements

The culture of HEK-293S cells in a stirred tank bioreactor for adenoviral vectors production for gene therapy is studied. Process monitoring using oxygen uptake rate (OUR) was performed. The OUR was determined on-line by the dynamic method, providing good information of the process evolution. OUR enabled cell activity monitoring, facilitating as well the determination of the feeding rate in perfusion cultures and when to infect the culture. Batch cultures were used to validate the monitoring methodology. A cell density of 10 × 10⁵ cell/mL was infected, producing 1.3 × 10⁹ infectious viral particles/mL (IVP/mL).To increase cell density values maintaining cell specific productivity, perfusion cultures, based on tangential flow filtration, were studied. In this case, OUR measurements were used to optimize the dynamic culture medium feeding strategy, addressed to avoid any potential nutrient limitation. Furthermore, the infection protocol was defined in order to optimize the use of the viral inoculum, minimizing the uncontrolled release of particles through the filter unit mesh. All these developments enabled an infection at 78 × 10⁵ cell/mL with the consequent production of 44 × 10⁹ IVP/mL, representing a cell specific productivity 4.3 times higher than for the batch culture.     

Revisiting Verhulst and Monod models: analysis of batch and fed-batch cultures

The paper re-evaluates Verhulst and Monod models. It has been claimed that standard logistic equation cannot describe the decline phase of mammalian cells in batch and fed-batch cultures and in some cases it fails to fit somatic growth data. In the present work Verhulst, population-based mechanistic growth model was revisited to describe successfully viable cell density (VCD) in exponential and decline phases of batch and fed-batch cultures of three different CHO cell lines. Verhulst model constants, K, carrying capacity (VCD/ml or μg/ml) and r, intrinsic growth factor (h⁻¹) have physical meaning and they are of biological significance. These two parameters together define the course of growth and productivity and therefore, they are valuable in optimisation of culture media, developing feeding strategies and selection of cell lines for productivity. The Verhulst growth model approach was extended to develop productivity models for batch and fed-batch cultures. All Verhulst models were validated against blind data (R² > 0.95). Critical examination of theoretical approaches concluded that Monod parameters have no physical meaning. Monod-hybrid (pseudo-mechanistic) batch models were validated against specific growth rates of respective bolus and continuous fed-batch cultures (R² ≈ 0.90). The reduced form of Monod-hybrid model C_L/(K_L + C_L) describes specific growth rate during metabolic shift (R² ≈ 0.95). Verhulst substrate-based growth models compared favourably with Monod-hybrid models. Thus, experimental evidence implies that the constants in the Monod-hybrid model may not have physical meaning but they behave similarly to the biological constants in Michaelis–Menten enzyme kinetics, the basis of the Monod growth model.     

Real-time monitoring of adherent Vero cell density and apoptosis in bioreactor processes

This study proposes an easy to use in situ device, based on multi-frequency permittivity measurements, to monitor the growth and death of attached Vero cells cultivated on microporous microcarriers, without any cell sampling. Vero cell densities were on-line quantified up to 10⁶ cell mL⁻¹. Some parameters which could potentially impact Vero cell morphological and physiological states were assessed through different culture operating conditions, such as media formulation or medium feed-harvest during cell growth phase. A new method of in situ cell death detection with dielectric spectroscopy was also successfully implemented. Thus, through permittivity frequency scanning, major rises of the apoptotic cell population in bioreactor cultures were detected by monitoring the characteristic frequency of the cell population, f_c, which is one of the culture dielectric parameters. Both cell density quantification and cell apoptosis detection are strategic information in cell-based production processes as they are involved in major events of the process, such as scale-up or choice of the viral infection conditions. This new application of dielectric spectroscopy to adherent cell culture processes makes it a very promising tool for risk-mitigation strategy in industrial processes. Therefore, our results contribute to the development of Process Analytical Technology in cell-based industrial processes.     

A new method for on-line measurement of the volumetric oxygen uptake rate in membrane aerated animal cell cultures

Oxygen is a key substrate in animal cell metabolism and its consumption is thus a parameter of great interest for bioprocess monitoring and control. A system for measuring it based on an oxygen balance on the liquid phase was developed. The use of a gas-permeable membrane offered the possibility to provide the required quantity of oxygen into the culture, while avoiding problems of foaming or shear stress generally linked to sparging. This aeration system allowed moreover to keep a known and constant k(L)a value through cultures up to 400 h. Oxygen uptake rate (OUR) was measured on-line with a very good accuracy of +/-5%, and the specific OUR for a CHO cell line was determined during batch (growth phase) and continuous culture as, respectively, equal to 2. 85x10(-13) and 2.54x10(-13) mol O(2) cell(-1) h(-1). It was also shown that OUR continuous monitoring gives actually more information about the metabolic state of the culture than the cell concentration itself, especially during transition phases like the end of the growth phase in a batch culture.     

Computer programs for modeling mammalian cell batch and fed-batch cultures using logistic equations

A MATLAB^® toolbox was developed for applying the logistic modeling approach to mammalian cell batch and fed-batch cultures. The programs in the toolbox encompass sensitivity analyses and simulations of the logistic equations in addition to cell specific rate estimation. The toolbox was first used to generate time courses of the sensitivity equations for characterizing the relationship between the logistic variable and the model parameters. Subsequently, the toolbox was used to describe CHO cell data from batch and fed-batch mammalian cell cultures. Cell density, product, glucose, lactate, glutamine, and ammonia data were analyzed for the batch culture while fed-batch analysis included cell density and product concentration. In all instances, experimental data were well described by the logistic equations and the resulting specific rate profiles were representative of the underlying cell physiology. The 6-variable batch culture data set was also used to compare the logistic specific rates with those from polynomial fitting and discrete derivative methods. The polynomial specific rates grossly misrepresented cell behavior in the initial and final stages of culture while those based on discrete derivatives had high variability due to computational artifacts. The utility of logistic specific rates to guide process development activities was demonstrated using specific protein productivity versus growth rate trajectories for the 3 cultures examined in this study. Overall, the computer programs developed in this study enable rapid and robust analysis of data from mammalian cell batch and fed-batch cultures which can help process development and metabolic flux estimation.     

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures was examined, using on-line off-gas analyses to measure the oxygen and carbon dioxide consumption rates continuously. A cell suspension from continuous cultures at steady state was used as the inoculum. It was observed that a dynamic phase occurred in the initial phase of the experiment. In this phase the bacterial ferrous iron oxidation and growth were uncoupled. After about 16 h the bacteria were adapted and achieved a pseudo-steady state, in which the specific growth rate and oxygen consumption rate were coupled and their relationship was described by the Pirt equation. In pseudo-steady state, the growth and oxidation kinetics were accurately described by the rate equation for competitive product inhibition. Bacterial substrate consumption is regarded as the primary process, which is described by the equation for competitive product inhibition. Subsequently the kinetic equation for the specific growth rate, μ, is derived by applying the Pirt equation for bacterial substrate consumption and growth. The maximum specific growth rate, μ _max, measured in the batch culture agrees with the dilution rate at which washout occurs in continuous cultures. The maximum oxygen consumption rate, q _O2,max, of the cell suspension in the batch culture was determined by respiration measurements in a biological oxygen monitor at excess ferrous iron, and showed changes of up to 20% during the course of the experiment. The kinetic constants determined in the batch culture slightly differ from those in continuous cultures, such that, at equal ferric to ferrous iron concentration ratios, biomass-specific rates are up to 1.3 times higher in continuous cultures. Received: 8 February 1999 / Accepted: 17 February 1999     

Specific monoclonal antibody productivity and the cell cycle-comparisons of batch,continuous and perfusion cultures

A selection of mouse hybridoma cell lines showed a variation of approximately two orders of magnitude in intracellular monoclonal antibody contents. The different levels directly influenced apparent specific monoclonal antibody productivity during the death phase but not during the growth phase of a batch culture. The pattern of changes in specific productivity during culture remained basically similar even though at different levels for all cell lines tested. Arresting the cells in the G₁ phase using thymidine increased the specific productivity, cell volume and intracellular antibody content but at the same time led to decreased viability. In continuous culture DNA synthesis decreased with decreasing dilution rate though without an accompanying change in cell cycle and cell size distributions. The data shows both the decrease in viability and intracellular antibody content to be important factors which influence the negative association between specific antibody productivity and growth rate. In high cell density perfusion culture, when the cell cycle was prolonged by slow growth, viability was low and dead, but not lysed, cells were retained in the system, the specific antibody productivity was nearly two fold higher than that obtained in either batch or continuous cultures. The results imply that the prolongation of G₁ phase and the increase in death rate of cells storing a large amount of antibody together cause an apparent increase in specific antibody productivity.     

Cell cycle- and growth phase-dependent variations in size distribution, antibody productivity, and oxygen demand in hybridoma cultures

Simultaneous determination of cell size and DNA content of hybridomas (HB-32) revealed a direct correlation between average cell volume and progression through the cell cycle. Pseudocontinuous experiments showed that G(1) cells, as estimated from cell size measurements, secreted monoclonal antibody at rates higher than those of cells in other stages of interphase and mitosis. Similarly, fed-batch and batch experiments suggested that specific oxygen uptake rate (qO(2)) is also a function of cell cycle, being minimum for cells in G(0) and G(1) phase. In batch cultures, HB-32 showed a rapid decrease in oxygen uptake rate (OUR) just prior to reaching maximum cell concentration. The OUR steadily increased from 0.01-0.05 to 0.5-0.7 mmol O(2)/L h as the cells went from the lag to the midexponential phase. The qO(2) increased from 0.3 x 10(-10)-0.9 x 10(-10) mmol O(2)/cell h at inoculation to 3.3 x 10(-10)-3.7 x 10(-10) mmol O(2)/cell h during the early exponential phase where it remained relatively constant. Several hours before maximum cell concentration was reached, OUR and qO(2) rapidly decreased to levels below those observed at inoculation. The time at which the shift in OUR and qO(2) occurred and the onset of decrease in the average cell size corresponded to the time of glutamine depletion. Based on monitoring OUR on-line in batch cultures, glutamine was supplemented, resulting in increased cell concentration, extension of culture viability, and increased MAb concentration.     

Effects of temperature shift on cell cycle, apoptosis and nucleotide pools in CHO cell batch cultues

Temperature reduction in CHO cell batch culture may be beneficial in the production of recombinant protein and in maintenance of viability. The effects on cell cycle, apoptosis and nucleotide pools were studied in cultures initiated at 37°C and temperature shifted to 30 °C after 48 hours. In control cultures maintained at 37 °C, viable cells continued to proliferate until the termination of the culture, however, temperature reduction caused a rapid decrease in the percent of cells in S phase and accumulation of cells in G-1. This was accompanied by a concurrent reduction in U ratio (UTO/UDP-GNAc), previously shown to be a sensitive indicator of growth rate. Culture viability was extended following temperature shift, as a result of delayed onset of apoptosis, however, once initiated, the rate and manner of cell death was similar to that observed at 37 °C. All nucleotide pools were similarly degraded at the time of apoptotic cell death. Temperature reduction to 30 °C did not decrease the energy charge of the cells, however, the overall rate of metabolism was reduced. The latter may be sufficient to extend culture viability via a reduction in toxic metabolites and/or limitation of nutrient deprivation. However, the possibility remains that the benefits of temperature reduction in terms of both viability and productivity are more directly associated with cultures spending extended time in G-1.     

微藻光密度与细胞密度及生物质的关系

以四种常见微藻,小球藻(Chlorella sp.XQ-20044)、栅藻(Scenedesmus sp.SS-200716)、绿球藻(Chlorococcum sp.)和螺旋藻(Spirulina sp.CH-164)为实验材料,用梯度稀释法测定对数生长期不同浓度藻液的光密度(OD)、细胞密度和生物质干重(DW),在光自养分批培养模式下对4种微藻进行OD-波长(350—800 nm)扫描,同时测定细胞密度和生物质干重,分析藻液OD与细胞密度、生物质干重的关系。结果表明:在任何波长下,对数生长期的4种微藻细胞密度与OD值、生物质干重与OD值的变化都不成比例,波长不同其拟合曲线偏离直线的程度不同。但是,在435 nm处这种关系最接近直线,可以用直线方程近似描述(R20.98),其它波长处细胞密度-OD、干重-OD的关系都可以用二项式方程很好地描述(R20.99)。因此,光密度法适用于连续和半连续培养,可以用435 nm处测得的OD值计算细胞密度与干重。但是在分批培养模式下,4种微藻DW/OD比值随着培养时间均逐渐上升。小球藻DW/OD540为0.19—0.44 g/L,栅藻DW/OD540为0.36—0.53 g/L,绿球藻DW/OD540为0.48—0.75 g/L,螺旋藻DW/OD560为0.46—0.74 g/L,因此分批培养模式下采用测定藻液OD值反映细胞密度和生物质的方法不适用,只有直接测定细胞密度和生物质才是准确的。研究结果为正确使用分光光度法监测微藻生长提供依据。