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  • On-line Measurements and Control of Viable Cell Density in Cell Culture Manufacturing Processes using Radio-frequency Impedance

    Author: John P. Carvell, Jason E. Dowd
    Publication date: 01-03-2006
    Abstract
    In this work, radio-frequency (RF) impedance is reviewed as a method for monitoring and controlling cell culture manufacturing processes. It is clear from the many publications cited that RF Impedance is regarded as an accurate and reliable method for measuring the live cell bio-volume both on-line and off-line and the technology is also suitable for animal cells in suspension, attached to micro-carriers or immobilized in fixed beds. In cGMP production, RF Impedance is being used in three main areas. Firstly, it is being used as a control instrument for maintaining consistent perfusion culture allowing the bioreactor to operate under optimum conditions for maximum production of recombinant proteins. In the second application it has not replaced traditional off-line live cell counting techniques but it is being used as an additional monitoring tool to check product conformance. Finally, RF Impedance is being used to monitor the concentration of live cells immobilized on micro-carriers or packed beds in cGMP processes where traditional off-line live cell counting methods are inaccurate or impossible to perform.
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  • The changing dielectric properties of CHO cells can be used to determine early apoptotic events in a bioprocess.

    Author: Katrin Braasch, Et al.
    Publication date: 01/01/70
    Abstract
    Cell density and viability are two important parameters that are monitored during a mammalian cell bioprocess. The predominant method used to determine the viability of a cell population is trypan blue exclusion. This marks the loss of viability by the inability of cells to exclude the dye as a result of membrane damage. However, the loss in cell membrane integrity can be regarded as a late-stage event in the demise of cells and often indicates the end of apoptosis. Early indicators of apoptosis are desirable in cell bioprocesses because the initial stages are reversible by appropriate intervention such as nutrient feeding. In our study five different and independent methods were compared for the determination of viable and/or total cell density as well as cell viability. These included the particle counter (Coulter Counter), image analyser (Cedex with trypan blue exclusion), an on-line capacitance probe (Aber-Instruments), an off-line flow cytometer (Guava Millipore) and a prototype dielectrophoretic (DEP) cytometer. The multiple techniques of cell monitoring were applied to the culture of a novel CHO cell line producing a human-camelid chimeric antibody (EG2) grown in a bench-top bioreactor. This is a unique study which determined the relationship between these techniques and may be applied to elucidate some important properties of cells during metabolic changes in a bioprocess.
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  • The Development of large scale continuous perfusion culture system for ES Cell expansion and cardiac differentiation

    Author: Masanori Wada, Et al.
    Publication date: 28-04-2012
    Abstract
    We have recently developed scalable expansion culture and differentiation system for mouse ES cells. This system allows to : - Produce mouse cardiomyocytes by continuous and industrial scale - Eliminate labor-intensive procedure of medium exchange and differentiation by using perfusion culture system - Show the usefulness of biomass monitor for process monitoring of ES cell culture
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  • Modelling real-time simultaneous saccharification and fermentation of lignocellulosic biomass and organic acid accumulation using dielectric spectroscopy

    Author: David N. Bryant, Stephen M. Morris
    Publication date: 02-10-2011
    Abstract
    Dielectric spectroscopy (DS) is routinely used in yeast and mammalian fermentations to quantitatively monitor viable biomass through the inherent capacitance of live cells; however, the use of DS to monitor the enzymatic break down of lignocellulosic biomass has not been reported. The aim of the current study was to examine the application of DS in monitoring the enzymatic saccharification of high sugar perennial ryegrass (HS-PRG) fibre and to relate the data to changes in chemical composition. DS was capable of both monitoring the on-line decrease in PRG fibre capacitance (C = 580 kHz) during enzymatic hydrolysis, together with the subsequent increase in conductivity (G = 580 kHz) resulting from the production of organic acids during microbial growth. Analysis of the fibre fractions revealed >50% of HS-PRG lignocellulose had undergone enzymatic hydrolysis. These data demonstrated the utility of DS biomass probes for on-line monitoring of simultaneous saccharification and fermentation (SSF).
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  • Application of dielectric spectroscopy for monitoring high cell density in monoclonal antibody producing CHO cell cultivations

    Author: Laszlo Parta, Denes Zalai, Sandor Borbely, Akos Putics
    Publication date: 25-06-2013
    Abstract
    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.
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